THE JOURNAL OF BIOLOGICAL CHEMISTRYVol. 256, No. 10, Issue of May 25, pp. 5204-5208, 1981Printed in U.S.A.

Kinetic Analysis of the Nonenzymatic Glycosylation of Hemoglobin*

(Received for publication, October 17, 1980, and in revised form, January 23, 1981)

Paul J. Higgins and H. Franklin BunnFrom the Laboratory of the Howard Hughes Medical Institute, Brigham and Women's Hospital, and Harvard MedicalSchool, Boston, Massachusetts 02115

The rate constants have been derived for (a) thecondensation of glucose with hemoglobin to form thelabile Schiffbase intermediate, pre-AI,; (b) the dissocia-tion of this complex to hemoglobin and glucose; (c) therearrangement of this complex to form the stable ke-toamine, Hb A,,. These measurements required the pu-rification of commercially available D-[`4C]glucose inorder to remove a rapidly reacting contaminant. Theinitial condensation reaction rate (k'1) was measuredby incubating column purified Hb Ao for up to 8 h underphysiologic conditions with purified D-['4C]glucose inthe presence of cyanoborohydride which traps theSchiff base and reduces it to a stable adduct. A parallelincubation utilizing Hb AIC revealed the contribution ofthe ,i-NH2-terminal amino group (k1) to the overallvalue for k'1. The reverse reaction rate (k-1) was deter-mined from incubations carried out in the absence ofcyanoborohydride. The rate of the Amadori rearrange-ment (k2) was determined from longer (6-21 days) in-cubations under identical conditions, followed by chro-matographic isolation ofHb AIC. This value for k2 agreeswell with one we previously obtained from in vivo data.These experiments provide direct chemical evidence

for an aldimine precursor in the nonenzymatic glyco-sylation of protein. Furthermore, the use of these rateconstants provides a reasonable estimate of the distri-bution of the labile aldimine (pre-AI) and the stableketoamine (Hb AIC) in normal and diabetic red cells.This information is useful in the interpretation of meas-urements of glycosylated hemoglobin in diabetic pa-tients.

A variety of proteins undergo nonenzymatic modificationby forming covalent linkages with glucose. The aldehydefunction of glucose condenses with amino groups to form areversible Schiff base or aldimine linkage which is capable ofrearranging to a more stable ketoamine. The best understoodexample of such a modification is hemoglobin AIC, the mostabundant minor component in normal human red cells. HbA,, is identical in structure to the major component, Hb A,except that glucose is attached to the NH2 terminus of the /1chain by a ketoamine linkage (1-4). The steps in the synthesisof Hb A,, are as shown in Scheme 1. Hb A,, is formed slowlyand nearly irreversibly during the 120-day life span of the redcell (5). The extent to which Hb A,, accumulates depends onthe average concentration of glucose in the plasma during thepreceding 2-3 months. Thus, Hb A,, has proved to be a

* This work was supported by the Howard Hughes Medical Insti-tute and by Grant AM-18223 from the National Institutes of Health.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be herebymarked "advertisement' in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

HCO0HCOH

HOCHHCOH

HCOH0H20t

glucose

HC=N-/AHCOH

k1 HOCH amodori.k-1 tHOOH k2

HCOH

H CH20Haldimine

(Schiff base)

CH2-N H2-/AC=O

HOCH

HCOH

OHCOHCH0

ketoomine

HbA _rapid

pre ACsow

Hb AlC

SCHEME 1

reliable index of diabetic control (6-9) and is measured rou-tinely in many diabetes clinics.

In order to gain a better understanding of the formation ofglycosylated hemoglobin, we have completed a kinetic analy-sis. Specifically, we have devised methods to determine theindividual rate constants for the formation (k1) and dissocia-tion (khl) of the aldimine and for the conversion of thealdimine to the ketoamine (k2). These experiments permit amuch clearer understanding of the chemical mechanism forthe formation of Hb AIC. This information should be useful inthe clinical interpretation of glycosylated hemoglobin.

