researcharticle enzymatic conversion of rbcs by -n...

Research ArticleEnzymatic Conversion of RBCs by 120572-N-Acetylgalactosaminidasefrom Spirosoma linguale

Thomas J Malinski 1 and Harkewal Singh2

1Department of Veterinary Pathobiology University of Missouri USA2Department of Chemistry University of Missouri USA

Correspondence should be addressed toThomas J Malinski thomasmalinskiboehringer-ingelheimcom

Received 25 January 2019 Accepted 6 March 2019 Published 2 May 2019

Academic Editor Qi-Zhuang Ye

Copyright copy 2019 Thomas J Malinski and Harkewal SinghThis is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Spirosoma linguale is a free-living nonpathogenic organism Like many other bacteria S linguale produces a cell-associated 120572-N-acetylgalactosaminidaseThis work was undertaken to elucidate the nature of this activityThe recombinant enzyme was producedpurified and examined for biochemical attributesThe purified enzymewassim50 kDa active as a homodimer in solution It catalyzedhydrolysis of 120572-N-acetylgalactosamine at pH 7 Calculated KM was 11 mMwith kcat of 173 s

minus1 The described enzyme belongs to theGH109 family

1 Introduction

Substantial effort has been placed on stabilizing the world-wide blood supply It is commonplace to see appeals for blooddonations because of low levels of supply Methods to addressmaintaining a stable blood supply or alternative procedurehave been in the research forefront Enzymatic conversionperfluorocarbons hemoglobin-based oxygen carriers andbonding RBC with polyethylene glycol methodologies havereceived a great amount of attention Stem cell research alsoholds promise however less work has been pursued in thisarea due to the political landscape Each strategy has meritbut they are not so without their technical challenges Allof these approaches in concert may eventually play a rolein stabilizing the blood supply The enzymatic conversionof RBC blood type has been a determined pursuit for overthree decades Several research groups have identified anumber of enzymes that have the potential to convert RBCblood type The enzymes targeted to amend the RBC toType O are 120572-N-acetylgalactosaminidase (A997888rarrO) and 120572-galactosidase (B997888rarrO) Presently we possess a novel 120572-N-acetylgalactosaminidase from Spirosoma linguale that con-verts Type A RBCs to Type O The emphasis of this researchinvolves enzymatically converting Type A RBC to O with a

recombinant 120572-NAGA originating from Spirosoma lingualeS linguale is a common free-living nonpathogenic organismwhich encodes an 120572-N-acetylgalactosaminidase (120572-NAGA)120572-NAGA is an exoglycosidase within the EC 32149 GH109family classification as described in the carbohydrate-activeenzyme database httpwwwcazyorg which specificallyhydrolyzes 120572-N-acetylgalactosamine from the nonreducingterminal sugar of glycoconjugates The ABO blood groupas described by Landsteiner [1] and characterized by KabatWatkins and Morgan [2 3] is rich in specific glycoconju-gates targeted by 120572-NAGA As indicated the human ABO(types A B AB and O) blood group system based on thepresence or absence of blood group antigens A and B ismainly differentiated by surface exposed carbohydrate groupsdesignated as ABH Serologically individuals with type Ablood group express Antigen A and antibody to AntigenB Similarly individuals with type B blood group expressAntigen B and antibody to Antigen A Blood group AB doesnot express antibodies in contrast type O individuals expressantibodies to both type A and type B antigens Thereforeindividuals with A antigen produce antibodies to type Bblood and vice versa and thus blood transfusions betweenincompatible blood types leads to immune response and canbe fatal

HindawiEnzyme ResearchVolume 2019 Article ID 6972835 27 pageshttpsdoiorg10115520196972835

2 Enzyme Research

Biochemically types A and B red blood cells (RBCs) havedifferent trisaccharide moieties attached to the respectiveglycoproteins and lipids on their surface Type A RBCs ortype A antigen has GalNAc120572-13-(Fuc120572-12)-Gal120573 trisaccha-rides that terminate in 120572-13-linked N-acetylgalactosamine(GalNAc) whereas the B-antigen consisted of Gal120572-13-(Fuc120572-12)-Gal120573 and terminates in 120572-13-linked galactose(Gal) monosaccharide Type O cells only terminate withFuc120572-12-Gal120573 disaccharide core possessing neither GalNAcnor Gal and are nonantigenic Consequently the majorbiochemical difference between type AB and O is theterminal monosaccharide moiety attached to the respectiveRBCs Thus enzymatic removal of terminal GalNAc or Galrepresents an attractive strategy to generate universal type Oblood

Goldstein and coworkers used120572 galactosidase from coffeebeans to convert type B blood to type O Unfortunately thisenzyme suffered from low turnover rate and was required incopious amounts for effective type O generation

This in mind 120572-NAGA is also under investigation toenzymatically convert blood group A to O A number ofenzymes have been identified that have attributes to convertRBC blood type [4ndash16] Two 120572-NAGAs that demonstratedconversion of Type A

2RBCs to blood group O originated

from chicken [17] and C perfringens [13] The enzymaticconversion conditions were not optimal acidic conditionsare required for the chicken enzyme and the C perfringensenzyme preparation comprised additional glycosidases facil-itating the conversion reaction Both of these enzymes havenot been able to demonstrate the enzymatic conversion ofType A

1RBCs To date conversion of Type A

1RBCs to O

has been the most challenging due to the added complexityof the antigen group [18] Currently there are relatively fewdescribed enzymes identifiedwith the desired characteristicsmarkedly efficient and active within the blood pH rangeand easily generated [19] Interestingly Liu et al discoveredfew efficient enzymes using synthetic substrates that functionat neutral pH Their study showed that nature is perhapsendowed with several naturally occurring enzymes capableof converting type A and type B blood However due tocomplicated biochemistry of type A antigens up to 60 mg ofenzyme was needed for efficient conversion Keeping this inmind we initiated our efforts in identifying and exploring thecatalytic properties of an 120572-NAGA enzyme to enzymaticallyconvert type A blood to type O blood In this report wedescribe the expression purification and characterizationof the 120572-NAGA from S linguale These studies evaluatedand characterized this 120572-NAGA to determine the enzymersquosoptimal conditions (pH temperature buffer effects ionicstrength and specific activity) for cleaving the terminal120572-N-acetylgalactosamine carbohydrate from type A RBCThis work also estimated an optimal 120572-NAGA concentrationper unit of packed RBC evaluated the feasibility of 120572-NAGA for converting RBC to a universal donor state andevaluated the effects on the RBC following the 120572-NAGAconversion

2 Materials and Methods

Spirosoma linguale 33905 was purchased from the AmericanType Culture Collection RockvilleMaryland Chromatogra-phy columns (HisTrap Q Sepharose and Zorbax GF-250)SDS-PAGE materials (gels buffers and protein markers)QuickChange Site-Directed Mutagenesis Kit (Stratagene)Western Blot materials (Nitrocellulose Bio Trace Pall LifeScience buffers and developing reagent) BCA and Bradfordprotein determination solutions BSA standards restrictionenzymes the competent cells E coli CD41 (DE3) and TEVprotease were obtained from Fisher Scientific WalthamMassachusetts Chromatography and assay reagents andmicrobiological media were obtained from Fisher ScientificWaltham Massachusetts or VWR Scientific Radnor Penn-sylvania Glycosidase conjugates and gel filtration molecularweight markers were obtained from Sigma-Aldrich Com-pany St Louis Missouri DNA primers were obtained fromIntegrated DNA Technologies Coralville Iowa The pCR-Blunt vector and the competent cells E coli DH5120572 and BL21(DE3) and gel chromatography column were obtained fromInvitrogen Carlsbad California The pKA8H vector wasobtained from theUniversity ofMissouri Gene amplificationwas completed using an Eppendorf Mastercycler pro ther-mal cycler Hauppauge New York Protein purification wasaccomplished with an AKTAprime plus chromatographysystem from GE Healthcare Piscataway New Jersey Gelchromatography was performed on a Shimadzu LC-10 sys-tem Columbia Maryland Protein and enzyme assays wereperformed on a FLUOstar Omega plate reader from BMGLABTECH Cary North Carolina Fluorescent tagged anti-bodies were obtained from Santa Cruz Biotechnology SantaCruz California Flow cell cytometry was performed on anAttune Acoustic Focusing Cytometer Carlsbad California

21 Cloning and Mutagenesis

S linguale 120572-NAGA The entire coding sequence of locusSlin 6637 was cloned from S linguale DNA primers weredesigned to incorporateNde1 and BamH1 restriction enzymesites for amplification of the target gene as described below

S linguale 120572-NAGA Primers

pKA8H Forward Primer CCCGGGCATATGCCTTCGCTGTACACGACCGCTCAG

pKA8H Reverse Primer CCCGGGGGATCCTCAGTACACGTCGGTAAGGCCAAAGATGGG

The S linguale nag gene was amplified by PCR with thefollowing cycling parameters 94∘C for 60 seconds 52∘C for60 seconds and 72∘C for 60 seconds increasing 10 secondsper cycle for 35 cycles and then maintained at 4∘C followingcompletionThe amplified gene productswere initially ligatedinto pCR-Blunt vector overnight in awater bath incubated at16∘CThe ligation product was transformed into E coliDH5120572and plated on LB Agar containing kanamycin (40 120583gmL)and then incubated overnight at 37∘C Following incuba-tion a single colony was picked and grown in LB media

Enzyme Research 3

supplemented with 40 120583gmL kanamycin These cultureswere incubated at 37∘C overnight by constant shaking at 250rpm in an incubator-shaker for further plasmid preparationDNAwas isolated double-digestedwithNde1BamH1 restric-tion enzymes and cloned into pKA8H vector Briefly thepKA8H vector codes for an N-terminal 8timesHis affinity tagand a tobacco etch virus protease site The cloned genes wereconfirmed by sequence analysis by the University ofMissouriDNA Core Facility

The Sl 120572-NAGA pKA8H plasmid was transformed intothe expression strain E coli BL21(DE3) or CD41(DE3) andplated on LB Agar containing kanamycin (50 120583gmL) andthen incubated overnight at 37∘C as described In additiona site-directed mutant of S linguale 120572-NAGA in which thepurported residue protonates the leaving group in conjunc-tion with NAD+ His225 was modified to Alanine (H225A)designed for the purpose of evaluating enzyme activity

S linguale 120572-NAGA H225A Mutation Primers

Forward Primer GATCTCTACCCCACGGCCGGTCTGGGGCCGGTG

Reverse Primer CACCGGCCCCAGACCGGCCGTGGGGTAGAGATC

The 120572-NAGA mutant was amplified by PCR using thefollowing cycling parameters 94∘C for 30 seconds 55∘C for60 seconds and 68∘C for 75 minutes for 20 cycles andthen maintained at 4∘C following completion The mutationwas introduced into the aforementioned plasmid using theQuickChange Site-Directed Mutagenesis Kit The clonedgenes were confirmed by sequence analysis by the Universityof Missouri DNA Core Facility

22 Protein Expression and Purification The Sl 120572-NAGA(cloned into pKA8H vector) plasmid was transformed intothe expression strain E coli BL21(DE3) and plated on LBAgar containing kanamycin (40 120583gmL) and then incubatedovernight at 37∘C Protein expression was carried out usingStudierrsquos autoinduction method [24] Briefly single coloniesof the transformants were picked to inoculate four 1000 mLcultures made of 1 (wv) tryptone 05 (wv) yeast extract50 mM phosphate 50 mM ammonium chloride 5 mMsulfate 2 mM magnesium 1x trace minerals 05 glycerol005 glucose 02 lactose and 50 120583gmL ampicillin Theculture was incubated at 37∘C with constant shaking at 300rpm overnight Cells were harvested approximately 20 hourslater by centrifugation at 4∘C and 2100 x g and resuspendedin 20 mM Tris 150 mM NaCl pH 75 or 20 mM sodiumphosphate 05 M NaCl pH 70 The cell pellet was frozenat -20∘C for later use Frozen cells were thawed at 4∘Cfollowed by disruption using sonication or French pressUnbroken cells and debris were removed by centrifugationat 4∘C for 60 min at 31000 x g Additional cellular debriswas removed by ultracentrifugation at 4∘C for 60 min at183960 x g Supernatant was loaded onto an immobilizedmetal-ion affinity chromatography column (Ni2+-chargedHisTRAP GE Healthcare) using 20 mM Tris 015 M NaClpH 75 (buffer A) The protein was eluted using buffer A

supplemented with 1M imidazole Fractions were pooled andcombined with TEV protease (1 mg TEVP for 40 mg of 120572-NAGA) and 1 mM Tris(3-hydroxypropyl)phosphine (THP)Samples were incubated for 8 hours at 20∘C and then dialyzedfor at least 20 hours in buffer A Dialyzed protein was loadedonto Ni++ immobilized column and tag free enzymes werecollected in the flow through Thus purified SlNAGA wasdialyzed into 20 mM Tris pH 75 overnight at 4∘C Upondialysis the proteinwas aliquoted into thin-walled PCR tubesquick-frozen in liquid nitrogen and stored at -70∘C Proteinconcentration was estimated using bicinchoninic acid (BCAProtein Assay Reagent) Thermo Fisher (Rockford IL) [25]or the Bradford assay (Bradford Ultra Coomassie-basedprotein quantitationmethod) and Expedeon (Harston Cam-bridgeshire) [26] Analytical size exclusion chromatographywas performed to assess the quality and oligomeric state ofthe protein

