Proc. Nat. Acad. Sci. USA Vol. 70, No. 5, pp. 1313-1315, May 1973 Acetylation of Sickle Cell Hemoglobin by Aspirin (acetylsalicylic acid/oxygenation/anemia/urea/ethanol) IRVING M. KLOTZ AND JOSEPH W. 0. TAM Biochemistry Division, Department of Chemistry, Northwestern University, Evanston, Illinois 60201 Contributed by Irving M. Klotz February 26, 1973 ABSTRACT Incubation of HbS (or HbA) with aspirin hemoglobin was precipitated by addition of cold 5% tri- leads to incorporation of acetyl groups into the protein. chloroacetic acid. Samples of 0.5-0.8 mg were collected on Incorporation was followed by the use of aspirin labeled with 14C in the acetyl group. The acetylated hemoglobins 0.45 ,gm Millipore filters and washed with cold trichloroacetic show an increase in oxygen affinity compared to the parent acid to remove any noncovalently bound acetate. Some proteins. If acetylation also occurs in vivo, administration hemolysate mixtures were also dialyzed against bis-tris- of aspirin might ameliorate the severity of sickle cell buffered saline, pH 7.6, at 40 for 24 hr before being precipi- disease. tated for radioactive assay. The hemoglobin precipitate was Recent studies (1, 2) have shown that carbamylation of the mixed with 0.2 ml of water and 10 ml of scintillation fluid N-terminus of the jB-chain (as well as of the a-chain) of HbS consisting of 6 g of 2,5-diphenyloxazole and 75 mg of 1,4- is obtained on exposure of sickle-cell hemoglobin to cyanate. bis-2-(5-phenyloxazolyl)-benzene dissolved in 1 liter of a Oxygenation curves and other properties of carbamylated 2:1 mixture of toluene and Triton X-100. To correct for HbS are modified in a direction that should decrease the quenching, internal standards were used. A duplicate count acuteness of clinical manifestations of the presence of this was taken 24 hr after the first determination. protein. The concentration of hemoglobin in solution was deter- Chemical blocking of the amino-terminal valine chains thus mined from the absorbance at 540 nm of the cyanomethemo- appears to be a promising procedure for ameliorating the globin derivative. severity of sickle cell anemia. We have been interested in Extents of incorporation of [14C]acetyl groups are listed in achieving the same goal by a blocking procedure using a sub- Table 1. stance pharmacologically more acceptable than cyanate. Oxygen dissociation curves of hemoglobin, in erythrocytes Recalling that aspirin, acetylsalicylic acid, is able to acetylate or in solution, were determined by standard (4, 5) procedures. serum albumin (3), we have performed experiments to see Pressures of oxygen, P50, at which half-saturation of hemo- whether this drug also acetylates hemoglobin. Our results globin is achieved, are summarized in Table 2. show that such acetylation does indeed occur. Furthermore, RESULTS the modified hemoglobin shows an increased oxygen affinity. Aspirin thus should be an attractive candidate as a substance As the data in Table 1 illustrate, acetylation of hemoglobin S that can prevent or inhibit the onset of erythrocyte sickling. by aspirin at 25° is very slow. No significant uptake was ob- served in a 90-min period, either at pH 6 or 7. In 24 hr at 250, MATERIALS AND METHODS 0.15 mol of [14C]acetate was incorporated per mol of Hb. Incorporation experiments were done with whole blood, with Much increased acetylation was achieved at 370, however. erythrocytes, and with hemolysates. Fresh, heparinized, whole blood was oxygenated and then centrifuged to remove plasma. The erythrocytes were washed several times with TABLE 1. Incorporation into hemoglobin of [14C]acetate phosphate-buffered saline, pH 7.3. Hemolysates were obtained from aspirin by the freeze-thaw procedure, followed by removal of Mol of [14C]acetate erythrocyte ghosts by centrifugation at 50,000 X g for 1 hr. Tem-Tem- .0 Radioactively-labeled aspirin (14C in the carboxyl group of pera- Hours of in~~corporated acetyl), 6.60 Ci/mol and warranted isotope purity of 98% Hemoglobin ture, incuba- Mol of Hb Tetramer (from Mallinckrodt Chemical Works), was diluted with nine sample 0C. tion at pH 6.0 at pH 7.0 parts by weight of unlabeled aspirin (Baker Chemical Co.). The diluted aspirin and hemoglobin were mixed to give con- HbS in erythrocytes 25 1.5 '0;.01 '0.01 and mM (of HbS in whole blood 25 1.5 -00.01 -0.01 centrations of 5-20 mM of the aspirin 0.5-1.0 HbS in hemolysate 25 1.5 -.O01 0.1 tetramer) of the hemoglobin. The pH was adjusted to physio- HbS in whole blood 25 24 0.15 0.15 logical. The mixtures were incubated at a constant tempera- HbS in erythrocytes 37 1.0 0.35 ture with slow stirring. HbS in hemolysate 37 1.0 0.40 The extent of incorporation of [14C]acetyl groups was HbA in erythrocytes 37 1.0 - 0.38 measured by liquid scintillation counting. Erythrocytes were HbA in