THE JOURNAL OP BIOLOGICAL CHEMISTRY Vol. 252, No 23, Issue of December 10, pp. 8542-8548, W7’7

Printed m U.S.A.

Schiff Base Adducts of Hemoglobin MODIFICATIONS THAT INHIBIT ERYTHROCYTE SICKLING*

(Received for publication, July 5, 1977)

ROBERT H. ZAUGG, JOSEPH A. WALDER, AND IRVING M. KLOTZ

From the Department of Biochemistry and Molecular Biology and the Department of Chemistry, Northwestern University, Evanston, Illinois 60201

Normal and sickle erythrocytes were exposed in vitro to millimolar concentrations of 31 different carbonyl com- pounds. Schiff base (imine) linkages were formed with amino groups of intracellular hemoglobin. Adducts were isolated by gel electrofocusing and could be dissociated by dialysis. Aromatic aldehydes proved more reactive than aliphatic aldehydes, and ketones were unreactive. The influ- ence of various ring substituents on the reactivity of aro- matic aldehydes was found to conform closely to traditional concepts regarding electronic and steric effects. Several of the aromatic aldehydes were shown to markedly increase the oxygen affinity of hemoglobins A and S. In particular, 2,4-dihydroxybenzaldehyde and o-vanillin, at concentra- tions of 5 mu, produced 2- to 3-fold reductions in the P,, (partial pressure of oxygen at half-saturation) of sickle hemoglobin in whole blood. Since low degrees of oxygen saturation promote erythrocyte sickling, compounds of this type significantly inhibit sickling at reduced partial pres- sures of oxygen.

Sickle cell disease is a hemolytic disorder in which deoxy- genated erythrocytes assume a variety of unusual shapes, most typically sickled forms. These abnormally shaped eryth- rocytes are less deformable than normal red blood cells and, as a result, tend to become trapped in and to occlude the microcirculation. These effects lead to tissue injury and necro- sis. The propensity of such cells to sickle is traceable to a markedly decreased solubility of deoxyhemoglobin S relative to the oxygenated form. Molecules of intracellular HbS’ aggre- gate upon deoxygenation and form helical fibers which distort the cell into characteristic sickle shapes (1, 2).

Chemical approaches toward modifying the behavior of hemoglobin S can be based on covalent or noncovalent modifi- cations. Among covalent modifications, blocking of amino groups seems particularly attractive since this can be achieved by relatively mild reagents that might be pharmacologically acceptable. One class of such reagents is exemplified by cyanate (3, 4) and by aspirins (57), the former producing a

* This investigation was supported in part by the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec- tion 1734 solely to indicate this fact.

1 The abbreviations used are: HbS, hemoglobin S; HbA, hemoglo- bin A.

carbamyl substituent and the latter an acyl adduct at amino group sites on the protein.

An alternative general procedure for attaching substituents at -NH2 sites takes advantage of Schiff base formation with aldehydes. In Go, the appearance of HbA,,, a glucose adduct of hemoglobin (8, 9), in the blood of normal individuals points strongly to Schiff base formation between glucose and this oxygen carrier. That such sugar adducts are formed in vztro has been demonstrated with hemoglobin (10-12) as well as with a Val-His peptide (13). Furthermore, extensive studies have shown that aldehydic pyridoxal compounds can also be coupled to hemoglobin in vitro (14-18). It has seemed appro- priate, therefore, to undertake a broader examination of the reactions of carbonyl compounds with hemoglobin and of the

effects of these compounds on sickling properties.

EXPERIMENTAL PROCEDURES

Materi&-All aldehydes and ketones were obtained from com- mercial sources. p-Dimethylaminobenzaldehyde was purchased from Lapine Scientific, pyridoxal and pyridoxal phosphate from Sigma, and others from Aldrich. These compounds were used as obtained or were purified by fractional distillation or recrystallization when necessary. Potassium cyanate was purchased from Baker.

Carrier ampholytes (pH 6 to 8) for isoelectric focusing were obtained from LKB, acrylamide and bisacrylamide from Aldrich, ammonium persulfate and N,N,N’,N’-tetramethylethylenediamine from Bio-Rad.

