T H E J O U R N A L OF BIOLOGICAL CHEMISTRY

Prrnted m L! S A. Vol. 255. No. 15. Issue of August IO. p p . 7218-224, I W I

A NEW PROCEDURE FOR THE DETERMINATION OF KETOAMINE ADDUCTS IN PROTEINS*

(Received for publication, January 28, 1980)

A. Seetharama Acharya and James M. Manning From The Rockefeller University, New York, New York 10021

Treatment of hemoglobin S with glyceraldehyde in- creases its minimum gelling concentration by covalent modification of the protein. Glyceraldehyde could be covalently bound to hemoglobin either iB aldimine or in ketoamine linkage. The adducts of glyceraldehyde with hemoglobin S have been found to be stabIe to dialysis and could be isolated by gel filtration. These results suggest that the linkage between glyceralde- hyde and hemoglobin S is not an aldimine type but that the initial Schiff base adduct had probably undergone Amadori rearrangement to a ketoamine. In a new pro- cedure for determination of ketoamine adducts, the globin prepared from [’4C)glyceraldehyde-hemoglobin A was treated with phenylhydrazine; the I4C label was not released from the protein on treatment with phen- ylhydrazine, but remained firmly bound even after gel filtration. Glyceraldehyde in aldimine linkage would have been released from the protein on treatment with phenylhydrazine. The globin preparation obtained after the gel filtration step was yellow in coIor with an absorption maximum around 350 nm, demonstrating the formation of the phenylhydrazone of the ketoamine adduct. The AS5,, of the globin sample (after treatment with phenylhydrazine) was directly proportional to the amount of glyceraldehyde bound to hemoglobin. Thus, the reaction of glyceraldehyde with HbS has similari- ties with the nonenzymic glucosylation of HbA. The phenylhydrazine reaction may be a general spectro- photometric method for the measurement of the extent of ketoamine adduct formation on condensation of a- hydroxyaldehydes with proteins.

The use of aldehydes as antisickling agents has been the subject of several recent studies (1-4). The results from this laboratory have shown that glyceraldehyde is an efficient antisickling agent that acts primarily at the stage of deoxy- hemoglobin S aggregation. In order to understand the molec- ular details of the mechanism by which the glyceraldehyde- hemoglobin adduct interferes with the aggregation of the protein and the sickling of cells, a detailed study on the sites of reaction of glyceraldehyde with carbonmonoxyhemoglobin S was undertaken (5); the major sites of reaction were found to be Lys-lG(a), Val-l(P), Lys-82(p), Lys-59(/3), and Lys- lZO(,B). In those structural studies the glyceraldehyde-hemo- globin S adduct was reduced with sodium borohydride (Fig. 1).

* These studies were supported in part by National Institutes of Health Grant HL-18819. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The finding that Val-l(P) was much more reactive with glyceraldehyde than Val-l(a) was reminiscent of a similar pattern of reactivity of glucose with hemoglobin to form HbAI, (6-8). The prolonged elevation of blood glucose levels, as in the case of patients with diabetes mellitus, results in increased amounts of nonenzymically glucosylated hemoglobin (9-13). Similar nonenzymic reactions of glucose with the amino groups of other proteins such as lens crystallin (14), red cell membrane proteins (15, 16), albumin (17), collagen (181, and basic myelin protein of nerve (19) have been observed. In all these nonenzymic glycosylation reactions, the general mech- anism involves the reaction of free amino groups of the pro- teins with the aldehyde function of the sugar residue to generate the Schiff base ( i e . the aldimine linkage). With simple aldehydes, this reaction is readily reversible. With glucose, which has a hydroxyl group at C-2, the aldimine linkage undergoes an Amadori rearrangement to form a more stable ketoamine adduct (20). The resulting stability accounts for the success in isolating glucosylated proteins without prior reduction with sodium borohydride.

In an effort to gain information on the stability of the glyceraldehyde-hemoglobin adduct, we present, in this com- munication, several approaches that demonstrate that a ke- toamine linkage is also present in this adduct.

MATERIALS AND METHODS

The preparation of cell lysates and the purification of HbS on columns of DE-52 were carried out as described earlier (5). The same procedure was used for the preparation of HbA. Hb concentrations are given as CO-Hb tetramer.

