Specific modification of the carboxyl groups of hemoglobin S.
The reactivity of the carboxyl groups of hemoglobin S to form amide bonds with glycine ethyl ester by carbodiimide-activated coupling, and the influence of this derivatization on the functional properties of the protein have been investigated. Incubation of carbonmonoxy or oxyhemoglobin S with 20 mM 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide in the presence of 100 mM [14C]glycine ethyl ester, at pH 6.0 and 23 degrees C for 1 h resulted in the modification of, on an average, three carboxyl groups of the protein. The Hill coefficient of the modified hemoglobin S was 2.7, indicating normal subunit interactions. The derivatization increased the oxygen affinity of the molecule (the P50 was lowered from 8.0 to 5.0). The derivatization also resulted in an increase in the minimum gelling concentration of hemoglobin S from 16 to 24 g/100 ml. The reaction conditions used for the derivatization of the carboxyl groups of hemoglobin S are very selective for the protein carboxyl groups; very little of the label is associated with the heme carboxyls. Tryptic peptide mapping of the modified hemoglobin S indicated that the peptide beta T5, i.e. the segment representing amino acid residues 41 to 59 of beta-chain, accounted for nearly 75% of the label associated with the globin, demonstrating the high selectivity of the derivatization. Sequence analysis of the derivatized beta T5 demonstrated that at least 65% of the label incorporated into hemoglobin S is targeted toward the carboxyl group of Glu-43(beta), identifying it as the most reactive carboxyl group in hemoglobins. The results suggest that modification of the carboxyl group of hemoglobins S, presumably the gamma-carboxyl of Glu-43(beta), reduces the propensity of deoxyhemoglobin S to polymerize.
D019794:2,3-Diphosphoglycerate; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D006451:Hemoglobin, Sickle / Q000378:metabolism*; D006801:Humans; D010100:Oxygen / Q000378:metabolism; D010108:Oxyhemoglobins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D004163:Diphosphoglyceric Acids; D006451:Hemoglobin, Sickle; D010108:Oxyhemoglobins; D019794:2,3-Diphosphoglycerate; C029775:oxyhemoglobin S; C022010:glycine ethyl ester; D014357:Trypsin; D010100:Oxygen; D005998:Glycine