23 Enzyme Activity Assay To assess the enzyme activity anddetermine the preferred synthetic substrate a preliminarycolorimetric screen was established Briefly 80 120583L of 20 mMsodium phosphate pH 70 10 120583L each of eleven substrates(see below) and 10 mM and 10 120583L of 120572-NAGA (Ec and Slseparately) were incubated at 37∘C for 30 minutes At theend of 30 minutes the reaction was quenched using 500 mMglycine pH 100 and corresponding samples were measuredat 410 nm using a BMG FLUOstar Optima plate readerSubstrates tested included 4-Nitrophenyl conjugates ofN-acetyl-120572-D-galactosaminide 120573-D-glucopyranoside 120572-D-galactopyranoside 120572-L-arabinopyranoside N-acetyl-120572-D-glucosamine N-acetyl-120573-D-galactosaminide 120572-D-glucopy-ranoside 120572-D-mannopyranoside 120573-D-galactopyranoside 120572-L-fucopyranoside and N-acetyl-120573-D-glucosamine OD at410 nm was used to assess enzymersquos relative preferencefor substrates Determination of the Michaelis-Menten con-stants was completed with 4-Nitrophenyl N-acetyl-120572-D-galactosaminide The assay utilized 80 120583L of 20 mM sodiumphosphate pH 70 or 74 10 120583L of the substrate dilutionand the automated addition of 10 120583L of diluted enzymeAssays to determine the pH optimum of the enzyme for 4-Nitrophenyl N-acetyl-120572-D-galactosaminide were performedin 20 mM sodium phosphate in the pH range of 60-80 50 mM sodium acetate in the pH range of 30-60or 100 mM bicine pH 90 The assays were performed aspreviously described using the appropriate buffer solutionand substrate and enzyme dilution Assays to determinetemperature response of the enzyme for 4-NitrophenylN-acetyl-120572-D-galactosaminide were performed in 20 mMsodium phosphate pH 74 using a temperature range of 20-45∘C in increments of 5∘C The assays were performed aspreviously described using the buffer and substrate solutionat the desired temperature with the automated addition ofdiluted enzyme Product inhibition assays were performed todetermine the effect of N-acetylgalactosamine accumulationThe assays were performed at room temperature (sim22∘C) in70 120583L of 20 mM sodium phosphate pH 70 buffer 10 120583Lof the substrate dilution 10 120583L of N-acetylgalactosamine atconcentrations of 25 50 or 100 mM and the automatedaddition of 10 120583L diluted enzyme

4 Enzyme Research

24 Chromatography Methods Sample analysis by gel fil-tration chromatography was done as follows approximately500 120583L of purified protein sample was pipetted into a chro-matography vialThe chromatography vials were loaded ontoa sample tray and placed in the autoinjector The sequenceused for data acquisition included a mixture of five proteins(standard) plus samples from each recombinant proteinThe proteins in the standard solution included 120573-amylasealcohol dehydrogenase albumin carbonic anhydrase andcytochrome C A Shimadzu LC-10A system was used to dothe protein analysis The system was equipped with a ZorbaxGF-250 4-micron particle size 46 X 250 mm (internaldiameter X length) column for assays The column wasmaintained at a temperature of 30∘C A TBS (50 mM Tris150 mM NaCl) pH 74 mobile phase flowing at 10 mL perminute was used during the analyses A UV-Vis detector setat 280 nm wavelength was used to detect the proteins Chro-matographic data were collected with TotalChrom softwareversion 62101040104 The molecular mass determinationof unknown proteins was made by comparing the ratio ofVeVo for the protein in question to the VeVo of proteinstandards of known molecular mass (Ve elution volumeand Vo void volume) A calibration curve was generated byplotting the logarithms of the known molecular mass of eachprotein standard versus their respective VeVo value

25 RBC Enzyme Conversion Methods The initial proof ofconcept experiment to evaluate enzyme activity in vivo wasset up as follows 1 mL of human RBCs in glycine buffer (200mM pH 68) [19] with 100 120583g of enzyme was maintainedat room temperature (sim22∘C) These samples were evaluatedwith standard ABO typing solution Conversion of RBCfollowed many of the experiments that were performedduring the in vitro characterization of the enzyme(s) In vivoevaluation of the enzyme(s) included enzyme dose titrationkinetic activity buffer system PCV pH temperature productinhibition and reverse enzyme activity Blood groups A

1

and A2RBC were used in the enzyme dose titration and

temperature experiments the remaining experiments werecompleted with A

2RBC With the exception of the PCV

experiment each test was conducted with 20-30 PCV Allexperiments were completed with 05 or 1 mL of sampleAll samples (except kinetic activity samples) were invertedto mix every 15 minutes to minimize RBC aggregation with10 120583L aliquots removed at 1 and 2 hours and placed in 200120583L of PBS to discontinue enzyme activity Samples wereinverted tomix prior to each aliquot collection for the kineticactivity experiment Several buffer solutions were evaluatedglycine alanine lysine and PBS Glycine alanine and lysinewere tested at 150-300 mM concentrations with 50 mMincrements Phosphate buffer was evaluated at 5 mM inaddition phosphate buffer was evaluated with added NaClconcentrations (ionic strength) ranging from 0 to 20 mMat 5 mM increments Each assay used 100 120583g of enzymemaintained at room temperature A 250 mM glycine solutionwas used to evaluate enzyme activity at various pH Thesolutions were pH adjusted and ranged from 55 to 80 at 05pH increments Each assay used 100120583g of enzymemaintainedat room temperature Samples containing 25 50 75 and 100

percent PCV were tested to evaluate the impact of red cellconcentration on enzyme activity Each assay used 100 120583g ofenzymemaintained at room temperature An enzyme kineticexperiment composed of samples collected at 0 1 5 10 1530 45 60 75 90 105 and 120 minutes after addition of 120572-NAGA was completed with 100 120583g of enzyme Enzyme dosetitration experiments were completed with varying 120572-NAGAconcentrations at both refrigerated (sim 4∘C) and room (sim22∘C) conditions Enzyme concentrations tested were 0 1 510 20 25 30 40 50 60 70 75 80 90 100 250 300 350 400450 500 or 1000 120583g The product N-acetylgalactosaminewas utilized at concentrations of 10 50 or 100 mM andwas tested with blood group O to evaluate reverse enzymeactivity using 250 120583g of protein Inhibition of enzyme activitywas evaluated with blood group A

1RBC infused with 500

120583g 120572-NAGA The experiment included a sample under theconditions outlined a sample that at 1 hr was centrifuged andthe supernatant was removed and then replaced with freshbuffer and a sample had 1X additional buffer added after 1 hrAll samples were evaluated by Flow Cytometry

26 Flow CytometryMethod To determine antigenic conver-sion of RBC each sample was evaluated by Flow Cytometryafter enzymatic conversion Samples were stainedwithmouseAnti-H FITC mouse Anti-A and goat Anti-mouse PEmonoclonal antibodies The initial staining included 1 120583LAnti-H FITC and 1 120583L Anti-A with 10 120583L RBC and 50 120583LPBSThe samplesweremixed and incubated for 20minutes inthe dark at room temperature An additional 200 120583L PBS wasadded at the end of the incubation period and centrifuged for5 minutes at 1500 RPM The supernatant was removed andthe pellet was washed 2 additional times with 200 120583L PBS1 120583L of the PE antibody was added to each sample with 50120583L PBS mixed and incubated in the dark for 20 minutes atroom temperature The samples were washed as describedwith a final volume of 200 120583L PBS added 150 120583L of eachsample was diluted into 3 mL of PBS followed by analysis10000 events collected at a flow rate of 100 120583L per minute onthe Attune Acoustic Focusing Cytometer Unstained FITCand PE stained compensation samples were analyzed prior toRBC analysis

27 RBC Immunological Methods Nine New Zealand Whiterabbits were challenged with Types A O or ECO-A RBCLaboratory animals were utilized with approval of the MerialInstitutional Animal Care and Use Committee APS 11-99M-0912 On Days 0 and 21 each rabbit was challenged asindicated in Table 1 Preparation of challenge material wasperformed the day of challenge Day 0 treatments wereemulsified in Freundrsquos adjuvant while Day 21 was emulsifiedin Incomplete Freundrsquos one

On Day 0 rabbits were bled for baseline antibodiesand immunized with the test antigens in CFA The rabbitswere observed for adverse reactions to the challenge Therabbits were boosted with the test antigens in IFA on Day21 Blood samples were collected from the ear on Day 28 Allrabbits were anesthetized with acepromazine prior to bloodcollections Blood samples were collected into a serum tubes

Enzyme Research 5

Table 1 Rabbit challenge treatment schedule

Group Vaccine Dose volume Route ofadministration

Frequency ofadministration No of animals

1 Type A bloodgroup

500000 RBCsim1mL Subcutaneous 2 doses Days 0 and

21 3

2 Type O bloodgroup


21 3

3 Type A-ECOblood group


21 3

processed into serum and kept frozen at le20∘C Sampleswere analyzed against blood groups A and O for reactivityto antibodies by Flow Cytometry Undiluted serum sampleswere also analyzed for induced antibody response to Slinguale 120572-NAGA by Western Blot

3 Results

31 Protein Expression Purification and Characterization 120572-NAGA overexpressions were achieved using the methodsby Studier [24] as described Cells were harvested and theoverexpressed enzyme was released by rupturing Cell debriswas removed using two-step centrifugation and resultingsupernatant was used for further purification Both enzymeswere purified using two-step purification schemes thatis Ni++-IMAC affinity chromatography followed by ionexchange chromatography Enzyme activity was measuredusing the end point assay described above His tag thuspurified enzyme was cleaved using TEVP and subsequentaffinity step resulted in high amounts of tag free protein (datanot shown) Tag free 120572-NAGA was further purified using ionexchange chromatography Purified fraction of Tag free 120572-NAGA was injected onto size exclusion chromatography foranalysis 120572-NAGA eluted at column volume correspondingto a molecular mass of 96430 Da which suggested that theenzyme may exist in solution as dimer

To assess the relative activity of SlNAGA asemiquantitative enzyme activity assay was performedagainst several substrates Specifically 4-Nitrophenyl N-acetyl-120572-D-galactosaminide4-Nitrophenyl 120573-D-glucopyra-noside 4-Nitrophenyl 120572-D-galactopyranoside 4-Nitro-phenyl 120572-L-arabinopyranoside 4-Nitrophenyl N-acetyl-120572-D-glucosamine 4-Nitrophenyl N-acetyl-120573-D-galactos-aminide 4-Nitrophenyl 120572-D-glucopyranoside 4-Nitro-phenyl 120572-D-mannopyranoside p-Nitrophenyl 120573-D-galac-topyranoside 4-Nitrophenyl 120572-L-fucopyranoside and4-Nitrophenyl N-acetyl-120573-D-glucosamine were testedusing the method described above SlNAGA exhibitedthe highest activity against 4-Nitrophenyl N-acetyl-120572-D-galactosaminide (Figure 1) Furthermore steady statekinetic parameters of SlNAGA were estimated using 4-Nitrophenyl N-acetyl-120572-D-galactosaminide substrate At20∘C Km is estimated to be 063 mM while kcat is 128 sand catalytic efficiency is 203 (sminus1mMminus1) Similar specificactivity was demonstrated by E meningoseptica [28 29] andC perfringens [30] 120572-NAGA to 120572-N-acetylgalactosaminesubstrate

The enzyme assays characterizing120572-NAGAproteins fromS linguale included steady state kinetics with variable sub-strate(s) pH temperatures and buffers The assays wereperformed with 80 120583L buffer 10 120583L conjugate substrate and10 120583L enzyme in 96-well plates consisting of 100 120583L totalvolume A BMG FLUOstar Optima plate reader with a 410nm reference filter was used to collect data VisualEnzymics2010 andor Origin Pro v8 software was used to calculate theMichaelis-Menten factors

For the purified recombinant protein enzyme activityresponse to temperature variances is shown in Figure 2 TheS linguale enzyme activity was levelled and decreased withelevated temperature Eyring plot shows that the enzyme hasa linear response to temperature change as shown in Figure 3The Eyring equation is a theoretical model based on thetransition state that describes the temperature dependence ofthe reaction rate [25] S linguale enzyme activity has a linearresponse in the direction of lower temperature To evaluatethis response the enzyme activity was compared at 4∘C and25∘C Enzyme activity was approximately 25 lower at 4∘Cas shown in Figure 4

Michaelis-Menten steady state parameters were calcu-lated to evaluate temperature enzyme response character-istics as shown in Table 2 Assays to determine temper-ature response of the enzyme for 4-Nitrophenyl N-acetyl-120572-D-galactosaminide were performed in 20 mM sodiumphosphate pH 74 using a temperature range of 20-45∘Cin increments of 5∘C The turnover rate kcat of the Slinguale enzymewas relatively flat over the temperature rangetested At the same time substrate affinity KM increased andthen declined over the temperature range while the catalyticefficiency kcatKM remained somewhat constant

Assays to determine the pHoptimumof the enzyme for 4-Nitrophenyl N-acetyl-120572-D-galactosaminide were performedin 20 mM sodium phosphate in the pH range of 60-80 50mM sodium acetate in the pH range of 30-60 or 100 mMbicine pH 90 Enzyme activity decreased at acidic pH andthen peaked to be close to neutral pH and again decreasedas pH became alkaline Enzyme activity of S linguale was thehighest approximately pH 70 as shown in Figure 5

Michaelis-Menten steady state parameters were calcu-lated to evaluate pH enzyme response characteristics asshown in Table 3 S linguale enzyme exhibited increasedturnover rate as the optimal pH was achieved with decliningrate in the acidic or alkaline range S linguale 120572-NAGAsubstrate affinity turnover rate and catalytic efficiency wereat the highest levels close to neutral pH Van Etten has

6 Enzyme Research

020406080

100120

R

elat

ive A

ctiv

ity

S linguale -NAGA Relative Activity

4-Nitro

phenyl N-ac

etyl-

-D-ga

lactosam

ine

4-Nitro

phenyl -D

-glucopyra

noside

4-Nitro

phenyl -D

-galac

topyranosid

e

4-Nitro

phenyl -L-ar

abinopyranosid

e

4-Nitro

phenyl N-ac

etyl-

-D-gl

ucosaminide

4-Nitro

phenyl N-ac

etyl-

-D-ga

lactosam

ine

4-Nitro

pheny -D

-glucopyra

noside

4-Nitro

phenyl -D

-man

nopyranosid

e

p-Nitro

phenyl -D

-galac

topyranosid

e

4-Nitro

phenyl -L-fu

copyranosid

e

4-Nitro

phenyl N-ac

etyl-

-D-gl

ucosaminide

Figure 1 Relative S linguale 120572-NAGA activity to carbohydrate substrates

Table 2 Temperature Michaelis-Menten steady state kinetics

Enzyme Temperature ∘C 120583mole N-Acetylgalactosamines KM (mM) kcats

minus1 kcatKM (sminus1mMminus1)

S linguale120572-NAGA 20 014 plusmn 0005 063 plusmn 005 128 203

25 019 plusmn 0007 107 plusmn 007 173 16330 018 plusmn 0012 109 plusmn 012 165 15235 017 plusmn 0014 094 plusmn 015 154 16340 014 plusmn 0012 067 plusmn 012 133 19845 013 plusmn 0006 079 plusmn 008 122 154

