Abstract
The chemical modification of hemoglobin by aspirin (ASA) has been studied, both in intact human red cells and in purified hemoglobin solutions. After incubation of red cells with 20 mM [acetyl-1minus14C]ASA, incorporation of radioactivity into hemoglobin was observed in agreement with the results of Klotz and Tam (1973. Proc. Natl. Acad. Sci. U. S. A. 70: 1313-1315). In contrast, no labeling of hemoglobin was seen when [carbosyl-14-C]ASA was used. These results indicate that ASA acetylates hemoglobin. The acetylated hemoglobin was readily separated from unmodified hemoglobin by both gel electrofocusing and by column chromatography. Quantitation of the extent of acetylation by densitometric scanning of gels agreed very well with estimates obtained from radioactivity measurements. Hemolysates prepared from red cells incubated with ASA showed normal oxygen affinity and heme-heme interaction. Purified acetylated hemoglobin had a slightly increased oxygen affinity and decreased heme-heme interaction. There was no difference in the rate of acetylation of oxy- and deoxyhemoglobin. ASA acetylated column-purified hemoglobin A more readily than hemoglobin in crude hemolysate, but less rapidly than purified human serum albumin. The rate of acetylation of hemoglobulin increased with pH up to approximately pH 8,5. Structural studies were done on hemoglobin incubated with 2.0 mM and 20 mM [acetyl-1-14-C]ASA. Alpha- and beta-chains were acetylated almost equally. Tryptic digests of purified acetylated subunits were fingerprinted on cellulose thin layer plates and autoradiographed. Both alpha- and beta-chains showed a number of radioactive spots that were either ninhydrin negative or weakly ninhydrin positive. These results indicate that hemoglobin is acetylated at a number of sites, probably at the epislon-amino group of lysine residues. To determine whether ASA acetylates hemoglobin in vivo, hemolysates of 14 patients on long-term high-dose ASA therapy were analyzed by gel electrofocusing and compared to specimens of individuals not receiving ASA. The ASA-treated group had a twofold increase in a minor hemoglobin component having an isoelectric point lower than that of hemoglobin A, and indistinguishable from the minoe component which appears when hemoglobin is incubated with ASA in vitro.