Erythrocyte Preparation-Whole blood was drawn by venipunc- ture from healthy adults. EDTA was used as anticoagulant. Sickle cell blood was obtained through the Hematology Department, Cook County Hospital, Chicago, Ill., as residual blood from homozygous SS individuals. Erythrocytes were washed twice in isotonic saline solution (0.154 M NaCl) and once in isotonic phosphate buffer (0.123 M sodium phosphate), pH 7.2. Experiments performed on normal whole blood required no prewashing of erythrocytes. Washed sickle cell erythrocytes were resuspended to original hematocrit in fresh, frozen human AB plasma for experiments involving whole sickle blood.

Chemical ModqGztions -All compounds were tested at a concen- tration of 5 rnsr in 20% (v/v) erythrocyte suspensions in isotonic phosphate buffer (pH 7.2). Supplemental tests were conducted in whole blood and in 6% solutions of cell-free hemoglobin. After addition of the test reagent, samples were incubated for i/z h at 37” in a water bath shaker. Incubations with each of several compounds for longer intervals demonstrated that the reaction attained equilib- rium within i/z h. Reactions involving erythrocyte suspensions were terminated by centrifugation of the cells, removal of the superna- tant, and rapid freezing of the layer of packed erythrocytes by immersion in a dry ice/methanol bath. Reactions with cell-free hemoglobin were ended by freezing the entire sample with dry ice/ methanol. In all experiments, controls lacking reagent were incu- bated under similar conditions.

8542

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Hemoglobin Modifications That Inhibit Sickling 8543

The extent of chemical modification of hemoglobin was assessed by isoelectric focusing as described by Drysdale et al. (19). Frozen erythrocytes were thawed and completely hemolyzed by addition of 5 volumes of ice cold distilled water containing 0.01 M NaCN. Cell- free hemoglobin was mixed with an equal volume of 0.01 M NaCN. The cyanide converts any methemoglobin (generally less than 10% of the total) to cyanomethemoglobin which co-electrophoreses with Fez+-hemoglobin (20). Gel electrofocusing was conducted in tubes (85 mm x 5 mm, inner diameter) containing 5% (w/v) acrylamide, 0.2% (w/v) bisacrylamide, 2% (v/v) carrier ampholytes, and 5% (v/v) glycerol. Solutions of 0.02 M phosphoric acid (pH 2.2) and 0.01 M NaOH (pH 12) served as anolyte and catholyte, respectively. The temperature of the jacketed electrophoresis chamber was main- tained at 4”. Following pre-electrophoresis for 10 min, 200 to 300 pg of Hb in 20 ~1 of 2% ampholytes was applied to each gel tube. A current of 1 mA/gel was maintained until the voltage reached 500 V, after which no further adjustments were made. Overall running time was 3 to 4 h, although the hemoglobin bands reached their equilibrium positions within ii/z h. In every case the modified species appeared in a position anodal to unmodified HbA.* After electrofocusing, the gels were removed from the tubes and fixed in 10% trichloroacetic acid to prevent diffusion of the focused bands. The extent of modification was quantitated by integration of peaks in densitometric scans of the gels.

Oxygen Binding Studies -Oxygen affinities of modified erythro- cytes were determined (a) in 20% cell suspensions of normal eryth- rocytes in isotonic phosphate buffer containing 3 rnsr compound, and (b) in whole sickle cell blood with 5 rnsr compound. After addition of the test reagent, adjustments in pH (in the range 7.2 to 7.4) were made as necessary so that control and treated suspensions were identical 10.02 unit. Cell suspensions (in lo-ml flasks) were incubated for i/z h at 37” in a water bath shaker. Gas mixtures containing 5, 4, 3, 2, and 1% oxygen in 5% CO, (Matheson Gas Products) were then administered successively, using a gas propor- tioner and flow meters supplied by Matheson. Erythrocytes were allowed l/z h to equilibrate at each oxygen level, after which a 0.5. ml aliquot was removed and its oxygen saturation measured on an IL 182 Co-Oximeter. In control experiments, this was shown to be sufficient time to attain equilibrium. The percentage of oxygen saturation was plotted against the partial pressure of oxygen (mm Hg). The P,, value (oxygen tension at 50% saturation) was deter- mined graphically for each of the treated and control suspensions. The following parameter was defined to describe the effect on oxygen binding of treatment with the various test compounds:

In this equation, P& and P&, are the partial pressures of oxygen at half-saturation for the control and treated suspensions, respectively.