MATERIALS AND METHODS

Red cell hemolysates were prepared as previously described (15),gassed with carbon monoxide, and chromatographed on Bio-Rex 70cation exchange resin (Bio-Rad, Inc.). Glycosylated hemoglobin com-ponents (Hbs AIai, AIa2, AIb, and AIC) were eluted by the method ofMcDonald et al. (10). The major component (Hb Ao) was eluted bya linear NaCI gradient from 0.1-1.0 M. Column fractions of Hb Aowere pooled, concentrated by pressure filtration (Amicon PM-10membrane), and dialyzed versus Krebs Ringer phosphate buffer, pH7.3.

Uniformly labeled D-[`4C]glucose (New England Nuclear) was pu-rified by preincubation with hemolysate for 4 h at 37 'C in thepresence of 20 mM NaCNBH3 (Aldrich). After incubation the un-reacted D-[14C]glucose was separated from the hemolysate by pressurefiltration (Amicon PM-10 membrane). The D-[14C]glucose solutionwas eluted through a cation exchange resin (Dowex 50W-X2, 200-400mesh, H' form) followed by an anion exchange resin (Dowex 1-X8,200-400 mesh, formate form) and then lyophilized. Alternatively, theD-[14C]glucose was purified by thin layer chromatography as describedbelow.

Purified Hb Ao was incubated with purified and unpurifiedD-[14C]glucose in a sterile solution of Krebs Ringer phosphate buffer,pH 7.3, at 37 'C. Incubations were carried out either in the presenceof 20 mM NaCNBH3 or without NaCNBH3. Concentration of glucoseranged between 15 and 50 mM. Aliquots were removed from theincubation solution at specific time intervals and unbound glucoseremoved by rapid passage through Sephadex G-25 (Pharmacia). In-corporation of D-['4C]glucose into Hb Ao was determined by measur-ing the hemoglobin concentration and radioactivity of each gel-fil-tered sample. To a 0.9-ml aliquot was added 1.5 ml of a 1:1 solutionof Protosol (New England Nuclear) and isopropanol, 0.2 ml of 30%hydrogen peroxide, and 10 ml of Liquiscint cocktail (National Diag-nostics, Somerville, NJ). Samples were counted for 10 min on anIsoCap/300 liquid scintillation counter (Searle Analytic Corp.).Counts were corrected using a standard quench curve.

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Kinetics ofNonenzymatic Glycosylation

Purified and unpurified D-[14C]glucose was chromatographed bythin layer chromatography (MN 300 CM-cellulose plates, Analtech,Inc.) in butanol:pyridine:water (6:4:3).' Autoradiograms of the plateswere prepared using Kodak XR-5 film. Glucose was measured enzy-matically by the glucose oxidase assay2 and colorimetrically by theferricyanide test (11).

CALCULATION OF RATE CONSTANTS

The second order rate constant for the formation of thelabile aldimine [H G] can be calculated from incubations ofpurified D-['4C]glucose and hemoglobin in the presence ofcyanoborohydride (Figs. 1 and 3). During the first severalhours, the reaction obeys pseudo-zero order kinetics since onlya trivial proportion of each reactant is consumed. k'1 can becalculated from the slope of the linear progress curve and theglucose concentration [G].

d[H=G] = k'-IH][G] = (k, + k1a + klE, + kiE2 ... )[H][G] (1)dt

where k'1 is the overall rate of condensation of glucose atseveral sites on hemoglobin (14, 15) as determined experimen-tally. ki (=-k1") is the rate of condensation at the /3-NH2terminus while k1i is the rate at the a-NH2 terminus and kiEl,k,2 etc., are the rates at certain lysine residues.

k'l = A[H=G]/[H1 (2)[GI,At

The first order rate constant (kh-) for the dissociation ofglucose from the aldimine complex [H0G] can be calculatedfrom incubations of purified D-[14C]glucose with hemoglobinin the absence of cyanoborohydride. As shown in Fig. 4, thereaction comes to equilibrium at approximately 6 h:

d[HdG] = k'i[H][G] - k-i[H=G] - k2[H=G1 = 0 (3)dt

Since k_ 1 » k2 (see below), at equilibrium (e)