0

50

100

150

200

15 25 35 45 55

kcat

(s-1

)

degCelsius

S linguale -NAGAEnzyme Activity vs Temperature

Figure 2 S linguale 120572-NAGA activity versus temperature

expressed concerns of evaluating pH optimum with con-jugated substrates due to effects of the pKa of the leavinggroup [26] The pKa of the leaving group of 4-NitrophenylN-acetyl-120572-D-galactosaminide is 76 With the leaving grouppKa within the pH range of the optimum of the enzyme thevalue of the conjugate data is uncertain

Inhibition analyses have been performed on similar120572-NAGA proteins Lui et al showed inhibition of Emeningoseptica enzyme activity with several divalent met-als Cu2+ Ni2+ and Zn2+ at concentrations of 1 or 10

S linguale -NAGA Eyring Plot

Eyring Plot

minus120

minus100

minus080

minus060

minus040

minus020

000

ln(k

T)

00032 00033 00034000311T

２2 = 097

Figure 3 Eyring plot S linguale 120572-NAGA activity versus tempera-ture

mM but EDTA did not impact enzyme activity at theseconcentrations [19] Undiluted Adsol solution tested againstthe C perfringens enzyme was the only solution to loweractivity as demonstrated by Hsieh and Smith [9] Productinhibition does not appear to be evaluated Because the goalis to use these enzymes to convert RBCs enzyme activity

Enzyme Research 7

Table 3 pH Michaelis-Menten steady state kinetics

Enzyme pH 120583mole N-Acetylgalactosamines KM (mM) kcats

minus1 kcatKM(sminus1mMminus1)


64 014 plusmn 002 089 plusmn 018 130 14570 050 plusmn 005 067 plusmn 012 459 68674 017 plusmn 001 020 plusmn 004 154 76580 008 plusmn 001 013 plusmn 003 75 579

Table 4 Michaelis-Menten single substrate inhibition kinetics

Enzyme 120583mole N-Acetyl-galactosamines KM (120583M) Ki (mM) kcats


S linguale120572-NAGA 015 plusmn 0006 080 plusmn 0069 018 plusmn 0024 138 173

Temperature

0

20

40

60

80

100

120

R

elat

ive A

ctiv

ity

4∘C 25∘C

Figure 4 S linguale 120572-NAGA activity 4∘C versus 25∘C

0

100

200

300

400

500

60 64 70 74 80

kcat

(s-1

)

pH

S linguale -NAGA Enzyme Activity vs pH

Figure 5 S linguale 120572-NAGA activity versus pH

brings about accumulation of product which may impactcomplete conversion of RBCs Approximately 04 120583ML ofN-acetylgalactosamine is present on RBCs The product N-acetylgalactosamine was used to test if increased productgeneration would inhibit enzyme activity The inhibitionassays were performed at room temperature (sim22∘C) in 70120583L of 20 mM sodium phosphate pH 70 buffer 10 120583L ofthe substrate dilution 10 120583L of N-acetylgalactosamine atconcentrations of 005 01 25 50 or 100 mM and the

minus1000

100200300400500600700800

minus10 0 10 20 30

1Ve

loci

ty

1[Substrate]

S linguale -NAGA N-Acetylgalactosamine Product Inhibition

No Inhibitor

0050 mM Inhibitor

010 mM Inhibitor

25 mM Inhibitor

Figure 6 Product inhibition S linguale 120572-NAGA activity

automated addition of 10 120583L diluted enzyme S linguale 120572-NAGA was affected by product generation The Lineweaver-Burk plots in Figure 6 provide an overview of productinhibition The S linguale enzyme appears to be sensitive toN-acetylgalactosamine concentrations as low as 50120583MThreeinhibition kinetic models competitive noncompetitive anduncompetitive were evaluated with the kinetic data usingVisualEnzymics 2010 to determine the appropriate model tocalculate the Ki VisualEnzymics allows the user to performan ANOVA comparing the modeled data to select the correctmodel procedure A competitive model was used to calculatethe Michaelis-Menten inhibition parameters of S linguale 120572-NAGAThe calculated parameters kcat and kcatKM indicatea marked difference compared to pH enzyme activity but toa lesser degree of the temperature data as shown in Table 4

A genetic mutation of the purported histidine cofactoramino acid 225 to alanine was introduced to evaluate Slinguale120572-NAGA activity Histidine 225 acts in concert with atightly integrated NAD+ cofactor during the hydrolysis reac-tion of the N-acetylgalactosamine molecule The mutation

8 Enzyme Research

15min

30min

1 hr 4 hr 7 hr 24 hr

Time

S linguale WT -NAGA vs H225A Activity

H225A

H225A wimidazoleWT -NAGA

0

05

1

15

2

25Ab

sorb

ance

410

nm

Figure 7 S linguale wild type versus H225A 120572-NAGA activity

0

100

200

300

400

500

600

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Time (Minutes)

280

nm (U

V)

Figure 8 S linguale 120572-NAGA purity by gel filtration chromatogra-phy

H225A eliminated enzymatic activity disrupting the ability ofthe enzyme to catalyze hydrolysis of N-acetylgalactosamineEnzymatic activity was monitored at selected intervals com-paring the wild type enzyme to H225A with or withoutsupplemental imidazole as shown in Figure 7 Imidazolesupplemented with wild type enzyme was not evaluatedbecause during purification steps it was visually noted thatenzyme activity was impeded

Purity of the S linguale enzyme by gel filtration chro-matography is shown in Figure 8 The estimated molecularweight of S linguale as determined by gel filtration chro-matography 96000 Da is shown in Table 5 and Figure 9 S

0

50

100

150

200

250

080 100 120 140 160

kDa

VeVo

Sl -NAGA

Figure 9Gel filtration chromatographymolecularmass estimation

3000

2500

2000

1500

1000

500

0

Cou

nt

minus105 minus103 104 106

PE Fluorescence

Figure 10 Flow cytometry profile of PE stained blood subgroups A1

and A2

linguale protein appears to act as dimer under the conditionstested

32 RBC Enzyme Conversion Results RBCs were stainedwith a fluorescent fluorophore phycoerythrin (PE) andfluorescein isothiocyanate (FITC) to determine blood groupstatus before andor after enzymatic conversion Blood groupA is composed of the subgroups A

1and A

2 each of which

generates a distinct PE flow cytometric histogram as shownin Figure 10

RBCs were subjected to S linguale 120572-NAGA to deter-mine the conversion of blood group A to O under severalconditions for example pH PCV temperature and enzymeamount (120583gmL) Four solutions were tested glycine alaninelysine and phosphate with varying amounts of NaCl todetermine an optimal conversion solution Glycine alanineand lysine solutions were tested at 150 200 250 300 and350 mM pH 70 The phosphate buffer solution was testedat 5 mM phosphate pH 70 with 0 5 10 15 20 or 25 mMNaCl The RBC conversion solutions glycine alanine andlysine figures are in 250 mM solutions S linguale 120572-NAGAwas evaluated under these conditions as shown in Figures11(a)ndash11(d) It appeared that conversion of blood group A to

Enzyme Research 9

PE Fluorescence

Cou

nt

1400

1200

1000

800

600

400

200

0100 101 102 103 106105104

Cou

nt

FITC Fluorescence

1400

1200

1000

800

600

400

200

0

100 101 102 103 104

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

105

104

102 103 106105104

(a) Glycine solution (Type O RBC and enzyme treated Type A2 RBC) FITC and PE fluorescence histograms and scattergram

Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104

FITC Fluorescence100 101 102 103

Cou

nt

1000

800

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0

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

102 103 106105104

105

104

(b) Alanine solution (Type O RBC and enzyme treated Type A2 RBC) FITC and PE fluorescence histograms and scattergram

Figure 11 Continued

10 Enzyme Research

FITC Fluorescence

Cou

nt1400

1200

1000

800

600

400

200

0

Cou

nt

1400

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PE Fluorescence100 101 102 103 106105104100 101 102 103 105104

FITC

Flu

ores

cenc

e

PE Fluorescence

102

103

106

105

104

102 103 106105104

(c) Lysine solution (Type O RBC and enzyme treated Type A2 RBC) FITC and PE fluorescence histograms and scattergram

Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106 107105104

(d) Phosphate buffer (TypeORBC and enzyme treated TypeA2 RBC) and PE fluorescence histogram (0 5 10 15 20 and25 mM NaCl)

Figure 11 S linguale 120572-NAGA blood group conversion by solution

Table 5 Gel filtration chromatography molecular mass estimation

S linguale 120572-NAGA(Da) Ve VeVo

120573-Amylase 200000 2451 100Alcohol dehydrogenase 150000 2776 113Albumin 66000 3030 124Carbonic anhydrase 29000 3157 129Cytochrome C 12400 3381 138 Monomer Dimer

Est (Da) Calculated (Da) Calculated (Da)S linguale 120572-NAGA 2939 120 96000 48215 96430

Enzyme Research 11

Cou

nt

PE Fluorescence100 101 102 103 106105104

1500

1000

500

0

S linguale -NAGA

Figure 12 250 mM glycine solution (Type O RBC Type A2RBC

and enzyme treated Type A2RBC) and PE fluorescence histogram

(55 60 65 70 75 and 80 pH)

O occurred in the glycine and alanine solutions but did notoccur in the lysine or phosphate buffer solutions following 1-hour incubation at room temperature

Enzymatic activity was determined in glycine and alaninesolutions Optimal pH enzyme activity was tested in 250 mMglycine solution pH 55 60 65 75 or 80 as shown inFigure 12 RBCs were incubated for 1 hour with S linguale120572-NAGA at room temperature

The effects of packed cell volume PCV related to enzymeactivity were tested with S linguale 120572-NAGA in 250 mMglycine pH 70 at room temperature with 25 50 or 75 percentPCV as shown in Figures 13(a) and 13(b) No additionalsolution was added to the 100 PCV sample

Various amounts of enzyme were used to determine theoptimal level to convert RBCs in solution The amount ofenzyme ranged from 1 to 1000 120583gmL A 250 mM glycine pH72 solution at room or refrigerated temperature was used toevaluate enzyme activity All Figures 14ndash21 will indicate theenzyme amounts and the testing conditions S linguale 120572-NAGA was tested at several enzyme concentrations rangingfrom 1 to 100 120583gmL of enzyme at room temperature and100 ndash 500 120583gmL of enzyme at refrigerated temperature oftreated Type A

2RBCs The initial test performed at room

temperature with 10 ndash 100 120583gmL of enzyme indicated con-version of RBCs without any apparent difference in enzymeconcentration required as shown in Figure 14 Additionaltesting with an enzyme concentration range of 1 ndash 100 120583gmLof enzyme provided definitive results At 1 hour of incubationapparent conversion of RBCs appeared between 50 and 100120583gmL of enzyme as shown in Figure 15 Allowing 2 hours ofincubation resulted in conversion of RBCs with between 25and 50 120583gmL of enzyme as shown in Figure 16 At refriger-ated temperatures approximately 8 times additional enzyme400 120583gmL is required to achieve conversion of RBCs asshown in Figure 17 TypeA

1RBCshave approximately 6 times

more antigen sites than A2RBCs 15 versus 025 million

respectively Initial titration of Type A1RBCs with 100 ndash 500

120583gmL of enzyme at refrigerated temperature did not convertthe RBCs to Type O At room temperature this enzyme titra-tion range yielded mixed results at the 500 120583gmL enzymeconcentration as shown in Figure 18 Several of the samples

have the Type A2flow cytometric signature indicating that

enzymatic conversion is occurring but is still incompleteBased on the in vitro data S linguale 120572-NAGA was shownto be sensitive to product accumulation An experiment toevaluate this potential event compared 3 samples set up asdescribed with 3 samples after 1-hour incubation the sampleswere centrifuged with the subsequent supernatant removedand replaced with fresh solution and with 3 samples after1-hour incubation additional solution was added effectivelydiluting the sample by two as shown in Figure 19 Nodifferences were noted from these two additional treatmentsWhile evaluating the data an unexpected result occurred onthe day glycine buffer was added to the Type A

1RBCs excess

material was prepared saved and used on two consecutivedays This involves the results noted in Figures 18 and 19These data were compared with the original data collected onDay 0 as shown in Figure 20(a) and the data collected onDay 1 overlaid with Day 0 data as shown in Figure 20(b)Thisdata suggests that pretreatment with the conversion solutionapproximately 24 hours prior to blood conversion enhancesenzymatic activity

In vivo kinetics of the S linguale 120572-NAGA was evaluatedwith Type A

2RBCs The experiment used 250 mM glycine

solution pH 70 with RBCs at approximately 30 PCV and100 120583gmL enzyme Samples were collected at preenzymetreatment (0 min) and 1 5 10 15 30 45 60 75 90 105 and120minutes after treatment Conversion of the Type A

2RBCs

appears to occur at approximately 15 minutes after enzymeaddition as shown in Figures 21(a)ndash21(c)

Plotting out the fluorescence response (events per sec-ond) over time appears to indicate saturation of the enzymeactivity during liberation of the 120572-N-acetylgalactosaminesugar from the A antigen as shown in Figure 22

Reverse enzyme activity was tested to determine anyresidual sugar addition to the antigen complex Type O RBCswere combined with enzyme 250 mM glycine solution pH72 and 0 10 50 or 100 mMN-acetylgalactosamine and thenincubated for 2 hours at room temperature Conversion toType A RBCs does not appear to occur in the presence of N-acetylgalactosamine as shown in Figure 23

In vitro S lingualeH225Amutant did not exhibit enzymeactivity The H225A mutant was also tested in vivo with TypeA1and A

2RBCs for the presence of enzyme activity Type A

1

and A2RBCs were combined with H225A enzyme and 250

mM glycine solution pH 72 and then incubated for 2 hoursat room temperature The H225A mutant does not appear toconvert Type A

1or A2RBCs to the Type O blood group as

shown in Figure 24

33 RBC Immunological Results Nine New Zealand Whiterabbits were challenged with Types A O or ECO-A RBCsLaboratory animals were utilized with approval of the MerialInstitutional Animal Care and Use Committee APS 11-99M-0912 On Days 0 and 21 each rabbit was challenged asindicated in Table 1 in Section 2 Preparation of challengematerial was performed the day of challenge and Day 0treatments were emulsified in complete Freundrsquos adjuvantwhile Day 21 was emulsified in incomplete Freundrsquos

