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Experiment 1

Protein Quantification by using absorbance,

Bradford and Lowry methods

Part 1: Absorbance Assay

PR !" PLE

Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm.The peak at 280 nm is mainly because of the amino acids with aromatic rings.

EQ# P$E!%

Li uid handling supplies! spectrophotometer! "# lamp and $uart% cuvette.

PR&"E'#RE

&arry out steps '() *280 nm only+ for a very rough estimate. &arry out all steps ifnucleic acid contamination is likely.

1. ,arm up the "# lamp *about '- min.+2. d/ust wavelength to 280 nm3. &alibrate to %ero absorbance with buffer solution only4. easure absorbance of the protein solution5. d/ust wavelength to 210 nm6. &alibrate to %ero absorbance with buffer solution only7. easure absorbance of the protein solution

"AL"#LA% &!(

Unknown proteins or protein mixtures. * Path length for most spectrometers is ' cm+

&oncentration *mg ml+ 3 bsorbance at 280 nm divided by path length *cm.+

Pure protein of known absorbance coefficient. *&oncentration is in mg ml! 4! or molaritydepending on which type coefficient is used+

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&oncentration 3 bsorbance at 280 nm divided by absorbance coefficient

Unknowns with possible nucleic acid contamination.

&oncentration *mg ml+ 3 *'.-- x 280+ ( 0.51 x 210+

!&%E( 6

1. This method is simple! and re uires an extremely small sample volume2. 7owever! the protein sample must be pure and does not contain any non(protein components

with the same absorption spectrum! such as the contamination of nucleic acids .

3. This method is uickest! but error(prone.

Part ): Bradford $ethod

PR !" PLE

The assay is based on the observation that the absorbance maximum for an acidicsolution of &oomassie rilliant lue 9(2-0 shifts from )1- nm to -:- nm when binding toprotein occurs. oth hydrophobic and ionic interactions stabili%e the anionic form of thedye! causing a visible color change. The assay is useful since the extinction coefficient of adye(albumin complex solution is constant over a '0(fold concentration range.

EQ# P$E!%

;n addition to standard li uid handling supplies a visible light spectrophotometer is needed!with maximum transmission in the region of -:- nm! on the border of the visible spectrum*no special lamp or filter usually needed+. 9lass or polystyrene *cheap+ cuvettes may beused! however the color reagent stains both.

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concentrate is expensive! but the lots of dye used have

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apparently been screened for maximum effectiveness. B7omemadeB reagent works uitewell but is usually not as sensitive as the io(Aad product.

PR&"E'#RE

Assay

1. ,arm up the spectrophotometer before use.2.

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Part +: artree-Lowry Assay

PR !" PLE

"nder alkaline conditions the divalent copper ion forms a complex with peptide bonds inwhich it is reduced to a monovalent ion. onovalent copper ion and the radical groups oftyrosine! tryptophan! and cysteine react with @olin reagent to produce an unstable productthat becomes reduced to molybdenum tungsten blue.

EQ# P$E!%

;n addition to standard li uid handling supplies a spectrophotometer with infrared lampand filter is re uired. 9lass or polystyrene *cheap+ cuvettes may be used.

REA*E!%(

1. Aeagent consists of 2 gm sodium potassium tartrate x ) 720! '00 gm sodiumcarbonate! -00 ml '? ?a>7! 720 to one liter *that is! 5m ?a(E tartrate! 0.8'sodium carbonate! 0.-? ?a>7 final concentration+. Eeeps 2 to F months.

2. Aeagent consists of 2 gm sodium potassium tartrate x ) 720! ' gm copper sulfate*&uG>) x -720+! :0 ml 720! '0 ml '? ?a>7 *final concentrations 50 m ?a(Etartrate! )0 m copper sulfate+. Eeeps 2 to F months.

3. Aeagent & consists of ' vol @olin(&iocalteau reagent diluted with '- vols water.

PR&"E'#RE

1. Prepare a series of dilutions of 0.F mg ml bovine serum albumin in the same buffer containing theunknowns! to give concentrations of F0 to '-0 micrograms ml *0.0F to 0.'- mg ml+.