Isolation of Glyceraldehyde-Hemoglobin Adducts-Purified HbS or HbA in the carbonmonoxy form was dialyzed against phosphate- buffered saline (PBS)’ pH 7.4; ~~-[’~C]glyceraldehyde (custom syn- thesis by New England Nuclear a t 150 Ci/mol) was diluted with unlabeled DL-glyceraldehyde (Sigma) to attain the desired specific radioactivity. This [‘4C]glyceraldehyde, previously incubated at 37°C in PBS, was added to HbS or HbA solution (also incubated at 37°C) to a final concentration of 10 m. At different time intervals, appro- priate aliquots were removed and passed through a column of Seph- adex G-25 (1.5 X 30 cm) equilibrated and eluted with 10 m~ phosphate buffer, pH 7.4. This gel filtration separated the glyceraldehyde-he- moglobin adduct from the unreacted glyceraldehyde.

Preparation of Globin by Acid/Acetone Extraction-This prepa- ration was done essentially by the method of Rossi-Fanelli et al. (21). HbS or the glyceraldehyde adduct of HbS or HbA in 10 mM phosphate buffer, pH 7.0, was added slowly with stirring to 10 volumes of cold acetone, 0.2% in HC1 at 0°C. The globin precipitated and the heme remained in the acid/acetone phase. The sample was allowed to stand at 0°C for 10 min, and the precipitated globin was isolated by centrifugation of the sample at 2500 X g at 4°C. When [“C]glyceral- dehyde-HbA adducts were used, very little of the I4C label went into the acid/acetone phase, and almost all of the radioactivity was present in the precipitated globin. This precipitated globin was diisolved in

I The abbreviation used is: PBS, phosphate-buffered saline.

7218

Glyceraldehyde-Hemoglobin Adducts

HQ

&OH + HZN-HbS I CHzOH

Glyceraldehyde Hernoglobm s

11 H,CIN-HbS

I

7219

L

FIG. 1. The reaction of the glycer- 7 CHZOH I) N H N H ~ aldehyde-hemoglobin adduct with H-F-NH HbS

either Dhenylhydrazine or sodium H-C-OH

Aldirntne (Schlf f base) phenylhydrazine Adduct

borohykde: - C H 2 0 H I I Arnadarl

H -C-N-HbS

c=o I

I C H Z O H

Ketoamlne Adduct

a NHNHz

Phenylhydrozlne

water to the original volume and was precipitated with acid/acetone. The reprecipitated globin was taken up to 0.1 M acetic acid and lyophilized.

Reaction of Globin Samples of Glyceraldehyde-HbA Adducts with Phenylhydrazine-The lyophilized preparation of globin (about 0.5 to 1.0 p o l ) was dissolved in 2 ml of 10 m phosphate buffer, pH 7.0, 6 M in guanidine hydrochloride; 0.2 ml of 1 M sodium acetate, pH 5.0, and an appropriate aliquot of 1 M phenylhydrazine solution in 0.2 M sodium acetate buffer, pH 5.0, were added to this solution so that the final concentration of phenylhydrazine was 100 mM. The treatment with phenylhydrazine was carried out for 15 min at 37°C. The sample was then placed on a Sephadex G-25 column equilibrated with 0.1 M acetic acid; gel filtration was carried out using 0.1 M acetic acid to separate the globin sample from unreacted phenylhydrazine.

In the preliminary studies on the reaction of phenylhydrazine with a glyceraldehyde-RNase A adduct, it was not necessary to use 6 M guanidine hydrochloride during the reaction, since the glyceralde- hyde-RNase A adduct was soluble. Similarly, for gel filtration after reaction of phenylhydrazine with RNase, Sephadex G-25 columns were eluted with 10 rrm phosphate buffer, pH 7.0, but no guanidine was present since the phenylhydrazine derivatives of glyceraldehyde- RNase A adducts were soluble at pH 7.0.

Spectral Measurements-All the spectral measurements were car- ried out with an Aminco DW-2 spectrophotometer. All the other methods used were described earlier, except that in the reduction of phenylhydrazine derivatives of glyceraldehyde-globin adducts with NaBH4 (5), it was necessary to have present 6 M guanidine hydro- chloride to keep the protein soluble.

RESULTS

Stability of the Glyceraldehyde-Hemoglobin Adduct to Di- alysis-In our earlier studies (5) on the identification of the amino acid residues of HbS modified by glyceraldehyde, we reduced the product to generate fully stable 2,3-dihydroxypro- pyl derivatives* since we had only limited knowledge of the stability of any of these adducts. In order to determine whether the glyceraldehyde-HbS adduct could be isolated without prior reduction with sodium borohydride, purified hemoglobin S was incubated with 10 mM [‘*C]glyceraldehyde at pH 7.4 (PBS) for 90 min, and the reaction mixture was passed through a column of Sephadex G-25 equilibrated and eluted with PBS at 4°C. The radioactivity was eluted in two peaks (Fig. 2), a small amount of radioactivity (about 10% of the total) eluted with HbS, and the rest was eluted in the low molecular weight region of the Sephadex column. When the adduct is isolated by this procedure, the amount of the

* The compounds now referred to as 2,3-dihydroxypropylvaline and 2,3-dihydroxypropyllysine were previously termed glycerolvaline and glycerollysine, respectively.