[H=G], (4)

The rate at which the aldimine at the /3-NH2 terminusrearranges to the more stable ketoamine (Hb AIC) can becalculated from the data shown in Fig. 5 where the formationof Hb A,, is linear over 16 days. Since the concentration ofglucose is constant, the concentration of aldimine [H G]rapidly reaches a constant level, just as in the incubationshown in Fig. 4.

d[HG] = k2[H=G]edt

k_ A[HG]/[H]k2=

k'i[GlAt

(5)

(6)

RESULTS

Determination ofki-In order to measure the rate at whichhemoglobin forms the aldimine (Schiff base) linkage withglucose (k'1), it was necessary to devise incubation conditionsin which all of the adduct formed was trapped so that thereverse reaction (the dissociation of the aldimine (k -)) couldnot take place. Cyanoborohydride readily reduces Schiff baselinkages but not ketone groups at neutral pH (12, 13) and isalso a less denaturing reducing agent than borohydride. Weshowed that during a 5 h incubation of glucose with 20 mMNaCNBH3 under conditions subsequently employed in the

'S. Kornfeld, personal communication.2 Sigma Technical Bulletin 510: 7-73.

(.k

Es

experiments described below, there was no loss of glucose, asdetermined by enzymatic assay. The rate of incorporation ofD-[`4C]glucose into hemoglobin was identical in 10 and 20 mMCNBH3. Therefore, at 20 mm all of the Schiff base that wasformed during the incubation was reduced by cyanoborohyd-ride before dissociation could take place. These preliminaryexperiments established the conditions for the reliable deter-mination of k'1.

Incubations were initially carried out using unpurified D-[14C]glucose. Incorporation of 14C by hemoglobin in theseexperiments showed a highly reproducible curvilinear functionwith time (Fig. 1). The moles of glucose incorporated/mol ofat3 dimer began to plateau at a point far below saturation ofreaction sites. This result could not be explained by straight-forward kinetic analysis. It seemed likely that the curvilinearfunction was due to inhomogeneity of the D-[`4C]glucose. Thedata could be explained by a small amount of radioactivecontaminant that reacted rapidly with the hemoglobin. Asshown in Fig. 2, autoradiography following thin layer chro-matography of the unpurified D-[`4C]glucose revealed smallamounts of streaking both above and below the spots corre-sponding to a- and /3-glucose. No such heterogeneity wasobserved when purified D-[`4C]glucose was chromatographed(Fig. 2). Subsequently, hemoglobin incubations were carriedout with purified D-[14C]glucose. As shown in Fig. 1, linearincorporation was observed, and indicates that the purifiedD-[`4C]glucose was homogeneous. D-[`4C]Glucose purified bythin layer chromatography also gave linear incorporation.From the data in Fig. 1, a k'l of 0.7 x 10` mm-1 h`1 wasdetermined. Replicate experiments invariably revealed linearprogress curves up to at least 8 h (Fig. 4). In 9 determinations,the mean value for k'1 was 0.9 ± 0.2 x 10-3 mm-' h` (± S.D.).The reliability of this approach to measuring k'l was cor-

roborated by two parallel experiments in which incubationsof purified D-[14C]glucose in the presence of unlabeled cyano-borohydride were compared with unlabeled glucose in the

HOURS OF /A'CUSA7ONVFIG. 1. Incorporation of radioactive glucose into hemoglo-

bin. Eleven mm glucose was incubated with 2 mM carboxyhemoglobinHb Ao (a/3) in Krebs-Ringer phosphate, pH 7.3, 37 'C, and 20 mMNaCNBH3. A A, unpurified D-[`4C]glucose from New EnglandNuclear (NEN); @-, purified D-[`4C]glucose. The lower curve(@-@) gives a value of 0.9 x 10-3 mm-'hh for k'i.