2. dd '.0 ml each dilution of standard! protein(containing unknown! or buffer *for the reference+ to 0.:0 ml reagent in separate test tubes and mix.

3. ;ncubate the tubes '0 min in a -0 degrees & bath! then cool to room temperature.4. dd 0.' ml reagent to each tube! mix! incubate '0 min at room temperature.5. Aapidly add F ml reagent & to each tube! mix! incubate '0 min in the -0 degree bath!

and cool to room temperature. @inal assay volume is - ml.6. easure absorbance at 1-0 nm in ' cm cuvettes.

"AL"#LA% &!(

Prepare a standard curve of absorbance versus micrograms protein *or vice versa +!and determine amounts from the curve.

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2. ?onetheless! the effect of these substances can be reduced by diluting out thesample but only if the protein concentration is sufficiently high I8J. oreover! it hasbeen shown that the time to perform this assay can be reduced through raisingtemperatures or using a microwave oven .

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Experiment . )Assay β of - */ucosidase*β ( 9lucosidase &ataly%ed 7ydrolysis of P?P9+

% E&R0

Βeta - */ucosidase is a glucosidase en%yme that acts upon β ' (K) bonds linking two glucose or glucose( substituted molecules and hydroly%es of glucosides. ;t catalyses the hydrolysis of terminal nonreducing residues in beta( F in '00 ml

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(1)

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Experiment . +

Assay of n8ertase Acti8ity

PR !" PLE

;nvertase β or ( fructofuranosidase cataly%es the hydrolysis of sucrose to yield invertsugar! resulting in a mixture of glcose and fructose. ;nverted sugar is sweeter thansucrose and has a much lesser tendency to crystalli%e. Hn%yme activity is assayedby estimating the amount of glucose* reducing sugar+ produced by

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×

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Experiment . 9

Assay of 0east A/coho/ 'ehydrogenase Acti8ity

PR !" PLE:

lcohol dehydrogenase catalyses the reaction6

A&7 2>7 N ? < N A&7> N ? neunit reduces one micromole of ? < N per minute at 2- o&.

Hn%yme activity is calculated by the following formula6 ∗

.

REA*E!%(:

1• 0.0F Godium pyrophosphate buffer *p7 8.8+2• 2 ethanol prepared by diluting '2.'2 ml of :-4 ethanol to '00 ml of distilled water3• 0.' Phosphate buffer! p7 5.-4• 0.0' Phosphate buffer! p7 5.-5• 0.02- ? < N

PR&"E'#RE:

'. The various constituents are pipetted into test tubes as follows6

@or lank *ml+ @or Test *ml+

Phosphate buffer *p7 8.8+ '.: '.8

? < N '.0 '.0

Hthanol 0.' 0.'

2. Pour the contents into a F ml cuvette and set the wavelength at F)0 nm.

3. The spectrophotometer should be set in the time drive mode.

4. easurement of en%yme activity is started as soon as 0.' ml of suitably diluted en%ymesample is added to the cuvette in the case of test sample.

5. The initial rate is measured for 2 minutes of reaction and the en%yme activity calculated asstated above.

!&%E(:

'. lcohol

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following preparation of solutions

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Experiment . ;

r!1 = + = 1 +

The kinetic parameters E m and # max can be determined by plotting a graph between ' # vs.' G referred to as the Lineweaver( urk Plot.

7owever! the above plot and others *Hadie(7ofstee+ don t allow accurate estimation of thekinetic constants.

Hisenthal and &ornish( owden ! suggested a modification of the ichaelis( enten e uation!which can also be expressed as6

= + = 1 +

Get up axes! E m and #! corresponding to the familiar x and y axes respectively. @or eachobservation *G!#+! mark off the points E m 3 (G on the E m axis and # max 3 v on the # max axis anddraw a line through the two points extending it to the first uadrant. ,hen this is done for allobservations! the lines intersect at a common point whose coordinate *E m! # max + provide thevalues of E m and # max that satisfy the ichaelis( enten e uation exactly for every observation.