2 8 c .-

E 6

a aJ

Phenylhydrazone of Glyceraldehyde ( l o w rnoleculor welqht product)

H I

H - C - N - H b S

l H C = N - N H ~ I

, C H 2 0 H

Phenylhydrazone o f Ketoamlne adduct (hlgh molecular

wetqht product)

Elut ion volume ( m I )

FIG. 2. Preparation of glyceraldehyde-hemoglobin adduct by gel filtration. Carbonmonoxyhemoglobin S (-1 mM in tetramer) was mixed with [“C]glyceraldehyde (final concentration 10 mM) at pH 7.4 in PBS at 37°C for 90 min. At the end of this incubation period, the reaction mixture was subjected to gel filtration on a Sephadex G-25 column (2 X 40 cm) equilibrated and eluted at 4°C with PBS saturated with CO, and the fractions were analyzed for Aero and for rahoactivity (0.1-ml aliquots). The fractions indicated, namely 54 to 66 ml were pooled, and used for the stability studies. The inset shows the kinetics of the release of [“Clglyceraldehyde from [“C] glyceraldehyde-hemoglobin S adduct when dialyzed against PBS at 23°C. The glyceraldehyde-hemoglobin S adduct isolated as described above was diluted to 20 ml with PBS and divided into 5 equal parts (4 ml each). One fraction was subjected to gel filtration on the Sephadex G-25 column as described above. The other 4 parts were dialyzed against PBS, removed after 3, 8, 20, 29 h of dialysis, respec- tively, and subjected to gel filtration on the Sephadex G-25 column. The I4C label that remains bound to HbS after various periods of dialysis against PBS was determined. The ’ C label present in the HbS sample that was not subjected to dialysis was taken as 100%.

[‘4C]glyceraldehyde bound to HbS is nearly the same as that obtained when the glyceraldehyde adduct was isolated after reduction with sodium borohydride (as described earlier (5)). Apparently, the linkage between the glyceraldehyde and pro- tein functional groups is sufficiently stable to permit isolation of the adduct by gel filtration. Similar results were obtained when gel filtration was performed at 23°C with 10 rn phos- phate buffer, pH 7.0, as the eluent.

To characterize further the stability of the glyceraldehyde-

7220 Glyceraldehyde-Hemoglobin Adducts

HbS adduct thus isolated, the appropriate fractions were pooled and dialyzed against PBS at room temperature (23°C); the kinetics of the release of protein-bound [‘4C]glyceralde- hyde upon dialysis was determined (Fig. 2, inset). If the glyceraldehyde were bound to HbS by a reversible aldimine linkage, incubation of the adduct at pH 7.4, 23°C (in the absence of any free glyceraldehyde), should have resulted in a release of the glyceraldehyde, which would have escaped from the dialysis bag. After 24 h of dialysis, nearly 90% of the glyceraldehyde remained bound to HbS. This observation suggested that the linkage between glyceraldehyde and he- moglobin S is not the aldimine type (Schiff-Base) since such a bond is reversible. Presumably, the initial aldimine product has rearranged to the more stable ketoamine product (Fig. 1). If the isolated glyceraldehyde-hemoglobin adduct is indeed a ketoamine derivative rather than the aldimine derivative, we reasoned that it should be possible to detect directly the protein-bound keto function.

Reaction of Protein- bound Glyceraldehyde with Phenyl- hydrazine-Preliminary studies on the reaction of phenylhy- drazine with RNase A that had been previously incubated with glyceraldehyde revealed that the glyceraldehyde RNase A adduct reacted with phenylhydrazine. After passage through a Sephadex column to remove excess phenylhydra- zine, the yellow protein derivative showed a new absorption band with a maximum around 350 nm in addition to its original absorption band around 280 nm. Native RNase A not treated with glyceraldehyde did not show the new absorption peak around 350 nm after treatment with phenylhydrazine. In addition, RNase A that had been reduced with sodium borohydride after treatment with glyceraldehyde did not ex- hibit the 350 nm band upon reaction with phenylhydrazine. These results suggested that phenylhydrazine may be useful for the detection of protein-bound ketoamine adducts.