Tests for in Vitro Antisickling Activity -A 4-ml sample of whole sickle cell blood containing 5 rnM reagent and an identical control sample lacking reagent were placed in lo-ml flasks and equilibrated successively with gas mixtures containing 5, 4, 3, 2, and 1% oxygen in 5% CO,. The blood was exposed to each oxygen mixture for 30 min after which an aliquot was removed and fixed in isotonic 2% formalin. A minimum of 300 fixed cells was counted using phase contrast optics at x 450 magnification. The extent of sickling was plotted as a function of oxygen partial pressure.

RESULTS

We have investigated the reactions of intracellular HbA with the 31 carbonyl compounds shown in Fig. 1. The extents of reaction, as assessed by isoelectric focusing, were quanti- tated by integration of densitometric scans of the gels after fixation in 10% trichloroacetic acid. As shown in Table I,

aromatic aldehydes (Compounds 1 to 25) generally effected substantial modification of intracellular hemoglobin. Ali- phatic aldehydes (Compounds 26 to 29) were less effective,

and ketones (Compounds 30 and 31) were unreactive. Carbonyl compounds react with free amino groups of pro-

’ Electrofocusing of hemolysate from untreated erythrocytes re- veals a faint band in this anodal region as well. This is due to HbA,, a glucose adduct of hemoglobin, normally present in trace amounts (Ref. 19).

TABLE I

Modifications of intracellular HbA with aldehydes and ketones;

effects on oxygen affinity

All reactions involved 20% (v/v) suspensions of normal erythro- cytes in isotonic phosphate buffer, pH 7.2, at 37”.

Comwund Modified” %APp;“”

Benzaldehydes 1 Benzaldehyde 2 Salicylaldehyde 3 3-Hydroxybenzaldehyde 4 4-Hydroxybenzaldehyde 5 o-Anisaldehyde 6 m-Anisaldehyde 7 p-Anisaldehyde 8 2-Carboxybenzaldehyde 9 4Carboxybenzaldehyde

10 2-Chlorobenzaldehyde 11 4-Cyanobenzaldehyde 12 4-Dimethylaminobenzaldehyde 13 Helicin 14 2,3-Dihydroxybenzaldehyde 15 2,4-Dihydroxybenzaldehyde 16 o-Vanillin 17 5-Nitrosalicylaldehyde 18 3,4-Dihydroxybenzaldehyde 19 Vanillin 20 Isovanillin 21 Veratraldehyde

%

60 5 45 50 60 45 30 20 55 40 65 30 25 10 15 5 20 0 so 35 90 55

0 0 10 15 40 75 70 60 55 75

0 55 30 15 50 30 30 35 50 40

Other aromatic aldehydes 22 trans-Cinnamaldehyde 23 Pyridoxal 24 Pyridoxal phosphate 25 Furfural

30 35 20 60

0 0 75 40

Aliphatic aldehydes 26 Valeraldehyde 27 Cyclohexanecarboxaldehyde 28 Glyceraldehyde 29 Glucose

25 0 25 0

5 0 0 0

Ketones 30 Fructose 31 2-Hvdroxvacetonhenone

0 0 0 0

n Percentage of total hemoglobin which is modified in the presence of 5 mM compound as assessed by isoelectric focusing.

b Percentage of change in P,, in the presence of 3 rnM compound:

teins to form reversible Schiff base linkages (21). That the modified species detected by isoelectric focusing in the present study were Schiff base adducts of hemoglobin is supported by the finding that the modification is reversible. For example, erythrocytes were exposed to 5 mM 4-cyanobenzaldehyde (Compound 11) for i/z h in isotonic phosphate buffer. Hemoglo- bin extracted from these erythrocytes was greater than 90% modified (Fig. 2, Gel 1). Upon repeated resuspension of treated erythrocytes in reagent-free buffer, the extent of modification was diminished (Fig. 2, Gels 2 and 3) and

ultimately reduced to zero (Fig. 2, Gel 4). Similar results were obtained with modified cell-free hemoglobin that was dialyzed against reagent-free buffer.