hemolysate 37 1.0 - 0.42 firstwashed several times with 0.9-1.2% NaCl, then hemolyzed. The ghosts were removed by centrifugation. In all solutions Aspirin concentration was 20 mM. 1313 Downloaded by guest on October 1, 2021 1314 Biochemistry: Klotz and Tam Proc. Nat. Acad. Sci. USA 70 (1973) TABLE 2. Effect of acetylation of sickle cell hemoglobin Carbamylation of hemoglobins by cyanate has been found, on its oxygen affinity by others (5, 15), to increase oxygen affinity and, as Table 2 illustrates, acetylation by aspirin has a similar effect. Thus, P50, oxygen these modifications lead to increased concentrations of pressure oxyhemoglobin under hypoxic conditions and should, (mm of Hg) there- Hemoglobin Aspirin Hours of at half- fore, defer the onset of sickling. Since the pharmacology of sample (mM) incubation pH saturation aspirin has been exhaustively studied for almost a century (16), it has advantages over a new drug with similar potential. In erythrocytes 0 1-2 7.30 31 Even its inhibiting effect on the formation of prostaglandin 12 2.0 7.30 26 (17) may be advantageous, for prostaglandin has been re- 22 1.0 7.30 21 ported to induce sickling (Johnson, M., Rabinowitz, I., In hemolysate 0 1.0 7.30 12 A. 0 * 7.22 11 Willis, L., and Wolf, P. L., personal communication). 8 1.0 7.22 6.7 Furthermore, since acetylated hemoglobin is found in signifi- 8 1.5 7.22 5.3 cant amounts even in normal blood (18-20), there seems to be little likelihood of any auto-immune response to the acetylated * This sample was dialyzed overnight at 4° against saline with Hb generated by aspirin. [bis - (2 - hydroxyethyl)imino] - tris - [ (hydroxymethyl)methanel Noncovalent modifications of HbS aimed at perturbing its (Bis-Tris) buffer, pH 7.6. behavior, particularly its solubility, are exemplified by the use of urea (21-23). The rationale given for its application is that urea breaks hydrophobic bonds. Experience with simple proteins, however, indicates that urea does not affect intra- or inter-molecular protein interactions until its concentration Furthermore, the extent of incorporation was essentially the is well above 1 M, whereas it is said to be clinically effective same for Hb within erythrocytes as for Hb in hemolysates. at 50 mM concentrations (22, 23). In any event, the concept Table 2 demonstrates the effect of acetylation on P50. of intruding in intermacromolecular interactions to increase Whether within erythrocytes or in a hemolysate, HbS macromolecule solubility is sound. Again we ask, therefore, develops an increased oxygen affinity when acetylated by this time in regard to noncovalent modifications, whether aspirin. there are pharmacologically more acceptable procedures than the use of high concentrations of urea. There are indeed other DISCUSSION solutes, ethanol among them, that are effective in intervening Chemical approaches toward modifying the behavior of HbS in intermolecular bonding. Aqueous solutions containing can be based on covalent or noncovalent modifications. Among 0.1 M ethanol markedly weaken the binding of uncharged covalent modifications, blocking of the N-terminal amino small molecules by serum albumin (Klotz, I. M. and Ayers, J., groups seems particularly attractive, since the Val-1 residues unpublished results). Somewhat lesser, but definite com- of the (3-chains are at the portal of the tetramer into which petitive effects, should be present at lower alcohol concen- 2,3-diphosphoglycerate fits and modulates the deoxy-oxy trations. Blood concentrations of 50 mM, corresponding to equilibrium (6, 7). Partial blocking of this portal and reduc- 200 mg of ethanol per 100 ml of blood, are only mildly in- tion of its positive charge should shift the hemoglobin con- toxicating (24). It would be worthwhile, therefore, to examine formational equilibrium toward the oxygenated state. One the solubility of deoxygenated HbS in aqueous ethanol of general procedure for attaching substituents at this position 50-100 mM concentration. Similarly, the effect of low con- would be to take advantage of Schiff base formation between centrations of ethanol on the aggregation of HbS molecules an aldehyde and the terminal a-NH2 group. In vitro, pyridoxal below saturation could be studied, by hydrodynamic or by phosphate forms such a linkage with hemoglobin (8). It seems thermodynamic techniques. These experiments would provide very likely that glucose (or other reducing sugars) will do so in vitro background for appropriate clinical trials. also. A Val-His peptide definitely couples with glucose (9). We thank Dr. M. Vye and Mrs. K. Y. Tam of Evanston In vivo, the appearance of HbAj0, a glucose adduct of Hb Hospital for supplying us with samples of blood. We are also (10, 11), in normal bloods points strongly to a reaction of indebted to Drs. L. Lorand, G. Means, and D.