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REA*E!%(:

1. &itrate buffer *-0 m ! p7 ).8+ prepared by ad/usting p7 of -0 m citric acid with ' ??a>7 *dissolve :.1 gm citric acid in water and ad/ust p7 with ?a>7 solution to makeup volume to ' l+

2. P?P9 *2.- m + stock solution in citrate buffer *-0 m ! p7 ).8+ *5.-F g in '0 ml+

3. ?a 2&> F *'?+ solution in distilled water *'0.1 g in '00 ml+

PR&"E'#RE:

1. Take '.8 ml of P?P9 solution containing five different substrate concentrations *0.-(2.- m + in - different test tubes

2. Prepare two sets to one add 0.2 ml suitable diluted en%yme and to the other add 0.2ml citrate buffer

3. ;ncubate the tubes at -0 o& in water bath for '- minutes4. Gtop the reaction by adding ' ml ?a 2&> F solution and measure )'0 nm.5.

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Experiment . >

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' # 3 ' # m*'N' E i+ N Em #m.' G

7ere both E m and # m are affected by inhibitor.

REA*E!%(:

1• &itrate buffer *-0 m ! p7 ).8+! prepared by ad/usting p7 of -0 m citric acid with '??a>7 *dissolve :.1 g citric acid in water and ad/ust p7 with ?a>7 solution to makeup volume to ' l+

2• P?P9 *2.- m + stock solution in citrate buffer *-0 m ! p7 ).8+ *F5.1- mg in -0 ml+3• ?a 2&> F *'?+ solution in distilled water *'0.1 g in '00ml+4• 9lucose '00 m *'.8 g in '00 ml+5• β (glucosidase *2.- mg in - ml+

PR&"E'#RE:1. Take '.8 ml of P?P9 solution containing five different substrate concentrations *0.-(

2.- m + in - different test tubes2. Prepare two sets to one add 0.2 ml suitable diluted en%yme and to the other add 0.2

ml citrate buffer

3. ;ncubate the tubes at -0 o& in water bath for '- minutes4. Gtop the reaction by adding ' ml ?a 2&> F solution and measure )'0 nm.5.

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Experiment .@

n8estigating the effect of p and temperature on en yme acti8ity

PR !" PLE:

;n this lab! we will explore some of the properties of en%ymes. Hn%ymes are sensitive tochanges in temperature and p7 which alter their shapes and can even destroy catalyticability *denaturing+. Hn%ymes have evolved to work most efficiently at the temperature andp7 found in the part of the organism where they are needed. any en%ymes in the human

body function most efficiently at F5 o& and at a p7 of 5.)

The en%yme you will investigate is called catalase. &atalase is found in tissues of manyorganisms *both plants and animals+ because it plays a very important role in protecting cells. ;tspurpose is to destroy toxic substances which may be introduced into cells. lso! some cells use

catalase to destroy cellular debris or worn out organalles. 7ydrogen peroxide is a normal by(product of cellular metabolism but it is also toxic to cells. under normal conditions organismsproduce the en%yme catalase that uickly changes hydrogen peroxide into two harmlesssubstances! oxygen and water. 7owever! the function of the en%yme is affected by changes inthe environment. &atalase works to breakdown hydrogen peroxide by the following reaction6

27 2> 2 (((((K 27 2> N > 2

$A%ER AL(:

&atalase *to be prepared by you+! '4 hydrogen peroxide! forceps! filter paper discs! water

baths! two large test tubes! : small test tubes$P&R%A!% !&%E:

The assay system used in the lab consists of a filter paper disc which is coated with theen%yme and then dropped into a test tube of substrate *hydrogen peroxide+. s the catalystbreaks down the hydrogen peroxide into water and oxygen gas! the bubbles of oxygencollect underneath the filter and make it rise to the surface of the hydrogen peroxide. Thetime it takes for the filter to rise will be ur indication of the rate of en%yme activity.

Aate of en%yme activity 3 distance *depth of hydrogen peroxide in mm+ time *in sec+

,e will assume that each filter is coated with the same amount of catalase.