These experiments do not preclude the possibility that the reversible adduct formed is something other than a Schiff base. Model studies suggest that the most likely alternative

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Hemoglobin Modifications That Inhibit Sickling 8544

CHO

CHO

b

coo-

0 8

CHO CHO

&OH &OH

2 3

CHO CHO CHO CHO

CHO CHO CHO CHO CHO

CHO CHO CHO CHO

0 0 OCH,

IO I IN\

CH, CH,

12

CHO

OCH,

OCH,

21

FHO

‘=a kH,

bH,

&H,

26

CHO

6 0%

7

O-6 -DGlc

CHO CHO

@OCH, @OH

OCH,

20

HO

25

CHO

H&OH

kH,0~

28

FHO HFOH

HOFH

HFOH

HFOH

CH,OH

29

FH,OH COCH,

F=O HOFH

HFOH

HFOH 31 CH,OH

30 FIG. 1. Chemical structures of carbonyl compounds used to modify hemoglobins A and S. Predominant ionic species at neutral pH are

shown.

is thiohemiacetal formation with -SH groups of available cysteine residues (22). This possibility was ruled out by pretreatment of cell-free hemoglobin with the thiol-specific blocking agent, N-ethylmaleimide, using the procedure of

Benesch and Benesch (23). Subsequent exposure of the -SH- blocked hemoglobin to aromatic aldehydes 1, 2, 11, and 15 provided modification identical in extent to that obtained in the absence of N-ethylmaleimide.

Normal whole blood was treated with aromatic aldehydes 1 to 4, 11, 15, 16, 19, and 20 to assess the influence of serum proteins on the modification of intracellular hemoglobin. In general, the extent of hemoglobin modification in whole blood

was 50 to 75% of that obtained from reactions conducted in isotonic buffer.

The effect on oxygen affinity of modifying intracellular hemoglobin with 3 mM concentration of each compound is shown in Table I. In general, aromatic aldehydes provided substantial increases in the oxygen affinity of treated eryth- rocytes, whereas aliphatic aldehydes and ketones elicited no effect. Typical oxygen-binding curves for treated and un-

treated normal erythrocytes suspended in isotonic phosphate buffer are shown in Fig. 3. Salicylaldehyde (21, o-vanillin (16), and vanillin (19) produced substantial increases in oxy- gen affinity, characterized by decreases in the P,,. Glyceral-

dehyde (28) at 3 mM concentration elicited no effect. From Table I, it is apparent that chemical modification of hemoglo- bin is necessary to increase its oxygen affinity. Thus, com- pounds which do not modify hemoglobin do not affect oxygen binding. This dependence upon modification was further dem- onstrated by washing treated erythrocytes repeatedly in re- agent-free buffer as described in Fig. 2. A decrease in oxygen affinity resulted which paralleled the decreasing extent of modification.

Nevertheless, there is no direct correlation between the extent of modification and the effects on oxygen affinity. In particular, aromatic aldehydes that contain an o-OH substit- uent (Compounds 2, 14 to 17, and 23) had a disproportionately large effect on oxygen binding, whereas benzaldehyde (11, despite substantial modification, had little effect on oxygen affinity. This general lack of correlation may be due to

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Hemoglobin Modifications That Inhibit Sickling 8545

modifications occurring at different sites on the hemoglobin molecule. Support for this was provided by competition exper- iments. Benzaldehyde (at 3 and 5 mM concentrations) failed to diminish the increase in oxygen affinity produced by 3 mM 2,4-dihydroxybenzaldehyde (15).