However, in order to apply the phenylhydrazine reaction to hemoglobin to detect the protein-bound ketoamine adduct, several modifications were necessary to circumvent absorption artifacts from the heme moiety. Thus, it is necessary to prepare globin from hemoglobin before the reaction with phenylhydrazine. In addition, the insolubility of the phenyl- hydrazone derivative of the glyceraldehyde-globin adduct made it necessary to carry out the phenylhydrazine reaction in the presence of 6 M guanidine hydrochloride and to use Sephadex columns equilibrated and eluted with 0.1 M acetic acid to separate the protein from the excess reagent.

The characteristic absorption spectra of the phenylhydra- zine reaction products of the globin sample (obtained from HbA that had been previously incubated with 50 mM glycer- aldehyde at 37°C in PBS for various times) are shown in Fig. 3. The absorption spectrum of globin (prepared from native HbA, but not treated with glyceraldehyde) that was incubated with phenylhydrazine under identical conditions is also given for comparison. Only the glyceraldehyde-treated samples give rise to the absorbance with a maximum around 350 nm and this absorbance increases as a function of incubation time.

In this experiment we could determine how much of the protein-bound [‘4C]glyceraldehyde was present as the Schiff base or as the ketoamine. Acid/acetone precipitation of glyc- eraldehyde-treated HbA for the preparation of globin samples generally resulted in the recovery of 85 to 95% of the 14C label still attached to globin. Upon gel filtration of the phenylhy- drazone derivatives of globin prepared from the glyceralde- hyde-Hb adduct on Sephadex G-25 (equilibrated and eluted with 0.1 M acetic acid), almost all (>95%) of the I4C label remained bound to the protein for each of the samples. Less than 5% of the I4C label was recovered in the low molecular weight region. If the glyceraldehyde adduct had been present

L

40 260 280 300 320 340 360 380 400 420 440 460 480 5C

Wavelength (nm)

FIG. 3. Absorption spectra of phenylhydrazone-globin prep- arations. Globin samples were prepared from the glyceraldehyde- hemoglobin adducts (isolated by Sephadex G-25 gel filtration after incubating HbA with 50 mM [“Clglyceraldehyde for various periods) by the acid/acetone precipitation procedure and treated with 100 mM phenylhydrazine (see “Materials and Methods”). The phenylhydra- zine derivatives of the various globin preparations were separated from the unreacted phenylhydrazine by gel filtration on columns of Sephadex G-25 equilibrated and eluted with 0.1 M acetic acid. The A ~ M of the phenylhydrazone-globin samples thus isolated was ad- justed to about 1.8 by appropriate dilution with 0.1 M acetic acid and the spectra were recorded on an Aminco DW-2 spectrophotometer. Curves a, b, c, d, e, and frefer to incubation times of 0,0.3, 1.0, 2.0, 3% and 5% h, respectively.

in Schiff base linkage, phenylhydrazine would have liberated the 14C label from the protein (Fig. 1) since such “transaldi- minization” reactions are known to proceed rapidly (22). Thus, it is clear that practically all of the glyceraldehyde associated with the globin preparation is in a linkage other than the aldimine type (Schiff base).

The color yield a t 350 nm did not change when the concen- tration of phenylhydrazine was increased from 100 rn to 200 mM, a result which suggests that all of the protein-bound glyceraldehyde that was capable of reacting with phenylhy- drazine did react when the sample was treated with 100 mM phenylhydrazine. As a further check on whether the phenyl- hydrazone formation was complete, the globin samples (HbA preincubated with glyceraldehyde) were subjected to boro- hydride reduction after reaction with phenylhydrazine; the amount of dihydroxypropyllysine in the sample was deter- mined by amino acid analysis. No dihydroxypropyllysine was found but there was some radioactivity in this region of the chromatogram that could have arisen from hydrolysis of the phenylhydrazone. Based upon these results, practically all of the protein-bound glyceraldehyde can react with phenylhy- drazine. These results clearly indicate that the phenylhydra- zine reaction could be used to quantitate the amount of ketoamine product present in a given sample.

In addition to the spectral changes in the 350 nm region, there are also spectral changes a t 280 nm and 245 nm (Fig. 31. We have shown that these changes are also due to phenyl- hydrazone formation but they can be ignored for quantitative analysis of the data (see below). Amino acid analysis of each sample has been carried out to determine the protein concen- tration, and the yield of absorbance at 350 nm as a function of globin concentration.