12 Enzyme Research

Cou

nt

6000

5000

4000

3000

2000

1000

0


PE Fluorescence

(a) Full scale

Cou

nt

2500

2000

1500

1000

500

0


PE Fluorescence

(b) Magnified

Figure 13 250 mM glycine buffer (Type O RBC Type A2RBC and enzyme treated Type A

2RBC) and PE fluorescence histogram (100 75

50 and 25 PCV)

Cou

nt

1500

1000

500

0

PE Fluorescence100 101 102 103 106105104

Figure 14 S linguale 120572-NAGA at room temperature (Type ORBC Type A

2RBC and enzyme treated Type A

2RBC) and PE

fluorescence histogram (10 20 30 40 50 60 70 80 90 and 100120583gmL enzyme)

On Day 0 rabbits were bled for baseline antibodiesand immunized with the test antigens in CFA The rabbitswere observed for adverse reactions to the challenge Therabbits were boosted with the test antigens in IFA on Day 21Blood samples were collected from the ear on Day 28 All


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500

Figure 15 S linguale 120572-NAGA at room temperature for 1-hourincubation (Type O RBC and enzyme treated Type A

2RBC) and

PE fluorescence histogram (0 1 10 25 50 and 100 120583gmL enzyme)

rabbits were anesthetized prior to blood collections Bloodsamples were collected into serum tubes processed intoserum and kept frozen at le20∘C Samples were analyzedagainst blood group A tetrasaccharide and H trisaccharide

Enzyme Research 13


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

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3500


2RBC) and


Cou

nt

2000

1000

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3000

4000


PE Fluorescence

Figure 17 S linguale 120572-NAGA at refrigerated temperature 2-hourincubation (Type O RBC and enzyme treated Type A

2RBC) and PE

fluorescence histogram (350 and 400 120583gmL enzyme)


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500

Figure 18 S linguale 120572-NAGA at room temperature for 2-hourincubation (enzyme treated Type A

1RBC) and PE fluorescence

histogram (500 120583gmL enzyme)

substrates for reactivity to antibodies by Flow Cytometry asshown in Figures 25 and 26 Two of three rabbits challengedwith Type A RBCs not only responded strongly to the Aantigen tetrasaccharide but also responded moderately to theH antigen trisaccharide The rabbits challenged with TypeO RBCs did not appear to have an induced response to


PE Fluorescence

Cou

nt

2000

1000

0

3000

4000



histogram (500 120583gmL enzyme) Enzyme conversion without addi-tional steps enzyme conversion with solution replacement enzymeconversion with additional solution added and Type O RBC

either antigen Two of the ECO-A rabbits had a weak-to-lowantibody response to the A antigen substrate and all threerabbits responded similarly to the H antigen as the rabbitschallengedwithTypeORBCs Serum samples from the ECO-A challenged rabbits were also analyzed for induced antibodyresponse to S linguale 120572-NAGAbyWestern Blot as shown inFigure 27 There was no apparent induced antibody responseto S linguale 120572-NAGA by the ECO-A challenged rabbits

4 Discussion

Enzymatic conversion of one blood group to another has beensought for over three decades A number of enzymes havebeen identified that have desirable attributes and convert RBCblood types A B and AB to O [4ndash15 31] Two 120572-NAGAsthat demonstrated conversion of Type A

2RBCs to blood

group O originated from chicken [17] and C perfringens[13] The enzymatic conversion conditions were not optimalacidic conditions are required for the chicken enzyme andthe C perfringens enzyme preparation included additionalglycosidases Both of these enzymes have not been able todemonstrate the enzymatic conversion of Type A

1RBCs To

date conversion of Type A1RBCs to O has been the most

challenging due to the added complexity of the antigen group[18] Currently there are relatively few described enzymesidentified with the desired characteristics of highly efficientand specific active within the blood pH range and beingeasily generated [19] The results from this research projectare consistent with the identification of 120572-NAGA from Slinguale that has excellent properties to convert blood Type Ato O Our data suggest that the S linguale 120572-NAGA enzymehas potential use as a tool to convert type specific RBCs touniversal RBCsThis has been demonstrated by the specificityof the enzyme for the target substrate high enzyme activityusing various buffers temperatures and optimal activity ata neutral pH Sequence alignment shows that S linguale 120572-NAGA has 52 identity to the E meningoseptica 120572-NAGAas shown in Figure 28

14 Enzyme Research

1 Sample converted at Day 0 2 Sample incomplete conversion at Day 0


PE Fluorescence


PE Fluorescence

Cou

nt 2000

1000

0

3000

Cou

nt

2500

2000

1500

1000

500

0

3000

(a) Day 0 sample preparation (results in 1 hour results in 2 hours and control)



PE Fluorescenceminus105 minus103 104 106

PE Fluorescence

Cou

nt

2000

1000

0

3000

4000C

ount

2000

1000

0

3000

4000

5000

(b) Day 0 versus Day 1 comparison (results at Day 0 (1 hour) results at Day 1 (1 hour) and control)

Figure 20 S linguale 120572-NAGA at room temperature (enzyme treated Type A1RBC) and PE fluorescence histogram (500 120583gmL enzyme)

As determined by the relative identity and proteinsequence of the investigated 120572-NAGA S linguale enzymefalls within the GH109 family classification The GH109family involves a NAD+ dependent hydrolysis mechanismof 120572-N-acetylgalactosamine as described by Koshland [32]The glycoside hydrolase family 109 uses a mechanism thatrequires an NAD+ cofactor which remains tightly boundthroughout catalysis The reaction begins with an anionictransition state with elimination and redox steps Thereis an initial oxidation of the hydroxyl group attached tocarbon 3 by the enzyme-bound NAD+ cofactor This causesthe acidity of carbon 2 to increase whereby the enzymaticbase for example histidine 225 removes a proton An 120572120573-unsaturated intermediate is formed followed by the additionof water at the anomeric carbon The ketone at C3 is reducedgenerating the hydrolyzed sugar resetting the enzyme Thisreaction sequence was determined by NMR studies kineticisotope properties equilibrium studies X-ray crystallogra-phy and UVVis spectrophotometry [33 34] The S linguale

120572-NAGA purported hydrolytic reaction involves histidine225 in conjunction of the NAD+ cofactor as the H225Amutation abrogates enzymatic activity in vitro conjugatedsubstrate and in vivo RBCs

Initial physicochemical substrate specificity of theenzymewith the reagent120572-N-acetylgalactosaminewas highlyselective compared to eleven additional substrates Similarspecific activity was demonstrated by E meningoseptica[19 27] and C perfringens [35] 120572-NAGA to 120572-N-acetylgalactosamine substrate Optimal enzyme activitywas demonstrated by S linguale in the pH range of 60 -80 well within the range RBCs are stored Comparison ofMichaelis-Menten kinetic parameters in Table 6 illustratesthe diverse activity and substrate affinity of this enzymeclass The catalytic efficiency of the S linguale 120572-NAGAindicates approximately a 25 greater efficiency to the Emeningoseptica 120572-NAGA

S linguale exhibited a linear response to tempera-ture The enzyme originates from a mesophilic organism

Enzyme Research 15

Cou

nt

2000

1000

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3000

4000


PE Fluorescence(a) 0 ndash 10 minutes (0 1 5 and 10 minutes)

Cou

nt

2000

1000

0

3000

4000

6000

5000


PE Fluorescence(b) 15 ndash 60 minutes (15 30 45 and 60 minutes and TypeO RBC)


PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000

(c) 75 ndash 120 minutes (75 90 105 and 120 minutes andType O RBC)


Table 6 120572-NAGAMichaelis-Menten kinetic factors

120572-NAGA source KM (mM) kcat (sminus1) kcatKM (sminus1mMminus1) Difference kcatKMlowast

Elizabethkingia meningoseptica [19] 0077 98 128 78Elizabethkingia meningoseptica [27] 0063 75 119 73Spirosoma linguale 11 173 163 100lowastRelative to S linguale

where the response of S linguale 120572-NAGA activity moder-ately decreased with increasing temperature S linguale 120572-NAGA demonstrated optimal enzyme activity at 25∘CWhenassessed at 4∘C the S linguale enzyme activity decreasedapproximately to 25 with Type A

2RBCs

Using a phosphate buffer pH 70 as a base to relatethe effect on enzyme activity solutions composed of 50mM acetate 10 mM citrate 10 mM tris and 500 mMEDTA appeared to reduce relative enzyme activity A glycinesolution used in studies by Liu et al [19] and Yu et al [27]appeared to reduce enzyme activity in vitro compared tothe phosphate solution Liu et al showed inhibition of Emeningosetica enzyme activity with several divalent metalsCu2+ Ni2+ and Zn2+ at concentrations of 1 or 10 mM butEDTA did not impact enzyme activity at these concentra-tions [19] Undiluted Adsol solution tested against the C

perfringens enzyme was the only solution to lower activity asdemonstrated by Hsieh and Smith [9] Product accumulationwas tested for enzyme inhibition during this research projectThe S linguale 120572-NAGA indicated a concerning level ofcompetitive inhibition during product accumulation Thereaction mechanism proposed by Liu et al [19] outlinedthat H228 was involved with the NAD+ cofactor during theenzyme hydrolysis reaction The H228 as aligned with the Slinguale sequence was compared to H225 Histidine 225 wasmutated to an alanine to assess enzyme activity The H225Amutation resulted in the production of an inactive enzymewhen compared to the wild type recombinant 120572-NAGA

Conversion of TypeA1RBCs to blood groupOhas proved

to be difficult The ability to convert Type A2and B RBCs has

been known for over three decades The difficulty with TypeA1RBCs stands out because of the number of antigen sites

16 Enzyme Research

Table 7 120572-NAGA conversion rate relating antigen number to turnover rate

Blood type Antigen number kcat (sminus1) Conversion rate

(Hr)E meningosepticum 120572-NAGA A

1 150E+06 10 42A2 250000 10 69

S linguale 120572-NAGA A1 150E+06 173 24

A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)

Figure 22 S linguale 120572-NAGA enzyme conversion of treated TypeA2RBCs over time

Cou

nt

2000

1000

0

3000

4000


PE Fluorescence

Figure 23 Reverse enzyme activity S linguale 120572-NAGA at roomtemperature (enzyme treated Type O RBC) PE fluorescence his-togram (250 120583gmL enzyme) 2-hour incubation and control 0 mMsubstrate 10mM substrate 50mM substrate and 100mM substrate

the linkage type of the carbohydrate chains and the numberof carbohydrate repeats of the antigen of this blood groupcompared to Type A

2or B RBCs Table 7 provides a picture

of the difference between Types A1and A

2 The difference

illustrated is only of the approximate antigen number of thetwo Type A subgroups Using the in vitro turnover rate kcat(sminus1) of the two enzymes of this research and two closelyrelated enzymes the significant difference to convert Type ARBCs to blood group O is astounding

The 120572-NAGAs from S linguale and E meningosepticahave both demonstrated the capacity to convert Type A RBCto blood group O in this research and by two groups Liu etal [19] and Yu et al [27] respectively The E meningoseptica


PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000

Figure 24 S linguale H225A 120572-NAGA at room temperature(enzyme treated Type A

1and A

2RBC) PE fluorescence histogram

(250 120583gmL enzyme) 2-hour incubation Type A2RBC Type A

1

RBC and Type O RBC

enzymes used [19 27] had very similar steady stateMichaelis-Menten kinetic parameters An alignment of the proteinsequences is identical with the exception of four amino acidsat positions 160GgtV 174HgtR 426FgtS and 434VgtA Themajor variance between works reported by Liu et al andYu et al is the enzyme amount required to convert RBCsFor instance Liu et al show that to convert 200 mL ofpacked RBCs enzyme required for Type A

160 mg is needed

and for A215 mg on the other hand Yu et al reported the

enzyme requirements as 30 mg for A1and 08 mg for A

2 a

95 difference The estimated concentration of S linguale 120572-NAGA required to convert 200 mL of packed Type A

1RBCs

is 80 mg and for A220 mg There may be an explanation for

the difference but it is not evidentEvaluation of four solutions demonstrated that glycine

or alanine could equally serve as buffer of choice to convertRBCs Phosphate buffer with varied ionic strength wasa unsuitable buffer selection to convert RBCs for eitherenzyme Smith and Walker [36] showed that a glycinesolution was a superior selection to enzymatically convertRBCs Glycine and alanine are known to disrupt the negativecharged environment around the red cell membrane [37]

Erythrocytes (negative charges) in suspension causing arearrangement of charges through the formation of two ioniclayers that generate an electrical potential difference betweenthem are called the zeta potential as shown in Figure 29

Glycine and alanine both serve as potentiators reducingthe zeta potential around theRBC enhancing enzyme activity

Enzyme Research 17

Group 1 Rabbits Challenged to Type A RBCs


PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

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200

0

100

101

102

103

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104

SSC-

A

100

101

102

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107

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104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0

7 PreTrt Cy Type A7 PostTrt Cy Type A






Figure 25 Continued

18 Enzyme Research

Group 2 Rabbits Challenged to Type O RBCs

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

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107

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104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19

Group 3 Rabbits Challenged to Type ECO RBCs

Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

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104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

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104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0







Figure 25 Rabbit antibody response to Type A tetrasaccharide

20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

1 PreTrt Cy Type O1 PostTrt Cy Type O






Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21

Rabbit Antibody Response to Type O RBCsGroup 2 Rabbits Challenged to Type O RBCs

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research

Rabbit Antibody Response to Type O RBCs


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

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100

0

Cou

ntC

ount

250

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100

50

0







Figure 26 Rabbit antibody response to Type H antigen trisaccharide

Enzyme Research 23

195

142

96

71

48

33

28

Figure 27Western Blot of the S linguale 120572-NAGA ECO-A challenged rabbits Lanes 1 3 and 5 protein standard markers (kDa) lane 2 rabbit2 lane 4 rabbit 5 and lane 6 rabbit 9

Figure 28 120572-NAGA sequence alignment S linguale and E meningoseptica [20 21]

24 Enzyme Research

Negative NetErythrocyte membrane

Compact Layer

Diffuse Layer

Shear PlaneZeta Potential

Figure 29 Illustration of the zeta potential of RBCs [22]

+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2

OHminus (equal units)

pI = 597pH

pK1 = 234

pK2 = 960

Figure 30 Illustration of the titration of glycine [23]