Since the effects on oxygen affinity obtained with normal

FIG. 2. Electrofocused gels demonstrating reversibility of chemi- cal modification. Erythrocytes were treated with 5 mM 4-cyano- benzaldehyde (11) for l/z h at 37” in isotonic phosphate buffer, pH 7.2. Cells were then washed free of excess compound and resus- pended in reagent-free buffer. This procedure was repeated at hourly intervals for 6 h. Aliquots were removed and frozen at each interval. Isoelectric focusing was performed as described under “Experimental Procedures.” C, untreated Hb; 1 to 4, treated Hb after 0, 1, 2, and 6 resuspensions, respectively.

erythrocytes were in a favorable direction for the treatment of sickle cell disease, we studied the reactions of a number of aromatic aldehydes with intracellular HbS. Both the extent of modification and the influence on oxygen binding were very similar to those observed with normal erythrocytes. Fig. 4 shows oxygen-binding curves for treated and untreated whole sickle cell blood. With 5 mM salicylaldehyde (2) and o- vanillin (161, large increases in oxygen affinity were obtained. Potassium cyanate at this concentration had little effect on oxygen binding.

In vitro sickling tests were conducted on whole sickle cell blood containing 5 mM salicylaldehyde (21, o-vanillin (161, and potassium cyanate. The extent of sickling is plotted as a function of the partial pressure of oxygen in Fig. 5. The two aromatic aldehydes tested were approximately lo-fold more effective than cyanate in reducing the extent of sickling at equivalent oxygen tensions.

DISCUSSION

This report presents the results of a systematic examination of the influence of molecular structure on the reactions of carbonyl compounds with intracellular hemoglobins A and S. We have shown that aromatic aldehydes in particular form relatively stable adducts with hemoglobin which can be re- solved by isoelectric focusing. The modified species are focused at positions slightly anodal to unmodified hemoglobin (see Fig. 21, the position suggesting that a positive charge (e.g. E- NH2 of lysine) has been partially neutralized. Conjugate acids of Schiff base (imine) linkages between aromatic aldehydes and amino groups typically have pK, values near 7.0 (24). At neutral pH, therefore, the Schiff base adduct will carry on the average a half-positive charge producing a hemoglobin species which is slightly more anionic than unmodified pro- tein.

Further support for the existence of Schiff base linkages between aldehyde and hemoglobin was provided by the dem- onstration that adduct formation is reversible (see Fig. 2). In addition, the possibility that a reversible thiohemiacetal link- age formed was excluded by showing that modification with aldehydes was unaltered by pretreatment of hemoglobin with the thiol-specific agent, N-ethylmaleimide. loo a” # :“# i;;m 50 _ 8 50 -.. . . . . . . . . . . . . . . . . . . . . . . . . . . . IO - IO-

0 I I I I I I , I 0 lo 20 30

0 0 0 IO 20 30 40 0 IO 20 20 40

PO, hnl) ~0~ (mm) pot hnml

FIG. 3 (left). Oxygen-binding curves for normal erythrocytes 2,4-dihydroxybenzaldehyde (A-A), o-vanillin (V-V), or un- in isotonic phosphate buffer treated with 3 mM glyceraldehyde treated (O-O). (O-O), vanillin (&---a), salicylaldehyde (A---A), o-vanillin FIG. 5 (rig&). Sickling curves for whole sickle blood treated (V-V), or untreated (0-O). with 5 rnM potassium cyanate (O-O), o-vanillin (+---MI, sali-

FIG. 4 (center). Oxygen-binding curves for whole sickle blood cylaldehyde (A-A), or untreated (O-O). treated with 5 mM potassium cyanate (O--O), vanillin (m----m),

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8546 Hemoglobin Modifications That Inhibit Sickling

In Schiff base formation, an equilibrium of the following type is established:

Hb-NH, + R--CHO : Hb-N-CH-R + H,O (1)