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Kinetics ofNonenzymatic Glycosylation

Cl -Glucoses

-Gucose X

2 3 4 5

Fur ifed Unpurified

4 J4c) - GAUcosE-FIG. 2. Autoradiogram of purified and unpurified n-['4C]glu-

cose. Rechromatographv of each of the two major spots revealed aand P3 glucose in the same distribution as in the original autoradi-ogram.

presence of [1H]cvanoborohydridex Somewhat lower xvaluesfor h', were obtained with [ H]CNBH3, perhaps because of a

small tritium isotope effect.These experiments do not distinguish among the sites on

the hemoglobin molecule that are capable of forming ketoam-ine linkages with glucose. In normal red cells, about 4%4 ofhemoglobin (a/ dimers) is glycosylated at the 3-NH) termi-nus. In addition we have shown that about 8% of hemoglobin(a/ dimers) contains ketoamine-linked glucose at certain ly-sines as well as the NH> terminus of the a chain (14, 15).

Therefore h', determined from the above experiments is a

composite representing the sum of the rates of adduct for-mation at several sites. We attempted to localize the sites ofglycosylation involved in measurement of kI' (e.g. Figs. 1, 3,and 4) by structural analysis. Unfortunately, a prohibitivelylarge amount of purified D-[`4C]glucose was required toachieve levels of radioactivity adequate for determination ofsites of glvcosvlation. Partial information on the sites ofglycosylation was obtained by comparing the reactivities ofHb Al and Hb A1,, which has blocked /-NH -terminal aminogroups. As shown in Fig. 3, the rate of condensation ofD-['`C]glucose with Hb AII was that of Hb A. The contri-

bution of the /-NH, terminus to the rate of condensation ofglucose with hemoglobin is comparable to the ratio of Hb Al,to total glycosylated hemoglobin in normal red cells. Thus,we estimate that the rate of condensation of the 3-NH.terminus (ki) is 0.3 x 10-3 mm-' h`.

In order to apply this kinetic analysis to in t,it,o conditionswe determined the effect ofpH and hemoglobin concentrationon IC The rise in k'i with increasing pH is not surprising

since glucose condenses only with nonprotonated aminogroups. However, as shown in Fig. 4, the slope of h', cersuspH is less steep than what would be expected from the pK,,values of the reactive amino groups and probably reflectsadditional pH dependent factors. As Table I shows, kI' was

not significantly affected by increasing the concentration ofhemoglobin to about two-thirds the level found in normal redcells. This experiment was done under solvent conditions thatmimic the milieu inside the red blood cell.Determination oftk l-The reverse reaction H -G H +

G can be examined bv incubations of purified D-[ TC]glucoseand hemoglobin A1 in the absence of cyanoborohydride. Asshown in Fig. 5 the incorporation of glucose into hemoglobinwas (a) curvilinear, (b) lower than that in the presence ofNaCNBH3,, and (c) level after 4 h. From this progress curve,

kI1 could be calculated as explained above (see "Calculationof Rate Constants"). The curve shown in Fig. 4 is a theoretical

; H. F. Bunn and P. J. Higgins, Science, in press.

i 006/

HbAO

0104X

(Z)

-42

0 1 2 3 4

HOURS OF INCUBATION

FIG. 3. Comparison of the rate of incorporation of purifiedi)-[14C]glucose into Hb Ao (@-@) and Hb A,, ( ) in thepresence of 20 nnm NaCNBH3. Glucose concentratioin, 15 mM.

Otherwise, conditions are as described in Fig. 1. The data for Hb A,give a value of 0.8 x 10 ` mm h for k'.