The titration of glycine provides us with an understanding ofwhy it serves as a potentiator as shown in Figure 30 Becauseglycine and alanine are zwitterions within the pH range of4-8 they have a net zero charge This characteristic equallyallows both amino acids to neutralize both positive andnegative charges For this same reason lysine and phosphatesolutions having either a positive or a negative overall chargedo not create an environment for the enzyme to interact withRBCs because of the inability of reducing the zeta potential

This modified environment appears to improve the 120572-NAGA RBC interface leading to the hydrolysis of 120572-N-acetylgalactosamine An observation made during the con-version of RBCs involved pretreating cells in glycine bufferthe day prior to adding enzymeThis led to additional enzymeefficiency specifically for Type A

1RBC conversion Due

to the reduced zeta potential by glycine additional care ofthe blood sample is required when using these potentiatorsbecause agglutination of RBCs is augmented Jan and Chienshowed the effect of altering the charged environment aroundthe RBC impacted aggregation [38] Due to the effects ofglycine S linguale 120572-NAGA exhibited significant enzymeactivity in vivo across a broad pH range 55-80 The enzymedemonstrated efficient activity at both room and refrigeratedtemperatures but requires approximately 10X more enzymeswhen used at 4∘C Packed cell volume approaching 50has a negative impact on enzyme activity A packed cellvolume range used in this research was 20-35 In vitrotesting suggested that product accumulation would be com-petitive during enzyme activity Competitive inhibition dueto product accrual was not noted during any experimentExperimental data suggested the potential for reverse enzymeactivity when evaluating Flow Cytometry results collectedat 1 and 2 hours Testing with Type O RBCs and 120572-N-acetylgalactosamine indicated that conversion toTypeARBCdid not occur In vivo testing with the mutant H225A did notaccount for any conversion of Type A RBCs

Stability of the RBCs after enzymatic conversion isparamount if it is to advance beyond the research labora-tory environment On the surface the conversion processof RBCs seems to be minimal cleaving the terminal 120572-N-acetylgalactosamine Nevertheless this is a multiple step pro-cedure thatmay place stress on the RBCThis research projectinspected the stability of enzyme converted RBCs RBCcount hemoglobin concentration and hematocrit showedstatistical significance between Type A RBCs and enzymeconverted cells Visual hemolysis was observed in the enzymeconverted RBC samples The primary cause of hemolysisappears to be linked to the centrifugation steps in which theRBCs were subjected The Type A unconverted cells did notundergo centrifugation when prepared Graphically the datafrom the three groups Type A and cells converted at roomor refrigerated temperature appear similar Clinically theRBC samples may behave not differently Goldstein showedthat enzyme converted Type B cells behaved and survivedsimilarly to unconverted RBCs [12] Vosnidou et al incurredsimilar results as this research when comparing convertedand nonconverted RBCs [14] Kruskall et al noted thathemoglobin concentration decreased from approximately 74gdL after enzyme conversionwhichwas thought to be relatedto the washing steps following conversion of RBC [39]Within the same study RBC survival was found to be similarcomparing converted and nonconverted blood Additionalstudies should be conducted to fully evaluate the presence orabsence of cellular stress to converted RBCs

Typically foreign host proteins induce an immuneresponse when injected into the system The S linguale 120572-NAGA did not appear to induce an immunological reactionin the rabbit model when challenged with enzyme converted

Enzyme Research 25

RBCs A follow-up blood transfusion study will need tobe accomplished to provide a definitive indication whetherS linguale 120572-NAGA has immunological properties Twostudies using 120572ndashgalactosidase from coffee beans triggeredan immune response after transfusion of enzyme convertedblood in Rhesus monkeys and humans [15 39] Each of thestudies did not observe any adverse events relating to thisantibody responseThe two studies involved single transfusedunits of blood Transfusion of multiple units of blood overtime has not been investigated in subjects to evaluate for apotential increased immune response andor reaction

Infusing larger amounts of the enzymes to evaluateimmunologicaltoxicological concerns is currently sparse inthe literature A 150 120583gkg level of 120572ndashgalactosidase fromcoffee beans has been demonstrated to be safe in Rhesusmonkeys [15] Zhang determined that this concentrationrepresented approximately 2500X by body weight (120583gkg)safe concentrations of enzyme The Zhang study indicatedthat approximately 10 ngmL of enzyme remained afterconversion and washing of the RBCs An approximate 1Xdose of enzyme based on the Zhang study would equate to60 ngkg of120572-NAGANotwithstanding the immune responseto residual enzyme following conversion of the RBCs theresponse of blood transfusion reactions begets a higher levelof concern to the recipient

Nine New Zealand White rabbits were challenged withTypes A O or ECO-A RBCs Each rabbit was challengedon Days 0 and 21 according to group assignment Baselineand postchallenge blood samples were collected to deter-mine induced antibody response The samples were analyzedagainst blood group A tetrasaccharide and H trisaccharidesubstrates for reactivity to ABO blood antibodies by FlowCytometry Two of the rabbits challenged with Type A RBCsnot only responded strongly to the A antigen tetrasaccharidebut also respondedmoderately to theH antigen trisaccharideThe rabbits challenged with Type O RBCs did not appearto have an induced response to either antigen Two of theECO-A rabbits had aweak-to-low induced antibody responseto the A antigen substrate and all three rabbits respondedsimilarly to the H antigen as the rabbits challenged with TypeO RBCs This initial experiment provided valuable mixeddata but additional studies to evaluate transfused enzymeconverted blood to test subjects need to be completed todetermine the extent of an antibody responseThere has beenlimited experimental work to determine the effects of enzymeconverted transfused blood

In 1991 Lenny et al successfully transfused a smallvolume of B blood converted to O using a 120572-galactosidasefrom green coffee beans [40] In 1994 and 1995 Lenny et alsuccessfully transfused full unit(s) of converted B to O bloodinto volunteers [41 42] No clinical reactions were observedin the volunteers nor were there abnormal laboratory testresults from blood samples taken during the study In 2000Kruskall et al successfully transfused converted B to Oblood into 21 patients [39] No adverse reactions were notedin any of the patients Red blood cell survival was com-parable between patients transfused with converted versusnonconverted RBCs in all but two patients one patient had

gastrointestinal bleeding at the time of the transfusion andanother patientrsquos serumwas incompatible with the transfusedred blood cells Posttransfusion monitoring of the patientsnoted that 5 of 19 developed an increased anti-B titer Theseachievements not only have provided a step forward but alsohave raised additional questions Better understanding of theanti-B immune response and the cross-matching compatibil-ity issue with serum or plasma are pivotal to this aim Asstated these studies used 120572-galactosidase from green coffeebeans Currently additional immunological data that mayexist is not available regarding enzymes under investigation

5 Summary

This research program characterized the physicochemicalattributes of S linguale 120572-NAGA The initial process toconvert RBCs with the S linguale 120572-NAGA was establishedThe experimental testing provided confirmation to the con-version ofRBCsQuality and storage stability of the convertedRBCs was exhibited Initial data was produced to evaluatethe immunological properties of S linguale 120572-NAGA Theconcept and ground work has been established to convertRBCs with S linguale 120572-NAGA

Data Availability

The research data used to support the findings of this studyare included within the article

Disclosure

The present address of Harkewal Singh is Kaneka USInnovation Center 7979 Gateway Blvd Newark CA 94560The present address of Thomas J Malinski is Boehringer-Ingelheim Animal Health USA Inc Missouri Research Cen-ter 6498 Jade Road Fulton MO 65251

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] R Owen ldquoKarl Landsteiner and the first human marker locusrdquoGenetics vol 155 no 3 pp 995ndash998 2000

[2] W T J Morgan andW M Watkins ldquoUnravelling the biochem-ical basis of blood group ABO and Lewis antigenic specificityrdquoGlycoconjugate Journal vol 17 no 7-9 pp 501ndash530 2000

[3] E A Kabat and A E Bezer ldquoImmunochemical studies onblood groups I Estimation of a and b isoantibodies in humanserum by the quantitative preclpitin methodrdquo e Journal ofExperimental Medicine vol 82 no 3 pp 207ndash215 1945

[4] I Y Bakunina R A Kuhlmann L M Likhosherstov et al ldquo120572-N-acetylgalactosaminidase from marine bacterium Arenibac-ter latericius KMM 426T removing blood type specificity of A-erythrocytesrdquo Biochemistry (Moscow) vol 67 no 6 pp 689ndash695 2002

[5] S Dybus and D Aminoff ldquoAction of 120572-galactosidase fromClostridium sporogenes and coffee beans on blood group B

26 Enzyme Research

antigen of erythrocytes The effect on the viability of erythro-cytes in circulationrdquo Transfusion vol 23 no 3 pp 244ndash2471983

[6] M Harun-Or-Rashid T Matsuzawa Y Satoh et al ldquoPurifi-cation and characterization of 120572-N-Acetylgalactosaminidasesi and II from the starfish asterina amurensisrdquo BioscienceBiotechnology and Biochemistry vol 74 no 2 pp 256ndash2612010

[7] D J Hata and D S Smith ldquoBlood group B degrading activityof Ruminococcus gnavus120572-galactosidaserdquoArtificial Cells BloodSubstitutes and Biotechnology vol 32 no 2 pp 263ndash274 2004

[8] L C Hoskins E T Boulding and G Larson ldquoPurification andcharacterization of blood group A-degrading isoforms of 120572-N-acetylgalactosaminidase fromRuminococcus torques strain IX-70rdquo e Journal of Biological Chemistry vol 272 no 12 pp7932ndash7939 1997

[9] H Hsieh and D S Smith ldquoSeroconversion of A to O red cellmembranes with a-N-acetylgalactosaminidase from clostrid-ium perfringensrdquo Transfusion vol 41 pp 103Sndash103S 2001

[10] T Itoh and Y Uda ldquo120572-N-acetylgalactosaminidase from squidliver Purification and characterization of two enzymesrdquo eJournal of Biochemistry vol 95 no 4 pp 959ndash970 1984

[11] S Kadowaki T Ueda K Yamamoto H Kumagai and TTochikura ldquoIsolation and Characterization of a Blood GroupA Substance-degrading 120572-N-Acetylgalactosaminidase From anAcremonium sprdquo Agricultural and Biological Chemistry vol 53no 1 pp 111ndash120 1989

[12] J Goldstein G Siviglia RHurst L Lenny and L Reich ldquoGroupB erythrocytes enzymatically converted to group O survivenormally in A B and O individualsrdquo Science vol 215 no 4529pp 168ndash170 1982

[13] G N Levy and D Aminoff ldquoPurification and properties of 120572-N-acetylgalactosaminidase from Clostridium perfringensrdquoeJournal of Biological Chemistry vol 255 no 24 pp 11737ndash117421980

[14] N Vosnidou S Johnson M M Mitra et al ldquoSeroconversion oftype B to O erythrocytes using recombinant Glycine max 120572-D-galactosidaserdquo IUBMB Life vol 46 no 1 pp 175ndash186 1998

[15] Y-P Zhang F Gong G-Q Bao et al ldquoB to O erythrocyte con-version by the recombinant 120572-galactosidaserdquo Chinese MedicalJournal vol 120 no 13 pp 1145ndash1150 2007

[16] A Zhu C Monahan Z-K Wang and J Goldstein ldquoExpres-sion purification and characterization of recombinant 120572-N-acetylgalactosaminidase produced in the yeast Pichia pastorisrdquoProtein Expression and Purification vol 8 no 4 pp 456ndash4621996

[17] J Goldstein J Y Kuo L Lenny and G Siviglia EnzymaticConversion of Red Cells for Transfusion Defense TechnicalInformation Center 1985

[18] M L Olsson C A Hill H De La Vega et al ldquoUniversal redblood cells - Enzymatic conversion of blood group A and Bantigensrdquo Transfusion Clinique et Biologique vol 11 no 1 pp33ndash39 2004

[19] Q P Liu G Sulzenbacher H Yuan et al ldquoBacterial glycosidasesfor the production of universal red blood cellsrdquoNature Biotech-nology vol 25 no 4 pp 454ndash464 2007

[20] R Chenna H Sugawara T Koike et al ldquoMultiple sequencealignment with the Clustal series of programsrdquo Nucleic AcidsResearch vol 31 no 13 pp 3497ndash3500 2003

[21] P Gouet X Robert and E Courcelle ldquoESPriptENDscriptextracting and rendering sequence and 3D information from

atomic structures of proteinsrdquo Nucleic Acids Research vol 31no 13 pp 3320ndash3323 2003

[22] H P Fernandes C L Cesar and M D L Barjas-Castro ldquoElec-trical properties of the red blood cell membrane and immuno-hematological investigationrdquo Revista Brasileira de Hematologiae Hemoterapia vol 33 no 4 pp 297ndash301 2011

[23] D Nelson and M Cox Lehninger Principles of BiochemistryWH Freeman and Company 4th edition 2005

[24] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquo Analytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[25] R Chang Physical Chemistry for the Biosciences UniversityScience Books USA 2005

[26] R L V Etten ldquoHuman prostatic acid phosphatase a histidinephosphataserdquo Annals of the New York Academy of Sciences vol390 no 1 pp 27ndash51 1982

[27] C Yu H Xu L Wang J Zhang and Y Zhang ldquoHumanRBCs blood group conversion from A to O using a novel 120572-N-acetylgalactosaminidase of high specific activityrdquo ChineseScience Bulletin vol 53 no 13 pp 2008ndash2016 2008

[28] F W Studier ldquoProtein production by auto-induction in high-density shaking culturesrdquo Protein Expression and Purificationvol 41 no 1 pp 207ndash234 2005

[29] B I Cantarel P M Coutinho C Rancurel T Bernard V Lom-bard and B Henrissat ldquoThe Carbohydrate-Active EnZymesdatabase (CAZy) an expert resource for glycogenomicsrdquoNucleic Acids Research vol 37 no 1 pp D233ndashD238 2009

[30] D A Comfort K S Bobrov D R Ivanen et al ldquoBiochem-ical analysis of Thermotoga maritima GH36 120572-galactosidase(TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolasesrdquo Biochemistry vol 46 no 11 pp 3319ndash3330 2007

[31] A Zhu L Leng CMonahan et al ldquoCharacterization of recom-binant 120572-galactosidase for use in seroconversion from bloodgroup B to O of human erythrocytesrdquo Archives of Biochemistryand Biophysics vol 327 no 2 pp 324ndash329 1996

[32] D E Koshland ldquoStereochemistry and the mechanism of enzy-matic reactionsrdquo Biological Reviews of the Cambridge Philosoph-ical Society vol 28 no 4 pp 416ndash436 1953