An equilibrium between modified and unmodified hemoglobin should also exist within the electrofocusing gel. At a given concentration of free aldehyde, the extent of Schiff base formation should be the same within the gel as it is in solution. However, since little, if any, free aldehyde placed on the gel will migrate to the isoelectric positions of the hemoglo- bin species, the free concentration of aldehyde at those posi- tions will be derived solely from dissociation of the adduct. Hence, the concentration of free aldehyde, as well as the extent of modification observed in the gel, is a lower limit to the equilibrium value that exists within the erythrocyte. If the free aldehyde is uncharged at the isoelectric positions of the hemoglobin species, then its local concentration would be depleted only by diffusion. Since this is a relatively slow process within the gel, the Schiff base adduct should be comparatively stable. If, on the other hand, the free aldehyde is charged at the isoelectric positions of the hemoglobin species, it will migrate away from the site of focusing in response to the electric field, and the migration will cause the equilibrium in Equation 1 to shift progressively to the left.

This dissociating effect of an electric field on an adduct was demonstrated with the complex formed between HbA and 2,4- dihydroxybenzaldehyde (Compound 15). Inspection of the fo- cused Hb bands 2 h after onset of electrophoresis showed that about 90% of the hemoglobin was formed into a complex with aldehyde. Inspection of the gels after 3l12 and 5 h of focusing revealed that the extent of adduct formation had decreased to 70 and 50%, respectively. The first pK, for the ionization of the hydroxyl groups of 15 was determined by titration to be 7.45. Thus, at the p1 of the HbA.Schiff base complex (6.901, the free aldehyde will be about 20% ionized. As a result, the free aldehyde will migrate from the position of the hemoglobin species toward the anode, this migration leading to net disso- ciation of the Schiff base complex. Parallel tests with the adduct of hemoglobin and benzaldehyde (11, which lacks an

ionizable group, showed no change in the extent of modifica- tion with time of electrofocusing. These results demonstrate both the reversibility of Schiff base formation within the gel and the effect of the electric field on the stability of the equilibrium when the aldehyde carries a net charge.

The above analysis of electric field effects explains the paradoxical results obtained with 5nitrosalicylaldehyde (Compound 1’7). Treatment of erythrocytes with this com-

pound caused a significant increase in the oxygen affinity of hemoglobin (%AP,,, = 55) although no modification was de- tected by electrofocusing (see Table I). Clearly, a certain extent of chemical modification must be occurring if we are to account for the observed changes in the oxygen-binding prop- erties of the protein. The hydroxyl group of 17 is ortho to a formyl group and para to a nitro group and would be expected to have a very low pK, relative to phenol. This expectation was confirmed by spectrophotometric examination of 0.1 mM 5-nitrosalicylaldehyde in a series of 0.05 M acetic acid/sodium acetate buffers, pH 4.0 to 5.5. The pK, of the phenolic -OH was found to be 5.30. At the isoelectric pH of the Hb.Schiff base complex (6.901, the free aldehyde, being more than 97% ionized, would migrate more rapidly than 2,4-dihydroxyben- zaldehyde. As a result, the free aldehyde concentration would be depleted before a stable adduct could be observed.

Aside from Compounds 8,9, 17, and 24, all other compounds

studied possessing ionizable groups have pK, values equal to or greater than that of 2,4-dihydroxybenzaldehyde. Hence, these compounds should form relatively stable equilibria within the gels, and the observed levels of modification should parallel the equilibrium values that exist within the erythro- cyte. In support of this, we find that the relative extents of Schiff base formation with hemoglobin are in general agree- ment with predictions based on studies of model amines.