Kinetics of Incorporation of Glyceraldehyde into Hb as Determined by Phenylhydrazone Formation-In an attempt to determine whether there is a correlation between the amount of glyceraldehyde incorporated into Hb and the

Glyceraldehyde- Hemoglo bin Adducts 7221

amount that is present as the ketoamine derivative as deter- mined by the formation of phenylhydrazone, a detailed anal- ysis of the amount of glyceraldehyde incorporated and the extent of spectral change has been carried out. The samples for analysis were the Same as those whose spectra are given in Fig. 3. A time-dependent incorporation of ['4C]glyceraldehyde into protein during the 5%-h incubation period was found (Fig. 4). During the initial 2-h period, the incorporation of glycer- aldehyde into protein was nearly linear, with nearly 60% of the total glyceraldehyde incorporated during this time; incu- bation for another 3% h resulted in another 40% of glyceral- dehyde incorporated into the protein. We presume that this differential reactivity is probably due to different groups re- acting with glyceraldehyde at different rates, since we know that the extent of reaction at a given site is variable (5). The slowly reacting groups of HbA could either be functional groups other than the amino groups or amino groups that slowly become accessible to the aldehyde subsequent to mod- ification of the rapidly reacting amino groups.

In order to correlate the A350 of the various samples directly with the moles of glyceraldehyde incorporated into the pro- tein, a sample of glyceraldehyde-hemoglobin adduct (not treated with phenylhydrazine) was reduced with sodium bor- ohydride and analyzed on an amino acid analyzer equipped with a scintillation flow cell. The specific activity of glyceral- dehyde was assumed to be the same as the specific radioactiv- ity of product, dihydroxypropyllysine. We consider the radio- activity of the product to be a more accurate estimate of the specific radioactivity of the glyceraldehyde and this value was used to calculate the moles of glyceraldehyde incorporated into the protein. The relation between the amount of glycer-

0 '

5 i / oozu Z 4 6 8 1 0 1 2

incorooroledlmale HbA Mole3 glyceraldehyde

u 5 6

Y I 2 3 4

1 6

1.4 - 0

1.2 - a

1.0 ~

D I 0 0

0.8 2 ? 0

0.6 $ a

0.4

0.2

Time in hours FIG. 4. Kinetics of incorporation of ["Clglyceraldehyde into

hemoglobin A as determined by phenylhydrazone formation. HbA (1 mM) was incubated with 50 mM glyceraldehyde at 37°C. and suitable aliquots were removed at 0.3 h, 1 h, 2 h, 3% h, and 5% h and subjected to gel filtration on Sephadex G-25 column equilibrated and eluted with 10 mM phosphate buffer, pH 7.0 at 23°C. I4C label associated with the protein peak was determined and used to calculate the counts per min per ASO. The globin prepared from these samples, was treated with phenylhydrazine and the samples were isolated by second Sephadex filtration step. A350 of the various samples was determined. To calculate the A~%/Aso of HbA, amino acid analysis of the globin samples was carried out. From the arginine content of a given sample, A m was calculated as a measure of protein concentra- tion. The inset shows the correlation between the moles of glyceral- dehyde incorporated into HbA and the development of absorbance ( A ~ w per nmol of HbA per ml) upon reaction of the glyceraldehyde- HbA adduct with phenylhydrazine.

aldehyde incorporated into the protein and the A350 generated on reaction with phenylhydrazine is presented in Fig. 4, inset. Two aspects of the reaction of glyceraldehyde with HbA are readily apparent; namely, that the amount of phenylhydra- zone formed is directly related to the amount of the glyceral- dehyde incorporated, and that no lag period is evident be- tween the reaction of glyceraldehyde with HbA and the reac- tivity of the protein-bound glyceraldehyde with phenylhydra- zine. The absence of a lag period indicates that there is very little, if any, Schiff base (aldimine) adduct of glyceraldehyde with hemoglobin. Thus, under the present experimental con- ditions almost all of the protein-bound glyceraldehyde is present as the ketoamine product. Since we have shown above that the reaction of phenylhydrazine with the ketoamine product is nearly complete, from Fig. 4 one can calculate the molar extinction coefficient of the phenylhydrazone of the ketoamine product to be about 7500.

Since the absorption band with the maximum around 350 nm is generated on reaction of the ketoamine-glyceraldehyde adduct with phenylhydrazine, if all of the bound glyceralde- hyde is reactive toward phenylhydrazine, a constant value should be obtained when one calculates the specific radioac- tivity of globin per A350. Table I gives those values at various periods of reaction. For most samples, this value is nearly the same except for the 20-min sample for which the value is about 15% lower.