1.0

KI

N-

0

0

0

0

06k-

04

02

n5 6 7 8

pH

FIG. 4. Effect of pH on k'l. Buffer was titrate(d to various pHvalues prior to addition of glucose. Otherwise, conditions are asdescribed in Fig. 1.

plot based on a k i value of 0.35 h `. There is excellentagreement between this theoretical plot and the experimentalpoints. In a second determination of ki a value of 0.30 h`was obtained.Determination of k. -The rate of svnthesis of the stable

ketoamine form of Hb A,, was measured by sterile incubationsof D-[ 4C]glucose with Hb Al in the absence of NaCNBH andseparation of the Hb A,, and Hb A, bccation exchangechromatography (10). The results of two of these incubationsare shown in Fig. 6 for two concentrations of glucose (15 and50 mM). In view of the length of the chromatographic sepa-ration and our estimate of kt , it is likely that nearly all of thealdimine form of Hb A1, was lost during elution through thecation exchange column. Therefore, the rate of formation ofthe ketoamine, Hb A,1, can be calculated from these data. No

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Kinetics ofNonenzymatic Glycosylation

TABLE IEffect ofhemoglobin concentration on the rate of condensation

with glucose (k'l)These incubations were done in a buffer designed to simulate as

closely as possible the intracellular milieu of the human red bloodcell. The final composition of the incubation mixture was: carboxy-hemoglobin Ao: 0.7-6.7 mM (a/i); K', 126 mM; Na+, 13 mM; Mg2+, 3.5mM; Cl-, 120 mM; HCO3, 13 mM; PO4 3 mM; PCO2, 40 mm of Hg; pH7.2. The values for k'i obtained with this buffer (0.87 ± 0.05 x 10-3MM-1 h'1) were not different from those determined from otherexperiments utilizing Krebs ringer phosphate (0.9 ± 0.2 x 10-3 mm-1

h'1).Hemoglobin concentration k x 103

mg/mi mm-'h~23 0.8958 0.80115 0.86214 0.92

tLu(j~

0~

01

O 1 2 3 4 5 6 7 8HOURS OF/NCUBA7OAN

FIG. 5. Incorporation of purified D-[14C]glucose into hemo-globin in the presence (-4) and absence (O-O) of 20 mmNaCNBH3. Conditions were the same as those in Fig. 1 except thatthe glucose concentration was 14 mM. -* gives a value for k'i of0.7 x 10-3 mm-1 h`l. The lower curve was calculated by integratingEquation 3 using a value for k'l of 0.7 mMm- h`1 and value of k_1 of0.35 h`.

6

0 4

2

"0 2 4 6 8DA YS

10 12 14 16

FIG. 6. Rate of formation of Hb A,, during prolonged incu-bation of RIb Ao with glucose in absence ofNaCNBH3. Conditionsare described in Fig. 1. These data give values for k2 of 0.0053 h`l (15mM glucose) and 0.0044 h`1 (50 mM glucose).

methemoglobin or precipitation was observed during theseprolonged incubations. Results from six experiments are

shown in Table II. We obtained a mean value for k2 of 0.00550.0010 h-'. This in vitro rate is in good agreement with a

value of k2 of 0.0060 h-1 calculated from our in vivo iron

TABLE IIFormation ofHb A,, from prolonged incubations of Hb Ao and

glucoseThe experimental conditions are described in the legend to Fig. 1.

Hb Ao Glucose Incubation Hb A,, k2timemM mM days % h 1

1.1 15 21 4.4 0.00632.8 15 15 2.8 0.00572.0 15 11 2.0 0.00552.6 15 6 1.4 0.00712.6 17.5 19 3.0 0.00412.0 50 11 5.3 0.0044

Mean ± 1 S.D. 0.0055 ± 0.0010

kinetic data (5). Viewed another way, the three rate constantswhich we have derived predict that normal human red cellshaving an average glucose concentration of 5 mM and surviv-ing 120 days would have a level of Hb AIC (ketoamine) of 3.7%compared with a measured value of 4%.