For simple amines, imine formation decreases in the order: aromatic aldehydes = aliphatic aldehydes > aliphatic ketones > aromatic ketones (25). Within each of these classes, elec- tron-withdrawing substituents enhance Schiff base formation while sterically bulky substituents adjacent to the carbonyl group disfavor adduct formation (26). We found that neither aliphatic nor aromatic ketones (Compounds 30 and 31) formed Schiff base adducts with hemoglobin. On the other hand, aldehydes readily formed Schiff bases with hemoglobin, and aromatic derivatives showed a 2- to 3-fold increased reactivity over aliphatic aldehydes. Weakly electron-donating substitu- ents, such as p-OH and p-OCH, groups (Compounds 4 and 71, caused a lowered reactivity relative to benzaldehyde (see Table I). These same substituents in the meta position either are neutral (m-OH) or slightly electron-withdrawing (m- OCH,,) and Compounds 3 and 6 provided modification of HbA that was equivalent to or slightly greater than 1. Strong electron-withdrawing groups, such as 2-chloro and 4-cyano, make 10 and 11 the most reactive compounds tested, whereas strong electron donation by the p-dimethylamino group in 12 reduced the reactivity of that compound to zero. The p-carbox- ylate group is slightly electron-withdrawing, but 9 provided greatly reduced modification relative to 1 rather than the expected increase. The presence on 9 of a fixed negative charge at neutral pH should reduce the extent of modification detected electrophoretically in a manner analogous to that observed with 5-nitrosalicylaldehyde. However, treatment of erythrocytes with 9 caused no change in the oxygen-binding properties of hemoglobin. Thus, if considerable undetected adduct formed with 9, the modification must have taken place at a site that does not influence oxygen binding.3 Helicin (13), a highly soluble /J-n-glucoside of salicylaldehyde, achieved only slight modification of intracellular HbA. This behavior is probably a reflection of the steric hindrance imposed by the bulky o-substituent.

The modification data shown in Table I for the 3,4-disubsti- tuted benzaldehydes (18 to 21) are consistent with the findings just described for monosubstituted compounds. The electron withdrawal provided by the m-OCH, group in 19 and 21 increased the reactivity of these compounds relative to 18 and 20. The latter compounds carry a m-OH group which is neither a withdrawing nor a donating substituent. Both -OH and -OCH, groups are electron-donating in the para position, however, and this effect lowered the reactivity of all four compounds relative to benzaldehyde.

Resonance stabilization of the allenic substituent in cinna- maldehyde (22) should disfavor adduct formation and cause decreased modification relative to 1, as is indeed observed. Pyridoxal (23), pyridoxal phosphate (241, and furfural (25) all contain highly reactive carbonyl groups by virtue of strong

electron-withdrawing ring systems, but of these only 25 pro- vided increased modification. As a dianion at neutral pH, 24

3 Treatment of cell-free hemolysate with 9 indicated that the lack of modification was not due to a failure of the compound to permeate the erythrocyte membrane.

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Hemoglobin Modifications That Inhibit Sickling 8547

cannot permeate the erythrocyte membrane and is therefore nonreactive. The low level of observed modification with pyridoxal, which is membrane-permeable, is difficult to ex- plain, especially in light of its potent effect on oxygen binding. At neutral pH, 23 is less than 5% ionized (27) so that the low level of modification cannot be explained by an electric field effect as in the case of 17. Other factors must be responsible for the large discrepancy between extent of modification and oxygen binding in this case.

The available evidence from model studies indicates that Schiff base formation with small primary amines occurs somewhat more readily with aromatic aldehydes than with aliphatic. Hine et al. (28), in comparing Schiff base equilibria between methylamine and isobutyraldehyde or 4-pyridinecar- boxaldehyde, found the aromatic imine to have a lo-fold greater equilibrium constant (87 M-‘) than the aliphatic imine (8.5 Mm’). Pesek and Frost (29) obtained qualitatively similar results upon equilibrating n-butylamine with isobutyralde- hyde (K,, = 16 M-‘I, cyclohexanecarboxaldehyde (K,, = 28 Mm’), and benzaldehyde (Kc, = 40 M-I).

Extents of hemoglobin modification obtained in the present study (see Table I) can be readily converted to apparent equilibrium constants. A typical aromatic aldehyde (at a free concentration of 5 mM) eliciting 50% modification would have

a K, = 220 Mm’, whereas aliphatic aldehydes 26 and 27, providing 25% modification, have a K,, = 70 M-‘. These values, which represent lower limits to the true equilibrium values, are 3- to &fold greater than typical Schiff base equilib- rium constants determined with small molecule amines. This enhancement may reflect a macromolecular environment which favors the adduct relative to the starting materials, perhaps by excluding water as it forms.