Is the Amadori Rearrangement an Artifact of Acid/Ace- tone Precipitation-In all reactions of phenylhydrazine with HbA, the globin is isolated by the acid/acetone precipitation procedure. Since it is known that the Amadori rearrangement is an acid-catalyzed reaction (20), the question arises whether the glyceraldehyde bound to hemoglobin was actually present as a Schiff base but underwent rearrangement to the ketoam- ine product during acid/acetone precipitation. However, the fact that the glyceraldehyde-HbS adduct is quite stable against dialysis at neutral pH, 23"C, up to at least 24 h, argues against such an artifact. Furthermore, when glyceraldehyde- treated RNase A was reacted with phenylhydrazine, the pres- ence of the ketoamine product was evident. (This RNase A preparation was not treated with acid/acetone.)

Further studies were performed to exclude the possibility that the conditions of low pH used in the preparation of globin facilitated the rearrangement. The glyceraldehyde-hemoglo- bin adduct (2-h reaction product of Fig. 4) was lyophilized without prior preparation of globin. This sample in 10 mM phosphate (pH 7.0) and 7 M guanidine hydrochloride, was treated with phenylhydrazine. The product was placed on a Sephadex G-25 column equilibrated and eluted with 0.1 M acetic acid (Fig. 5A). Three components were present. The first was the protein peak that contained almost all of the radioactivity originally present in the glyceraldehyde-hemo- globin adduct. The second component appeared to be deriv- atives of heme and the third component was the unreacted phenylhydrazine. The specific activity of the globin (per Aam) was about 3.2 X lo5, much lower than that found when globin itself was treated with phenylhydrazine (Table I). We rea- soned that the protein peak was contaminated with some nonradioactive component having some absorbance at 350 nm, perhaps heme. Indeed, the spectrum of this protein sam- ple showed an absorption peak around 405 nm (probably a derivative of heme (23)) in addition to significant absorbance at 350 nm (Fig. 5, inset). The 405 absorption band could be removed from the protein-phenylhydrazone derivative by acid/acetone precipitation. The acid/acetone-precipitated globin was taken in 7 M guanidine hydrochloride (pH 7.0) and was again subjected to gel filtration on Sephadex G-25 (Fig. 5B). The protein spectrum at 350 nm was very similar to the

7222 Glyceraldehyde-Hemoglobin Adducts

TABLE I Specific radioactivity of the phenylhydrazone-globins

These data were calculated from the time points in Fig. 4. Time of glyceraldehyde reaction Counts per min X 10”/A:lm

h 0.3 1.0 2.0 3.5 5.5

3.9 4.6 4.9 5.0 4.8

O E

0:

0 4

0:

0 2

- 01 I Y

2 0 5 a

0

0 4

0.3

02

01

t

t

ir

320 360 400 440 Wovelength ( n m i

60 120 180 240 300 -I

Elutton volume ( m l 1

3000

2500

2000

1500

loo0 1 -

500 - c 3 - E

a 2500

u) c

2000 5 V 0

1500

1000

500

FIG. 5. Gel filtration of phenylhydrazine-treated [14C]glyc- eraldehyde-HbA adduct on Sephadex G-26. In A, the product was prepared and treated as described in the text. The globin sample which was yellow and had all of the I4C label showed a broad absorption band in the region of 320 to 440 nm with absorbance peaks around 350 and 405 nm (Curve A in the inset). As shown in B, this globin preparation was subjected to acid/acetone precipitation, and the precipitated protein taken in 7 M guanidine HCl and passed through the same Sephadex column ( E ) . The absorption spectrum of the yellow globin preparation thus obtained is given by Curve b in the inset. Note the absence of 405 nm band in this sample.

spectrum of the phenylhydrazone derivative of globin from the glyceraldehyde-Hb adduct and did not show any absorp- tion band around 405 nm (Fig. 5, inset). The specific activity of the globin per A3% was increased to 4.5 X lo5, a value very close to that found when globin prepared by the acid/acetone procedure was treated with phenylhydrazine (Table I). These results indicate that the glyceraldehyde-hemoglobin adduct is present as the ketoamine adduct even before it is subjected to acid/acetone precipitation for the preparation of globin.

DISCUSSION

The results presented in this communication establish that most, if not all, of the glyceraldehyde bound to HbA is present as the ketoamine adduct. Three types of evidence support this conclusion: 1) the stability of the glyceraldehyde bound to Hb against dialysis at pH 7.4 and 23°C; 2) the failure of the

glyceraldehyde bound to Hb to be released from the protein by phenylhydrazine; and 3 ) the ability of the glyceraldehyde bound to Hb to form a yellow protein derivative with phen- ylhydrazine.