[33] S S Rajan X Yang F Collart et al ldquoNovel Catalytic Mech-anism of Glycoside Hydrolysis Based on the Structure of anNAD+Mn2+-Dependent Phospho-120572-Glucosidase from Bacil-lus subtilisrdquo Structure vol 12 no 9 pp 1619ndash1629 2004

[34] V L Yip AVarrot G J Davies et al ldquoAnunusualmechanismofglycoside hydrolysis involving redox and elimination steps by afamily 4 beta-glycosidase fromThermotoga maritimardquo Journalof the American Chemical Society vol 126 article no 15237973pp 8354-8355

[35] H-Y Hsieh M J Calcutt L F Chapman M Mitra and DS Smith ldquoPurification and characterization of a recombinant120572-N- acetylgalactosaminidase from Clostridium perfringensrdquoProtein Expression and Purification vol 32 no 2 pp 309ndash3162003

[36] D S Smith and J C Walker Buffer System for IncreasingSeroconversion Efficiency U S Patent Ed The Curators of theUniversity of Missouri 1997

[37] Issit ldquoForces that hold red cells apartrdquo in Applied Blood GroupSerology pp 35-36 Montgomery (Scientific Publications)Miami Fl USA 1985

Enzyme Research 27

[38] K M Jan and S Chien ldquoRole of surface electric charge in redblood cell interactionsrdquoe Journal of General Physiology vol61 no 5 pp 638ndash654 1973

[39] M S Kruskall J P Aubuchon K YAnthony et al ldquoTransfusionto blood groupA andOpatients of groupBRBCs that have beenenzymatically converted to groupOrdquoTransfusion vol 40 no 11pp 1290ndash1298 2000

[40] L L Lenny R Hurst J Goldstein L J Benjamin and RL Jones ldquoSingle-unit transfusions of RBC enzymatically con-verted from group B to group O to A and O normal volunteersrdquoBlood vol 77 no 6 pp 1383ndash1388 1991

[41] L L Lenny R Hurst J Goldstein and R A GalbraithldquoTransfusions to group O subjects of 2 units of red cellsenzymatically converted from group B to groupOrdquoTransfusionvol 34 no 3 pp 209ndash214 1994

[42] L L Lenny R Hurst A Zhu J Goldstein and R A GalbraithldquoMultiple-unit and second transfusions of red cells enzymati-cally converted from group B to group O report on the end ofphase 1 trialsrdquo Transfusion vol 35 no 11 pp 899ndash902 1995

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

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Journal of Parasitology Research

GenomicsInternational Journal of


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The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

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Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



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ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

2 Enzyme Research

Biochemically types A and B red blood cells (RBCs) havedifferent trisaccharide moieties attached to the respectiveglycoproteins and lipids on their surface Type A RBCs ortype A antigen has GalNAc120572-13-(Fuc120572-12)-Gal120573 trisaccha-rides that terminate in 120572-13-linked N-acetylgalactosamine(GalNAc) whereas the B-antigen consisted of Gal120572-13-(Fuc120572-12)-Gal120573 and terminates in 120572-13-linked galactose(Gal) monosaccharide Type O cells only terminate withFuc120572-12-Gal120573 disaccharide core possessing neither GalNAcnor Gal and are nonantigenic Consequently the majorbiochemical difference between type AB and O is theterminal monosaccharide moiety attached to the respectiveRBCs Thus enzymatic removal of terminal GalNAc or Galrepresents an attractive strategy to generate universal type Oblood

Goldstein and coworkers used120572 galactosidase from coffeebeans to convert type B blood to type O Unfortunately thisenzyme suffered from low turnover rate and was required incopious amounts for effective type O generation

This in mind 120572-NAGA is also under investigation toenzymatically convert blood group A to O A number ofenzymes have been identified that have attributes to convertRBC blood type [4ndash16] Two 120572-NAGAs that demonstratedconversion of Type A

2RBCs to blood group O originated

from chicken [17] and C perfringens [13] The enzymaticconversion conditions were not optimal acidic conditionsare required for the chicken enzyme and the C perfringensenzyme preparation comprised additional glycosidases facil-itating the conversion reaction Both of these enzymes havenot been able to demonstrate the enzymatic conversion ofType A

1RBCs To date conversion of Type A

1RBCs to O

has been the most challenging due to the added complexityof the antigen group [18] Currently there are relatively fewdescribed enzymes identifiedwith the desired characteristicsmarkedly efficient and active within the blood pH rangeand easily generated [19] Interestingly Liu et al discoveredfew efficient enzymes using synthetic substrates that functionat neutral pH Their study showed that nature is perhapsendowed with several naturally occurring enzymes capableof converting type A and type B blood However due tocomplicated biochemistry of type A antigens up to 60 mg ofenzyme was needed for efficient conversion Keeping this inmind we initiated our efforts in identifying and exploring thecatalytic properties of an 120572-NAGA enzyme to enzymaticallyconvert type A blood to type O blood In this report wedescribe the expression purification and characterizationof the 120572-NAGA from S linguale These studies evaluatedand characterized this 120572-NAGA to determine the enzymersquosoptimal conditions (pH temperature buffer effects ionicstrength and specific activity) for cleaving the terminal120572-N-acetylgalactosamine carbohydrate from type A RBCThis work also estimated an optimal 120572-NAGA concentrationper unit of packed RBC evaluated the feasibility of 120572-NAGA for converting RBC to a universal donor state andevaluated the effects on the RBC following the 120572-NAGAconversion

2 Materials and Methods

Spirosoma linguale 33905 was purchased from the AmericanType Culture Collection RockvilleMaryland Chromatogra-phy columns (HisTrap Q Sepharose and Zorbax GF-250)SDS-PAGE materials (gels buffers and protein markers)QuickChange Site-Directed Mutagenesis Kit (Stratagene)Western Blot materials (Nitrocellulose Bio Trace Pall LifeScience buffers and developing reagent) BCA and Bradfordprotein determination solutions BSA standards restrictionenzymes the competent cells E coli CD41 (DE3) and TEVprotease were obtained from Fisher Scientific WalthamMassachusetts Chromatography and assay reagents andmicrobiological media were obtained from Fisher ScientificWaltham Massachusetts or VWR Scientific Radnor Penn-sylvania Glycosidase conjugates and gel filtration molecularweight markers were obtained from Sigma-Aldrich Com-pany St Louis Missouri DNA primers were obtained fromIntegrated DNA Technologies Coralville Iowa The pCR-Blunt vector and the competent cells E coli DH5120572 and BL21(DE3) and gel chromatography column were obtained fromInvitrogen Carlsbad California The pKA8H vector wasobtained from theUniversity ofMissouri Gene amplificationwas completed using an Eppendorf Mastercycler pro ther-mal cycler Hauppauge New York Protein purification wasaccomplished with an AKTAprime plus chromatographysystem from GE Healthcare Piscataway New Jersey Gelchromatography was performed on a Shimadzu LC-10 sys-tem Columbia Maryland Protein and enzyme assays wereperformed on a FLUOstar Omega plate reader from BMGLABTECH Cary North Carolina Fluorescent tagged anti-bodies were obtained from Santa Cruz Biotechnology SantaCruz California Flow cell cytometry was performed on anAttune Acoustic Focusing Cytometer Carlsbad California

21 Cloning and Mutagenesis

S linguale 120572-NAGA The entire coding sequence of locusSlin 6637 was cloned from S linguale DNA primers weredesigned to incorporateNde1 and BamH1 restriction enzymesites for amplification of the target gene as described below

S linguale 120572-NAGA Primers

pKA8H Forward Primer CCCGGGCATATGCCTTCGCTGTACACGACCGCTCAG

pKA8H Reverse Primer CCCGGGGGATCCTCAGTACACGTCGGTAAGGCCAAAGATGGG

The S linguale nag gene was amplified by PCR with thefollowing cycling parameters 94∘C for 60 seconds 52∘C for60 seconds and 72∘C for 60 seconds increasing 10 secondsper cycle for 35 cycles and then maintained at 4∘C followingcompletionThe amplified gene productswere initially ligatedinto pCR-Blunt vector overnight in awater bath incubated at16∘CThe ligation product was transformed into E coliDH5120572and plated on LB Agar containing kanamycin (40 120583gmL)and then incubated overnight at 37∘C Following incuba-tion a single colony was picked and grown in LB media

Enzyme Research 3










4 Enzyme Research



1











Enzyme Research 5




1 Type A bloodgroup


21 3

2 Type O bloodgroup


21 3



21 3


3 Results








6 Enzyme Research

020406080

100120

R

elat

ive A

ctiv

ity


4-Nitro

phenyl N-ac

etyl-

-D-ga

lactosam

ine

4-Nitro

phenyl -D

-glucopyra

noside

4-Nitro

phenyl -D

-galac

topyranosid

e

4-Nitro

phenyl -L-ar

abinopyranosid

e

4-Nitro

phenyl N-ac

etyl-

-D-gl

ucosaminide

4-Nitro

phenyl N-ac

etyl-

-D-ga

lactosam

ine

4-Nitro

pheny -D

-glucopyra

noside

4-Nitro

phenyl -D

-man

nopyranosid

e

p-Nitro

phenyl -D

-galac

topyranosid

e

4-Nitro

phenyl -L-fu

copyranosid

e

4-Nitro

phenyl N-ac

etyl-

-D-gl

ucosaminide







0

50

100

150

200

15 25 35 45 55

kcat

(s-1

)

degCelsius






Eyring Plot

minus120

minus100

minus080

minus060

minus040

minus020

000

ln(k

T)

00032 00033 00034000311T

２2 = 097



Enzyme Research 7










Temperature

0

20

40

60

80

100

120

R

elat

ive A

ctiv

ity

4∘C 25∘C


0

100

200

300

400

500

60 64 70 74 80

kcat

(s-1

)

pH




minus1000

100200300400500600700800

minus10 0 10 20 30

1Ve

loci

ty

1[Substrate]


No Inhibitor

0050 mM Inhibitor

010 mM Inhibitor

25 mM Inhibitor




8 Enzyme Research

15min

30min


Time


H225A


0

05

1

15

2

25Ab

sorb

ance

410

nm


0

100

200

300

400

500

600

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Time (Minutes)

280

nm (U

V)




0

50

100

150

200

250

080 100 120 140 160

kDa

VeVo

Sl -NAGA


3000

2500

2000

1500

1000

500

0

Cou

nt


PE Fluorescence


and A2



1and A

2 each of which



Enzyme Research 9

PE Fluorescence

Cou

nt

1400

1200

1000

800

600

400

200

0100 101 102 103 106105104

Cou

nt

FITC Fluorescence

1400

1200

1000

800

600

400

200

0

100 101 102 103 104

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104


Cou

nt

1000

800

600

400

200

0

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

102 103 106105104

105

104


Figure 11 Continued

10 Enzyme Research

FITC Fluorescence

Cou

nt1400

1200

1000

800

600

400

200

0

Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104100 101 102 103 105104

FITC

Flu

ores

cenc

e

PE Fluorescence

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106 107105104







Enzyme Research 11

Cou

nt

PE Fluorescence100 101 102 103 106105104

1500

1000

500

0

S linguale -NAGA



(55 60 65 70 75 and 80 pH)













1RBCs excess





2RBCs






1




shown in Figure 24


12 Enzyme Research

Cou

nt

6000

5000

4000

3000

2000

1000

0


PE Fluorescence

(a) Full scale

Cou

nt

2500

2000

1500

1000

500

0


PE Fluorescence

(b) Magnified



50 and 25 PCV)

Cou

nt

1500

1000

500

0

PE Fluorescence100 101 102 103 106105104



2RBC) and PE




PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and



Enzyme Research 13


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence


2RBC) and PE



PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500






PE Fluorescence

Cou

nt

2000

1000

0

3000

4000





4 Discussion


2RBCs to blood


1RBCs To



14 Enzyme Research



PE Fluorescence


PE Fluorescence

Cou

nt 2000

1000

0

3000

Cou

nt

2500

2000

1500

1000

500

0

3000





PE Fluorescence

Cou

nt

2000

1000

0

3000

4000C

ount

2000

1000

0

3000

4000

5000







Enzyme Research 15

Cou

nt

2000

1000

0

3000

4000



Cou

nt

2000

1000

0

3000

4000

6000

5000




PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000







2RBCs






16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Enzyme Research 3










4 Enzyme Research



1











Enzyme Research 5




1 Type A bloodgroup


21 3

2 Type O bloodgroup


21 3



21 3


3 Results








6 Enzyme Research

020406080

100120

R

elat

ive A

ctiv

ity


4-Nitro

phenyl N-ac

etyl-

-D-ga

lactosam

ine

4-Nitro

phenyl -D

-glucopyra

noside

4-Nitro

phenyl -D

-galac

topyranosid

e

4-Nitro

phenyl -L-ar

abinopyranosid

e

4-Nitro

phenyl N-ac

etyl-

-D-gl

ucosaminide

4-Nitro

phenyl N-ac

etyl-

-D-ga

lactosam

ine

4-Nitro

pheny -D

-glucopyra

noside

4-Nitro

phenyl -D

-man

nopyranosid

e

p-Nitro

phenyl -D

-galac

topyranosid

e

4-Nitro

phenyl -L-fu

copyranosid

e

4-Nitro

phenyl N-ac

etyl-

-D-gl

ucosaminide







0

50

100

150

200

15 25 35 45 55

kcat

(s-1

)

degCelsius






Eyring Plot

minus120

minus100

minus080

minus060

minus040

minus020

000

ln(k

T)

00032 00033 00034000311T

２2 = 097



Enzyme Research 7










Temperature

0

20

40

60

80

100

120

R

elat

ive A

ctiv

ity

4∘C 25∘C


0

100

200

300

400

500

60 64 70 74 80

kcat

(s-1

)

pH




minus1000

100200300400500600700800

minus10 0 10 20 30

1Ve

loci

ty

1[Substrate]


No Inhibitor

0050 mM Inhibitor

010 mM Inhibitor

25 mM Inhibitor




8 Enzyme Research

15min

30min


Time


H225A


0

05

1

15

2

25Ab

sorb

ance

410

nm


0

100

200

300

400

500

600

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Time (Minutes)

280

nm (U

V)