Normal erythrocytes showed marked increases in oxygen affinity when exposed to single, low doses of certain aromatic aldehydes (see Table I). Aliphatic aldehydes and ketones manifested no effect on oxygen binding despite significant adduct formation in certain instances. Although modification by aromatic aldehydes was necessary to influence oxygen binding, it was not a sufficient condition. Thus, benzaldehyde, a potent modifier, had little or no effect on oxygen affinity.

The most potent effecters of oxygen binding were those aromatic aldehydes which contain an o-hydroxyl group (2, 14 to 17, and 23). This substituent may direct these compounds to specific sites on hemoglobin which strongly influence the

OXY e deoxy equilibrium. In support of this, we found that benzaldehyde (at 3 or 5 mM) did not compete with 2,4-dihy- droxybenzaldehyde (3 mM) to diminish the increased oxygen affinity elicited by the latter.

Two sites at which modification should increase the oxygen affinity are the NH,-terminal amino groups of the a-chains. Benesch et al. (18) have recently reported increases in oxygen affinity upon attachment of 5’-deoxypyridoxal at these sites. Likewise, specific carbamylation of these amino groups by cyanate leads to an increased oxygen affinity (4). Alterna- tively, Schiff base formation by aromatic aldehydes in the region of the P-chain NH, termini could increase the oxygen affinity by preventing normal binding at this site of 2,3- diphosphoglycerate, a potent negative effector of oxygen bind- ing (30). Schiff base formation at Lys 4Oa, which forms a salt bridge in deoxyhemoglobin to the COOH-terminal carboxylate group of His 146p (311, may also tip the oxy ti deoxy equilibrium in favor of the oxy form.

Exposure of sickle erythrocytes suspended in isotonic phos- phate buffer to a variety of aldehydes produced modification

of intracellular hemoglobin, as well as mcreases m oxygen affinity, equivalent to those observed with normal erythro- cytes. Since studies conducted in whole normal blood did not reveal large reductions in the extents of modification relative to those obtained in buffer, the remaining studies with sickle erythrocytes were done in AB plasma.

Fig. 4 shows the effects of several aromatic aldehydes on the oxygen binding of sickle cell erythrocytes in whole blood. As for normal erythrocytes, o-OH-substituted benzaldehydes produce the greatest increases in oxygen affinity. The oxygen- binding curve for sickle cells treated with an equivalent concentration of potassium cyanate is also shown. At this concentration (5 mM), cyanate has little effect on oxygen binding. Comparison is made with cyanate since this com- pound inhibits sickling by increasing the oxygen affinity of HbS (32). Fig. 5 shows that aromatic aldehydes likewise inhibit erythrocyte sickling. Indeed, salicylaldehyde (2) and o-vanillin (16) are approximately lo-fold more effective than cyanate in reducing the extent of sickling at equivalent oxygen tensions. Convergence of the curves in Fig. 5 at low ~0, values suggests that these aldehydes have little effect on

sickling in the absence of oxygen. Thus, as is the case with cyanate, the anti-sickling effects of these compounds appear to be mediated through increases in the oxygen affinity of intracellular hemoglobin S.

Sickle erythrocytes have a diminished oxygen affinity rela- tive to normals (compare control curves in Figs. 3 and 4). In view of observations reported for 5’-deoxypyridoxal (18) and those described here for a variety of aldehydes, it seems reasonable to expect that artificial elevation of the oxygen affinity of sickle blood into the normal range by administra- tion of certain aromatic aldehydes could alleviate sickling UL uiuo. Cyanate inhibits sickling in uivo by this mechanism, but therapeutic levels give rise to adverse side effects (33). The greater efficacy of the present compounds may permit their use at lower dosages, well within an acceptable margin of safety.

Acknowledgments -We express our appreciation to Drs. Ashok Pate1 and K. R. P. Rao, Department of Hematology, Cook County Hospital, Chicago, for providing residual sickle cell blood used in this study and for helpful discussions. We are also indebted to Mrs. Janet Goranson for her assistance with the manuscript.

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R H Zaugg, J A Walder and I M KlotzSchiff base adducts of hemoglobin. Modifications that inhibit erythrocyte sickling.

1977, 252:8542-8548.J. Biol. Chem. 

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