DISCUSSION

These experiments provide direct chemical evidence thatnonenzymatic glycosylation of hemoglobin involves the initialformation of a reversible aldimine (Schiff base) precursorwhich slowly rearranges to a stable ketoamine. Although thevalues we have obtained for the individual rate constants arereproducible and internally consistent, a number of consider-ations limit the application of these results to the in vivophenomenon. First, it was necessary to remove a rapidlyreacting contaminant from the D-[14C]glucose. Recently,Trueb et al. (16) have reported the presence of variableamounts of this contaminant in 3H- and 14C-labeled glucosepreparations from a number of commercial suppliers. In thisreport, we describe two ways to purify the D-[14C]glucose inorder to obtain a compound that gives reliable second orderkinetics. Second, the conditions we have used depart some-what from those existing in the circulating red cell. Ourhemoglobin preparations were fully saturated with carbonmonoxide and had a final concentration 15% that of theerythrocyte. However, k'l was not appreciably affected byincreasing hemoglobin concentrations to a value approachingthat of the red cell (Table I). It is unlikely that the other tworate constants are affected by hemoglobin concentration. Thebuffer contained inorganic phosphate rather than the organicphosphate 2,3-diphosphoglycerate which is very important inmediating hemoglobin function. Finally, the "on" and "off"rates we have obtained for the aldimine (k'l and k-1) involvemore than one reactive site. Even though the NH2 terminusof the /3 chain is favored, other sites on the hemoglobinmolecule are also glycosylated (14, 15), although more slowly.The experiment depicted in Fig. 3 shows that the contributionof the /3-NH2 terminus to k'l is roughly proportional to theratio of Hb Al, to total glycosylated hemoglobin in red cells.This experiment permits an estimation of the rate of conden-sation at the /3-NH2 terminus (k1). Likewise, the value wehave obtained for k2 pertains only to the rate ofrearrangementat the fl-NH2 terminus. In contrast, we have no direct meas-urement of k_1 at this site, but assume that this rate is similarat all reactive sites.

Despite these reservations, the rate constants we haveobtained are likely to be reasonably close to those whichpertain in vivo and provide insights into the mechanismsresponsible for the nonenzymatic glycosylation ofhemoglobin.The results can be summarized as shown in Scheme 2. Duringthe incubation of hemoglobin with glucose, the labile aldimine(H=G) increases within a few hours to reach an equilibrium

50mM Glucose>/ a

- ~~~~~~15mM Glucose~0

rn E- .~.^^ Ld

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Kinetics ofNonenzymatic Glycosylation

ki = 03x10-3 mM1 hr1k' = 0.0055 hr>HH+G .3h' H=G k2~ HG

k-1 = 0.33 hr-

SCHEME 2

plateau (Fig. 5). During this time, only a minute amount ofketoamine (HG) is formed since the rate of the rearrangement(k2) is only l/60 that of the rate of dissociation ofH G back tohemoglobin and glucose (k-'). Likewise, in the circulatingerythrocyte, at any given concentration of glucose, there is arapidly equilibrating level of hemoglobin in the aldimine form.The approximate proportion of this labile adduct can becalculated from these rate constants. We estimate that, innormal red cells with an average glucose concentration of 5mM, about 0.5% of the total hemoglobin is pre-AI, (aldimine)or about 10% of the total Hb A,, (ketoamine and aldimine). Indiabetic red cells, the amount of the labile pre-AI, should beproportional to the level of blood glucose and therefore canvary widely depending on the degree of control. Several in-vestigators have reported a prompt fall in glycosylated he-moglobin upon institution of rigorous diabetic control4 (17-20). This finding can be readily explained by a rapid decreasein pre-AI,.The rate constants derived from the present in vitro incu-

bations are in full quantitative agreement with our previousin vivo study (5) and corroborate that Hb AIC, in contrast topre-AIc, is formed slowly and in an almost linear fashion.5 Thiskinetic pattern indicates that, unlike pre-Arc, the formation ofHb A,, is nearly irreversible.

Acknowledgments-We thank Diane Harris for her able secretarialassistance and Robert Shapiro and Paul Gallop for helpful discussions.

4 The measurement of glycosylated hemoglobin in these studiesincludes both the ketoamine and aldimine forms of Hb A,,.

5 In the present work, as well as in our previous in vivo study (5),column-purified Hb A,, contained little, if any, pre-A1,.

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P J Higgins and H F BunnKinetic analysis of the nonenzymatic glycosylation of hemoglobin.

1981, 256:5204-5208.J. Biol. Chem. 

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