0

50

100

150

200

250

080 100 120 140 160

kDa

VeVo

Sl -NAGA


3000

2500

2000

1500

1000

500

0

Cou

nt


PE Fluorescence


and A2



1and A

2 each of which



Enzyme Research 9

PE Fluorescence

Cou

nt

1400

1200

1000

800

600

400

200

0100 101 102 103 106105104

Cou

nt

FITC Fluorescence

1400

1200

1000

800

600

400

200

0

100 101 102 103 104

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104


Cou

nt

1000

800

600

400

200

0

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

102 103 106105104

105

104


Figure 11 Continued

10 Enzyme Research

FITC Fluorescence

Cou

nt1400

1200

1000

800

600

400

200

0

Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104100 101 102 103 105104

FITC

Flu

ores

cenc

e

PE Fluorescence

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106 107105104







Enzyme Research 11

Cou

nt

PE Fluorescence100 101 102 103 106105104

1500

1000

500

0

S linguale -NAGA



(55 60 65 70 75 and 80 pH)













1RBCs excess





2RBCs






1




shown in Figure 24


12 Enzyme Research

Cou

nt

6000

5000

4000

3000

2000

1000

0


PE Fluorescence

(a) Full scale

Cou

nt

2500

2000

1500

1000

500

0


PE Fluorescence

(b) Magnified



50 and 25 PCV)

Cou

nt

1500

1000

500

0

PE Fluorescence100 101 102 103 106105104



2RBC) and PE




PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and



Enzyme Research 13


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence


2RBC) and PE



PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500






PE Fluorescence

Cou

nt

2000

1000

0

3000

4000





4 Discussion


2RBCs to blood


1RBCs To



14 Enzyme Research



PE Fluorescence


PE Fluorescence

Cou

nt 2000

1000

0

3000

Cou

nt

2500

2000

1500

1000

500

0

3000





PE Fluorescence

Cou

nt

2000

1000

0

3000

4000C

ount

2000

1000

0

3000

4000

5000







Enzyme Research 15

Cou

nt

2000

1000

0

3000

4000



Cou

nt

2000

1000

0

3000

4000

6000

5000




PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000







2RBCs






16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


4 Enzyme Research



1











Enzyme Research 5




1 Type A bloodgroup


21 3

2 Type O bloodgroup


21 3



21 3


3 Results








6 Enzyme Research

020406080

100120

R

elat

ive A

ctiv

ity


4-Nitro

phenyl N-ac

etyl-

-D-ga

lactosam

ine

4-Nitro

phenyl -D

-glucopyra

noside

4-Nitro

phenyl -D

-galac

topyranosid

e

4-Nitro

phenyl -L-ar

abinopyranosid

e

4-Nitro

phenyl N-ac

etyl-

-D-gl

ucosaminide

4-Nitro

phenyl N-ac

etyl-

-D-ga

lactosam

ine

4-Nitro

pheny -D

-glucopyra

noside

4-Nitro

phenyl -D

-man

nopyranosid

e

p-Nitro

phenyl -D

-galac

topyranosid

e

4-Nitro

phenyl -L-fu

copyranosid

e

4-Nitro

phenyl N-ac

etyl-

-D-gl

ucosaminide







0

50

100

150

200

15 25 35 45 55

kcat

(s-1

)

degCelsius






Eyring Plot

minus120

minus100

minus080

minus060

minus040

minus020

000

ln(k

T)

00032 00033 00034000311T

２2 = 097



Enzyme Research 7










Temperature

0

20

40

60

80

100

120

R

elat

ive A

ctiv

ity

4∘C 25∘C


0

100

200

300

400

500

60 64 70 74 80

kcat

(s-1

)

pH




minus1000

100200300400500600700800

minus10 0 10 20 30

1Ve

loci

ty

1[Substrate]


No Inhibitor

0050 mM Inhibitor

010 mM Inhibitor

25 mM Inhibitor




8 Enzyme Research

15min

30min


Time


H225A


0

05

1

15

2

25Ab

sorb

ance

410

nm


0

100

200

300

400

500

600

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Time (Minutes)

280

nm (U

V)




0

50

100

150

200

250

080 100 120 140 160

kDa

VeVo

Sl -NAGA


3000

2500

2000

1500

1000

500

0

Cou

nt


PE Fluorescence


and A2



1and A

2 each of which



Enzyme Research 9

PE Fluorescence

Cou

nt

1400

1200

1000

800

600

400

200

0100 101 102 103 106105104

Cou

nt

FITC Fluorescence

1400

1200

1000

800

600

400

200

0

100 101 102 103 104

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104


Cou

nt

1000

800

600

400

200

0

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

102 103 106105104

105

104


Figure 11 Continued

10 Enzyme Research

FITC Fluorescence

Cou

nt1400

1200

1000

800

600

400

200

0

Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104100 101 102 103 105104

FITC

Flu

ores

cenc

e

PE Fluorescence

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106 107105104







Enzyme Research 11

Cou

nt

PE Fluorescence100 101 102 103 106105104

1500

1000

500

0

S linguale -NAGA



(55 60 65 70 75 and 80 pH)













1RBCs excess





2RBCs






1




shown in Figure 24


12 Enzyme Research

Cou

nt

6000

5000

4000

3000

2000

1000

0


PE Fluorescence

(a) Full scale

Cou

nt

2500

2000

1500

1000

500

0


PE Fluorescence

(b) Magnified



50 and 25 PCV)

Cou

nt

1500

1000

500

0

PE Fluorescence100 101 102 103 106105104



2RBC) and PE




PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and



Enzyme Research 13


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence


2RBC) and PE



PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500






PE Fluorescence

Cou

nt

2000

1000

0

3000

4000





4 Discussion


2RBCs to blood


1RBCs To



14 Enzyme Research



PE Fluorescence


PE Fluorescence

Cou

nt 2000

1000

0

3000

Cou

nt

2500

2000

1500

1000

500

0

3000





PE Fluorescence

Cou

nt

2000

1000

0

3000

4000C

ount

2000

1000

0

3000

4000

5000







Enzyme Research 15

Cou

nt

2000

1000

0

3000

4000



Cou

nt

2000

1000

0

3000

4000

6000

5000




PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000







2RBCs






16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Enzyme Research 5




1 Type A bloodgroup


21 3

2 Type O bloodgroup


21 3



21 3


3 Results








6 Enzyme Research

020406080

100120

R

elat

ive A

ctiv

ity


4-Nitro

phenyl N-ac

etyl-

-D-ga

lactosam

ine

4-Nitro

phenyl -D

-glucopyra

noside

4-Nitro

phenyl -D

-galac

topyranosid

e

4-Nitro

phenyl -L-ar

abinopyranosid

e

4-Nitro

phenyl N-ac

etyl-

-D-gl

ucosaminide

4-Nitro

phenyl N-ac

etyl-

-D-ga

lactosam

ine

4-Nitro

pheny -D

-glucopyra

noside

4-Nitro

phenyl -D

-man

nopyranosid

e

p-Nitro

phenyl -D

-galac

topyranosid

e

4-Nitro

phenyl -L-fu

copyranosid

e

4-Nitro

phenyl N-ac

etyl-

-D-gl

ucosaminide







0

50

100

150

200

15 25 35 45 55

kcat

(s-1

)

degCelsius






Eyring Plot

minus120

minus100

minus080

minus060

minus040

minus020

000

ln(k

T)

00032 00033 00034000311T

２2 = 097



Enzyme Research 7










Temperature

0

20

40

60

80

100

120

R

elat

ive A

ctiv

ity

4∘C 25∘C


0

100

200

300

400

500

60 64 70 74 80

kcat

(s-1

)

pH




minus1000

100200300400500600700800

minus10 0 10 20 30

1Ve

loci

ty

1[Substrate]


No Inhibitor

0050 mM Inhibitor

010 mM Inhibitor

25 mM Inhibitor




8 Enzyme Research

15min

30min


Time


H225A


0

05

1

15

2

25Ab

sorb

ance

410

nm


0

100

200

300

400

500

600

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Time (Minutes)

280

nm (U

V)




0

50

100

150

200

250

080 100 120 140 160

kDa

VeVo

Sl -NAGA


3000

2500

2000

1500

1000

500

0

Cou

nt


PE Fluorescence


and A2



1and A

2 each of which



Enzyme Research 9

PE Fluorescence

Cou

nt

1400

1200

1000

800

600

400

200

0100 101 102 103 106105104

Cou

nt

FITC Fluorescence

1400

1200

1000

800

600

400

200

0

100 101 102 103 104

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104


Cou

nt

1000

800

600

400

200

0

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

102 103 106105104

105

104


Figure 11 Continued

10 Enzyme Research

FITC Fluorescence

Cou

nt1400

1200

1000

800

600

400

200

0

Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104100 101 102 103 105104

FITC

Flu

ores

cenc

e

PE Fluorescence

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106 107105104







Enzyme Research 11

Cou

nt

PE Fluorescence100 101 102 103 106105104

1500

1000

500

0

S linguale -NAGA



(55 60 65 70 75 and 80 pH)













1RBCs excess





2RBCs






1




shown in Figure 24


12 Enzyme Research

Cou

nt

6000

5000

4000

3000

2000

1000

0


PE Fluorescence

(a) Full scale

Cou

nt

2500

2000

1500

1000

500

0


PE Fluorescence

(b) Magnified



50 and 25 PCV)

Cou

nt

1500

1000

500

0

PE Fluorescence100 101 102 103 106105104



2RBC) and PE




PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and



Enzyme Research 13


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence


2RBC) and PE



PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500






PE Fluorescence

Cou

nt

2000

1000

0

3000

4000





4 Discussion


2RBCs to blood


1RBCs To



14 Enzyme Research



PE Fluorescence


PE Fluorescence

Cou

nt 2000

1000

0

3000

Cou

nt

2500

2000

1500

1000

500

0

3000





PE Fluorescence

Cou

nt

2000

1000

0

3000

4000C

ount

2000

1000

0

3000

4000

5000







Enzyme Research 15

Cou

nt

2000

1000

0

3000

4000



Cou

nt

2000

1000

0

3000

4000

6000

5000




PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000







2RBCs






16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


6 Enzyme Research

020406080

100120

R

elat

ive A

ctiv

ity


4-Nitro

phenyl N-ac

etyl-

-D-ga

lactosam

ine

4-Nitro

phenyl -D

-glucopyra

noside

4-Nitro

phenyl -D

-galac

topyranosid

e

4-Nitro

phenyl -L-ar

abinopyranosid

e

4-Nitro

phenyl N-ac

etyl-

-D-gl

ucosaminide

4-Nitro

phenyl N-ac

etyl-

-D-ga

lactosam

ine

4-Nitro

pheny -D

-glucopyra

noside

4-Nitro

phenyl -D

-man

nopyranosid

e

p-Nitro

phenyl -D

-galac

topyranosid

e

4-Nitro

phenyl -L-fu

copyranosid

e

4-Nitro

phenyl N-ac

etyl-

-D-gl

ucosaminide







0

50

100

150

200

15 25 35 45 55

kcat

(s-1

)

degCelsius






Eyring Plot

minus120

minus100

minus080

minus060

minus040

minus020

000

ln(k

T)

00032 00033 00034000311T

２2 = 097



Enzyme Research 7










Temperature

0

20

40

60

80

100

120

R

elat

ive A

ctiv

ity

4∘C 25∘C


0

100

200

300

400

500

60 64 70 74 80

kcat

(s-1

)

pH




minus1000

100200300400500600700800

minus10 0 10 20 30

1Ve

loci

ty

1[Substrate]


No Inhibitor

0050 mM Inhibitor

010 mM Inhibitor

25 mM Inhibitor




8 Enzyme Research

15min

30min


Time


H225A


0

05

1

15

2

25Ab

sorb

ance

410

nm


0

100

200

300

400

500

600

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Time (Minutes)

280

nm (U

V)




0

50

100

150

200

250

080 100 120 140 160

kDa

VeVo

Sl -NAGA


3000

2500

2000

1500

1000

500

0

Cou

nt


PE Fluorescence


and A2



1and A

2 each of which



Enzyme Research 9

PE Fluorescence

Cou

nt

1400

1200

1000

800

600

400

200

0100 101 102 103 106105104

Cou

nt

FITC Fluorescence

1400

1200

1000

800

600

400

200

0

100 101 102 103 104

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104


Cou

nt

1000

800

600

400

200

0

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

102 103 106105104

105

104


Figure 11 Continued

10 Enzyme Research

FITC Fluorescence

Cou

nt1400

1200

1000

800

600

400

200

0

Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104100 101 102 103 105104

FITC

Flu

ores

cenc

e

PE Fluorescence

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106 107105104







Enzyme Research 11

Cou

nt

PE Fluorescence100 101 102 103 106105104

1500

1000

500

0

S linguale -NAGA



(55 60 65 70 75 and 80 pH)













1RBCs excess





2RBCs






1




shown in Figure 24


12 Enzyme Research

Cou

nt

6000

5000

4000

3000

2000

1000

0


PE Fluorescence

(a) Full scale

Cou

nt

2500

2000

1500

1000

500

0


PE Fluorescence

(b) Magnified



50 and 25 PCV)

Cou

nt

1500

1000

500

0

PE Fluorescence100 101 102 103 106105104



2RBC) and PE




PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and



Enzyme Research 13


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence


2RBC) and PE



PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500






PE Fluorescence

Cou

nt

2000

1000

0

3000

4000





4 Discussion


2RBCs to blood


1RBCs To



14 Enzyme Research



PE Fluorescence


PE Fluorescence

Cou

nt 2000

1000

0

3000

Cou

nt

2500

2000

1500

1000

500

0

3000





PE Fluorescence

Cou

nt

2000

1000

0

3000

4000C

ount

2000

1000

0

3000

4000

5000







Enzyme Research 15

Cou

nt

2000

1000

0

3000

4000



Cou

nt

2000

1000

0

3000

4000

6000

5000




PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000







2RBCs






16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Enzyme Research 7










Temperature

0

20

40

60

80

100

120

R

elat

ive A

ctiv

ity

4∘C 25∘C


0

100

200

300

400

500

60 64 70 74 80

kcat

(s-1

)

pH




minus1000

100200300400500600700800

minus10 0 10 20 30

1Ve

loci

ty

1[Substrate]


No Inhibitor

0050 mM Inhibitor

010 mM Inhibitor

25 mM Inhibitor




8 Enzyme Research

15min

30min


Time


H225A


0

05

1

15

2

25Ab

sorb

ance

410

nm


0

100

200

300

400

500

600

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Time (Minutes)