The reversibility of the aldimine (Schiff base) linkages (Fig. 1) has been well documented. Zaugg et al. (1) have studied the Schiff base adducts of hemoglobin formed on incubating erythrocytes with many aliphatic and aromatic aldehydes. They have shown that the Schiff bases formed with aromatic aldehydes are reversible. Thus, hemoglobin extracted from erythrocytes exposed to 5 m~ 4-cyanobenzaldehyde for 30 min was greater than 90% modified. However, upon repeated reauspension of the treated erythrocytes in aldehyde-free buffer, the extent of modification was diminished progres- sively, ultimately to zero. On the contrary, the glyceraldehyde- hemoglobin adduct is stable to dialysis against glyceralde- hyde-free phosphate-buffered saline. In addition, the glycer- aldehyde-bound Hb did not undergo any trans Schiff base reaction (22) when it was incubated with phenylhydrazine. This procedure is generally used to remove pyridoxal phos- phate from those proteins that contain this coenzyme bound as a Schiff base (24). Thus, the stability of the glyceraldehyde bound to HbA is much higher than is expected of a simple Schiff base linkage. The presence of the protein-bound keto groups in the glyceraldehyde adduct establishes that the glyceraldehyde present in the adduct has undergone the Amadori rearrangement to generate 3-hydroxy acetonyl he- moglobin.

In principle, the Amadori rearrangement is the isomeriza- tion of an aldosylamine to a 1-amino-1-deoxy-2-ketose (20). The reaction presumably involves the acceptance of a proton by the glycosylamine base, prototropic shifts, and the subse- quent discharge of a proton from the cation originally formed. Thus, the Amadori rearrangement is generally pictured as the ammono analog of aldose enolization, when the latter is cat- alyzed by acids. This Amadori rearrangement was first thought to apply exclusively to N-substituted derivatives of glucosylamine. But it is now recognized to be a general reac- tion that might occur with virtually all classes of aldosylam- ines. I t was suggested that the aldotrioses also might undergo the Amadori rearrangement since glyceraldehyde, when treated with rn-nitrobenzhydrazine in dilute acetic acid solu- tions, formed the osazone of pyruvaldehyde only if a primary aromatic amine or an aliphatic diamine were present (25). The present study establishes that the protein-bound glycer- aldehyde does indeed undergo an Amadori rearrangement.

The use of the phenylhydrazine reaction to detect and estimate the protein-bound keto groups is well documented. Fields and Dixon have used 2,4-dinitrophenylhydrazine to detect protein-bound carbonyl groups (26). Riley and Snell (27) have used the reaction of phenylhydrazine to establish the presence of covalently bound pyruvate as the prosthetic group of histidine decarboxylase. More recently, Seto (28) has used the same reaction to detect the presence of pyruvate in proline reductase. With histidine decarboxylase, the presence of a phenylhydrazone-protein adduct was recognized by the formation of a new absorption band around 323 nm (27). The reaction of phenylhydrazine with free pyruvate resulted in the formation of two overlapping ultraviolet absorption bands, with maxima around 288 nm and 312 nm. The molar extinction coefficient of the phenylhydrazone of pyruvate, as well as the position of the absorbance appears to be influenced by the carboxyl group of pyruvate. When this carboxyl group was involved in a peptide bond with phenylalanine, as in histidine decarboxylase, the absorbance at 288 nm decreased consider- ably and the second band exhibited a red shift to 323 with a concomitant slight decrease in its absorbance. In the present

Glyceraldehyde-Hemoglobin Adducts 7223

study (unpublished difference spectrum) the two absorption bands with the maximum around 285 nm and 355 nm gener- ated on reaction of phenylhydrazine with protein-bound glyc- eraldehyde (ketoamine product) are well separated as com- pared to that of pyruvoyl phenylalanine phenylhydrazone. With the glyceraldehyde-hemoglobin adduct a t lysine resi- dues, the linkage to the protein is through a secondary amine attached to a lysine side chain in contrast to the peptide bond present in pyruvoyl phenylalanine. The different absorption characteristics, compared with the phenylhydrazone of histi- dine decarboxylase, could also be due to the presence of a primary hydroxyl group vicinal to the keto group in the adduct of glyceraldehyde with hemoglobin.