&atalase can be prepared easily by the student as follows6

-0g of peeled potato was mixed with -0 ml cold distilled water and crushed ice andhomogeni%ed in a blender for F0 seconds. This extract was filtered through cheesecloth andcold distilled water was added to a total volume of '00 ml.

Part A: Effect of p

1. >btain ' large test tube of 20 ml 7 2> 2. easure and record the depth of 7 2> 2.

2. Label - small test tubes as p7F! p7-! p75! p7:! p7'' and asfollows6 p7F6 2 ml catalase N 2ml p7 F bufferp7-6 2 ml catalase N 2ml p7 - buffer

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;nclude two graphs.

1. p7 vs average rate

2. Temperature vs average rate

$uestions to consider in your analysis6

1. 7ow does temperature affect the activity of catalaseR Hxplain your observations by discussingthe effect of temperature on protein structure.

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Experiment .

En yme entrapment in ca/cium a/ginate beads

PR !" PLE:

;mmobili%ation of en%yme by gel entrapment involves the entrapment of the biocatalystwithin a polymeric network. The biocatalyst after mixing with the a ueous polymeric solutionis forced through a fine orifice *syringe+ into a salt solution that insolubili%es the mixturethrough ion exchange. The shape and si%e of beads can be controlled by choosing theorifice diameter and the distance of the no%%le from the li uid surface.

9el entrapment usually does not result in any adverse modification of the en%ymeconformation and can provide high yield of immobili%ation.

REA*E!%(:

1• cetate buffer *0.0- ! p7 -+2• Godium alginate )4 w v in acetate buffer3• Hn%yme source * aker s yeast permeabili%ed cells as source of invertase+4• 0.' sucrose in acetate buffer5• 0.- ? ?a>7

PR&"E'#RE:

1. ix the cell suspension to the sodium alginate solution *e ual volumes+ to bring the final

concentration of sodium alginate to 24 w v.2. dd this slurry dropwise into chilled calcium chloride solution under continuous stirring.3. &ure the calcium alginate beads in calcium chloride solution for F0 minutes and wash

thoroughly with the buffer.4. Guspend the gel beads in acetate buffer.5. ssay for the invertase activity in the beads and compare the same with that in the original cell

suspension on the basis of e ual weight of cell mass. Aeport the results in terms of yield of

immobili%ation! en%yme loading! bead diameter and packing density of the preparation.

En yme Assay

1. Take ' ml of suitably diluted en%yme preparation and add ' ml of 0.' sucrose solution.2. ;ncubate the mixture at room temperature for - min.3. fter incubation to ' ml of above mixture add ' ml of 0.0-? ?a>7 to stop the reaction.4. dd F ml of

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Experiment .

%o hydro/y e protein-based stains in fabrics into so/ub/eamino acidsC

PR !" PLE:

;n todaySs laundry detergents! en%ymes such as proteases and amylases are some of the active

ingredients. ;n the ".G.! about -04 of li uid detergents! 2-4 of powder detergents! and almost all

powdered bleach additives now contain en%ymes to help break down stains that are otherwise hard to

remove with conventional surfactants alone. @or example! amylase cataly%es the breakdown of starch(

based stains to smaller segments that make up the larger starch molecule. >ligosaccharides and dextrinsreleased from the en%ymeSs hydrolytic action are soluble thus! the stain is physically cut off from the

surface of the fabric piece by piece! with the en%yme acting as scissors. The action of proteases! as

implied by the name itself! is similar to that of amylase! except that a large protein molecule is hydroly%ed.

ther proteinaceous dirt includes perspiration! grass! and slime stains. This exercisedemonstrates that it takes protein to get out protein! as some television commercials claim.

$A%ER AL( REQ# RE':

1. Equipment

1• @lasks! 2-0 ml2• 9raduated cylinder3• 7ousehold clothing iron or drying oven4• Gpectrophotometer5• alance

2. Reagents

1• 7ousehold detergent! *to be supplied by the student+2• acterial protease3•

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PR&"E'#RE:

1.