280

nm (U

V)




0

50

100

150

200

250

080 100 120 140 160

kDa

VeVo

Sl -NAGA


3000

2500

2000

1500

1000

500

0

Cou

nt


PE Fluorescence


and A2



1and A

2 each of which



Enzyme Research 9

PE Fluorescence

Cou

nt

1400

1200

1000

800

600

400

200

0100 101 102 103 106105104

Cou

nt

FITC Fluorescence

1400

1200

1000

800

600

400

200

0

100 101 102 103 104

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104


Cou

nt

1000

800

600

400

200

0

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

102 103 106105104

105

104


Figure 11 Continued

10 Enzyme Research

FITC Fluorescence

Cou

nt1400

1200

1000

800

600

400

200

0

Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104100 101 102 103 105104

FITC

Flu

ores

cenc

e

PE Fluorescence

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106 107105104







Enzyme Research 11

Cou

nt

PE Fluorescence100 101 102 103 106105104

1500

1000

500

0

S linguale -NAGA



(55 60 65 70 75 and 80 pH)













1RBCs excess





2RBCs






1




shown in Figure 24


12 Enzyme Research

Cou

nt

6000

5000

4000

3000

2000

1000

0


PE Fluorescence

(a) Full scale

Cou

nt

2500

2000

1500

1000

500

0


PE Fluorescence

(b) Magnified



50 and 25 PCV)

Cou

nt

1500

1000

500

0

PE Fluorescence100 101 102 103 106105104



2RBC) and PE




PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and



Enzyme Research 13


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence


2RBC) and PE



PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500






PE Fluorescence

Cou

nt

2000

1000

0

3000

4000





4 Discussion


2RBCs to blood


1RBCs To



14 Enzyme Research



PE Fluorescence


PE Fluorescence

Cou

nt 2000

1000

0

3000

Cou

nt

2500

2000

1500

1000

500

0

3000





PE Fluorescence

Cou

nt

2000

1000

0

3000

4000C

ount

2000

1000

0

3000

4000

5000







Enzyme Research 15

Cou

nt

2000

1000

0

3000

4000



Cou

nt

2000

1000

0

3000

4000

6000

5000




PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000







2RBCs






16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


8 Enzyme Research

15min

30min


Time


H225A


0

05

1

15

2

25Ab

sorb

ance

410

nm


0

100

200

300

400

500

600

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Time (Minutes)

280

nm (U

V)




0

50

100

150

200

250

080 100 120 140 160

kDa

VeVo

Sl -NAGA


3000

2500

2000

1500

1000

500

0

Cou

nt


PE Fluorescence


and A2



1and A

2 each of which



Enzyme Research 9

PE Fluorescence

Cou

nt

1400

1200

1000

800

600

400

200

0100 101 102 103 106105104

Cou

nt

FITC Fluorescence

1400

1200

1000

800

600

400

200

0

100 101 102 103 104

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104


Cou

nt

1000

800

600

400

200

0

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

102 103 106105104

105

104


Figure 11 Continued

10 Enzyme Research

FITC Fluorescence

Cou

nt1400

1200

1000

800

600

400

200

0

Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104100 101 102 103 105104

FITC

Flu

ores

cenc

e

PE Fluorescence

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106 107105104







Enzyme Research 11

Cou

nt

PE Fluorescence100 101 102 103 106105104

1500

1000

500

0

S linguale -NAGA



(55 60 65 70 75 and 80 pH)













1RBCs excess





2RBCs






1




shown in Figure 24


12 Enzyme Research

Cou

nt

6000

5000

4000

3000

2000

1000

0


PE Fluorescence

(a) Full scale

Cou

nt

2500

2000

1500

1000

500

0


PE Fluorescence

(b) Magnified



50 and 25 PCV)

Cou

nt

1500

1000

500

0

PE Fluorescence100 101 102 103 106105104



2RBC) and PE




PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and



Enzyme Research 13


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence


2RBC) and PE



PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500






PE Fluorescence

Cou

nt

2000

1000

0

3000

4000





4 Discussion


2RBCs to blood


1RBCs To



14 Enzyme Research



PE Fluorescence


PE Fluorescence

Cou

nt 2000

1000

0

3000

Cou

nt

2500

2000

1500

1000

500

0

3000





PE Fluorescence

Cou

nt

2000

1000

0

3000

4000C

ount

2000

1000

0

3000

4000

5000







Enzyme Research 15

Cou

nt

2000

1000

0

3000

4000



Cou

nt

2000

1000

0

3000

4000

6000

5000




PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000







2RBCs






16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Enzyme Research 9

PE Fluorescence

Cou

nt

1400

1200

1000

800

600

400

200

0100 101 102 103 106105104

Cou

nt

FITC Fluorescence

1400

1200

1000

800

600

400

200

0

100 101 102 103 104

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104


Cou

nt

1000

800

600

400

200

0

PE Fluorescence

FITC

Flu

ores

cenc

e

102

103

106

102 103 106105104

105

104


Figure 11 Continued

10 Enzyme Research

FITC Fluorescence

Cou

nt1400

1200

1000

800

600

400

200

0

Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104100 101 102 103 105104

FITC

Flu

ores

cenc

e

PE Fluorescence

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106 107105104







Enzyme Research 11

Cou

nt

PE Fluorescence100 101 102 103 106105104

1500

1000

500

0

S linguale -NAGA



(55 60 65 70 75 and 80 pH)













1RBCs excess





2RBCs






1




shown in Figure 24


12 Enzyme Research

Cou

nt

6000

5000

4000

3000

2000

1000

0


PE Fluorescence

(a) Full scale

Cou

nt

2500

2000

1500

1000

500

0


PE Fluorescence

(b) Magnified



50 and 25 PCV)

Cou

nt

1500

1000

500

0

PE Fluorescence100 101 102 103 106105104



2RBC) and PE




PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and



Enzyme Research 13


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence


2RBC) and PE



PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500






PE Fluorescence

Cou

nt

2000

1000

0

3000

4000





4 Discussion


2RBCs to blood


1RBCs To



14 Enzyme Research



PE Fluorescence


PE Fluorescence

Cou

nt 2000

1000

0

3000

Cou

nt

2500

2000

1500

1000

500

0

3000





PE Fluorescence

Cou

nt

2000

1000

0

3000

4000C

ount

2000

1000

0

3000

4000

5000







Enzyme Research 15

Cou

nt

2000

1000

0

3000

4000



Cou

nt

2000

1000

0

3000

4000

6000

5000




PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000







2RBCs






16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


10 Enzyme Research

FITC Fluorescence

Cou

nt1400

1200

1000

800

600

400

200

0

Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106105104100 101 102 103 105104

FITC

Flu

ores

cenc

e

PE Fluorescence

102

103

106

105

104

102 103 106105104


Cou

nt

1400

1200

1000

800

600

400

200

0

PE Fluorescence100 101 102 103 106 107105104







Enzyme Research 11

Cou

nt

PE Fluorescence100 101 102 103 106105104

1500

1000

500

0

S linguale -NAGA



(55 60 65 70 75 and 80 pH)













1RBCs excess





2RBCs






1




shown in Figure 24


12 Enzyme Research

Cou

nt

6000

5000

4000

3000

2000

1000

0


PE Fluorescence

(a) Full scale

Cou

nt

2500

2000

1500

1000

500

0


PE Fluorescence

(b) Magnified



50 and 25 PCV)

Cou

nt

1500

1000

500

0

PE Fluorescence100 101 102 103 106105104



2RBC) and PE




PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and



Enzyme Research 13


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence


2RBC) and PE



PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500






PE Fluorescence

Cou

nt

2000

1000

0

3000

4000





4 Discussion


2RBCs to blood


1RBCs To



14 Enzyme Research



PE Fluorescence


PE Fluorescence

Cou

nt 2000

1000

0

3000

Cou

nt

2500

2000

1500

1000

500

0

3000





PE Fluorescence

Cou

nt

2000

1000

0

3000

4000C

ount

2000

1000

0

3000

4000

5000







Enzyme Research 15

Cou

nt

2000

1000

0

3000

4000



Cou

nt

2000

1000

0

3000

4000

6000

5000




PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000







2RBCs






16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Enzyme Research 11

Cou

nt

PE Fluorescence100 101 102 103 106105104

1500

1000

500

0

S linguale -NAGA



(55 60 65 70 75 and 80 pH)













1RBCs excess





2RBCs






1




shown in Figure 24


12 Enzyme Research

Cou

nt

6000

5000

4000

3000

2000

1000

0


PE Fluorescence

(a) Full scale

Cou

nt

2500

2000

1500

1000

500

0


PE Fluorescence

(b) Magnified



50 and 25 PCV)

Cou

nt

1500

1000

500

0

PE Fluorescence100 101 102 103 106105104



2RBC) and PE




PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and



Enzyme Research 13


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence


2RBC) and PE



PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500






PE Fluorescence

Cou

nt

2000

1000

0

3000

4000





4 Discussion


2RBCs to blood


1RBCs To



14 Enzyme Research



PE Fluorescence


PE Fluorescence

Cou

nt 2000

1000

0

3000

Cou

nt

2500

2000

1500

1000

500

0

3000





PE Fluorescence

Cou

nt

2000

1000

0

3000

4000C

ount

2000

1000

0

3000

4000

5000







Enzyme Research 15

Cou

nt

2000

1000

0

3000

4000



Cou

nt

2000

1000

0

3000

4000

6000

5000




PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000







2RBCs






16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


12 Enzyme Research

Cou

nt

6000

5000

4000

3000

2000

1000

0


PE Fluorescence

(a) Full scale

Cou

nt

2500

2000

1500

1000

500

0


PE Fluorescence

(b) Magnified



50 and 25 PCV)

Cou

nt

1500

1000

500

0

PE Fluorescence100 101 102 103 106105104



2RBC) and PE




PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and



Enzyme Research 13


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence


2RBC) and PE



PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500






PE Fluorescence

Cou

nt

2000

1000

0

3000

4000





4 Discussion


2RBCs to blood


1RBCs To



14 Enzyme Research



PE Fluorescence


PE Fluorescence

Cou

nt 2000

1000

0

3000

Cou

nt

2500

2000

1500

1000

500

0

3000





PE Fluorescence

Cou

nt

2000

1000

0

3000

4000C

ount

2000

1000

0

3000

4000

5000







Enzyme Research 15

Cou

nt

2000

1000

0

3000

4000



Cou

nt

2000

1000

0

3000

4000

6000

5000




PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000







2RBCs






16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Enzyme Research 13


PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500


2RBC) and


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence


2RBC) and PE



PE Fluorescence

Cou

nt

2500

2000

1500

1000

500

0

3000

3500






PE Fluorescence

Cou

nt

2000

1000

0

3000

4000





4 Discussion


2RBCs to blood


1RBCs To



14 Enzyme Research



PE Fluorescence


PE Fluorescence

Cou

nt 2000

1000

0

3000

Cou

nt

2500

2000

1500

1000

500

0

3000





PE Fluorescence

Cou

nt

2000

1000

0

3000

4000C

ount

2000

1000

0

3000

4000

5000







Enzyme Research 15

Cou

nt

2000

1000

0

3000

4000



Cou

nt

2000

1000

0

3000

4000

6000

5000




PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000







2RBCs






16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


14 Enzyme Research



PE Fluorescence


PE Fluorescence

Cou

nt 2000

1000

0

3000

Cou

nt

2500

2000

1500

1000

500

0

3000





PE Fluorescence

Cou

nt

2000

1000

0

3000

4000C

ount

2000

1000

0

3000

4000

5000







Enzyme Research 15

Cou

nt

2000

1000

0

3000

4000



Cou

nt

2000

1000

0

3000

4000

6000

5000




PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000







2RBCs






16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Enzyme Research 15

Cou

nt

2000

1000

0

3000

4000



Cou

nt

2000

1000

0

3000

4000

6000

5000




PE Fluorescence

Cou

nt2000

1000

0

3000

4000

5000







2RBCs






16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


16 Enzyme Research




1 150E+06 10 42A2 250000 10 69


A2 250000 173 04

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140

PE F

luor

esce

nce R

espo

nse

(Eve

nts p

er S

econ

d)

Time (min)


Cou

nt

2000

1000

0

3000

4000


PE Fluorescence





2 The difference




PE FluorescenceC

ount

2500

2000

1500

1000

500

0

3000


1and A



1

RBC and Type O RBC


160 mg is needed



2 a


1RBCs






Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Enzyme Research 17



PE Cy 55 PE Cy 55

SSC-

A

Cou

nt

1200

1000

800

600

400

200

0

Cou

nt

1200

1000

800

600

400

200

0

100

101

102

103

106

107

105

104

SSC-

A

100

101

102

103

106

107

105

104

100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

2500

2000

1500

1000

500

0







Figure 25 Continued

18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


18 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

Cou

nt

120

100

80

60

40

20

0

600

500

400

300

200

100

0

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

250

200

150

100

50

0







Figure 25 Continued

Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Enzyme Research 19


Cou

nt

250

300

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

250

200

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

90

80

70

60

50

40

30

20

10

0








20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


20 Enzyme Research


PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104







Cou

ntC

ount

200

100

0

300

400

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

900

800

700

600

500

400

300

200

100

0

Cou

nt

250

300

200

150

100

50

0

Figure 26 Continued

Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Enzyme Research 21


SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

Cou

ntC

ount

150

100

50

0

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

Cou

nt

250

300

200

150

100

50

0

120

100

80

60

40

20

0







Figure 26 Continued

22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


22 Enzyme Research



SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

PE Cy 55100 101 102 103 106 107105104

SSC-

A

100

101

102

103

106

107

105

104

PE Cy 55100 101 102 103 106 107105104

140

120

100

80

60

40

20

0

Cou

nt

400

300

200

100

0

Cou

ntC

ount

250

200

150

100

50

0








Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Enzyme Research 23

195

142

96

71

48

33

28



24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


24 Enzyme Research


Compact Layer

Diffuse Layer



+ +NH3

CH2 CH2

COOH

pK1

（3

pK2

（2

（2

COOminusCOOminus

GLYCINE TITRATION

7

00 05 1 15 2


pI = 597pH

pK1 = 234

pK2 = 960




1RBC conversion Due




Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Enzyme Research 25






5 Summary


Data Availability


Disclosure




References






26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


26 Enzyme Research



































Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Enzyme Research 27








Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


researcharticle enzymatic conversion of rbcs by -n...

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