We previously pointed out that the reactivity of glyceral- dehyde with Val-l(P) compared with Val-l(a) is reminiscent of glucose hemoglobin adduct, HbArc ( 5 ) . This similarity in the reaction of glucose with HbA and the reaction of glycer- aldehyde with HbS may now be extended further. Recently, Bunn and his associates have identified the €-amino groups of HbA modified by glucose (29). Interestingly, Lys-lG(a) is the most reactive eamino group of the a chain as is the case for the reaction of glyceraldehyde with HbA. In addition, prelim- inary studies on the chromatography of glyceraldehyde-HbS adducts (without borohydride reduction) on columns of CM- 52 indicate a separation of HbS that has been modified a t a- amino groups of valine from Hb modified on the €-amino groups of lysine residues, a finding similar to that observed with glucose reaction products of Hb. The present study clearly brings out the analogy in the reaction mechanism of these two sugar aldehydes with hemoglobin; both rearrange to the stable ketoamine product. However, one difference that does exist between the reaction of these two sugar aldehydes is the relative rates of reaction with the protein. The reaction of glucose with HbA is quite slow but the reaction of glycer- aldehyde with Hb is much faster, since this aldehyde exists as the open chain conformer. It remains to be seen whether this difference in the rate of reaction of glucose and glyceraldehyde is a reflection of any possible differences between the rate of rearrangement of the initial Schiff base adducts of these two aldehydes with HbA or simply related to the fact that D- glucose equilibrates among three structures, a anomer, ,L3 anomer, and the reactive aldehyde form, the latter form being present at a concentration of 0.003% (30).

The present demonstration that the Schiff base of glycer- aldehyde with Hb rearranges to the stable ketoamine product is significant with respect to the potential usefulness of glyc- eraldehyde as a possible therapeutic agent for sickle cell disease. The stability of the ketoamine product suggests that a reasonable half-life might be expected. Indeed, it is only in the structural studies for the identification of the amino groups of HbS modified by glyceraldehyde that it is necessary to reduce the glyceraldehyde-hemoglobin adduct with boro- hydride. On the contrary, in the functional studies performed to date, no attempts were made to reduce the glyceraldehyde- hemoglobin S adduct. The glyceraldehyde-hemoglobin S ad- duct appeared fairly stable because the antisickling effect was not reversed by repeated washing with buffer at O"C, and the minimum gelling concentrating as well as the electrophoretic mobility of the glyceraldehyde-hemoglobin adduct were un- changed after several weeks at 4°C (2, 3). The results of the present study provide an explanation for the stability of the glyceraldehyde-hemoglobin adducts observed in the earlier studies. However, it should be noted here that when ["CI- glyceraldehyde-hemoglobin S adduct was dialyzed against PBS (pH 7.4) at 23"C, nearly 10% of the label did come out of the dialysis bag. Furthermore, we have determined that the composite half-life of glyceraldehyde bound to lysines of HbS

is about 13% h when the latter was incubated at 37"C, pH 7.4, in the presence of red cell lysates (31). In view of the present demonstration that an Amadori rearrangement takes place, we are currently attempting to examine the properties of HbS that has glyceraldehyde bound by ketoamine linkage to Val- 1(P) or at specific lysine residues to determine the reversibility of this rearrangement at the a- and €-amino groups of hemo- globin.

In the present study where relatively high concentrations of glyceraldehyde (50 mM) were used for extended incubation periods (5% h) the glyceraldehyde reaction is limited to the amino functions of the proteins only during the initial stages of the in~ubat ion .~ All of the functional studies reported earlier (2, 3) have been carried out with samples of HbS that were subjected to short periods of treatment with lower concentra- tions of glyceraldehyde (10 mM) and the antisickling effect observed was most likely due to the modification of specific amino groups.

I t is of interest to note that about 4 lysine residues (of the total 44 groups in the molecule) of Hb are modified with glyceraldehyde; these 4 residues have recently been identified (5). Preliminary studies indicate that these eamino groups react with glyceraldehyde at least 5 times faster than with the €-amino group of a-t-butoxycarbonyllysine. It remains to be seen whether this selectivity is simply a function of the lower pK, of these amino groups, thereby rendering them more amenable for the formation of the initial aldimine linkage, or whether the selectivity is related to the presence of functional groups in the proximity of these amino groups that could catalyze or participate in the Amadori rearrangement of the initial aldimine linkage.

Acknowledgments-The facilities provided by Drs. Stanford Moore and William H. Stein are appreciated. Blood samples from patients with sickle cell disease were generously provided by Dr. Charles Peterson, and by Dr. John Bertles. The assistance of Ms. W. M. Jones and Ms. Debra Freidus during the initial stages of this investigation is gratefully acknowledged. The assistance of Ms. Bar- bara Curtopelle in the typing of the manuscript is appreciated.

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