Reversible reaction of 5,5'-dithiobis(2-nitrobenzoate) with the hemoglobins of the domestic cat: acetylation of NH3+ terminal group of the beta chain transforms the complex pH dependence of the forward apparent second order rate constant to a simple form.
We demonstrate kinetically that the reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl group of domestic cat hemoglobins is a reversible process. In the major hemoglobin, in which the NH3+ terminal group of GlyNA1[1]beta is free, kf, the apparent forward second order rate constant, has a complex pH dependence profile. In the minor hemoglobin, the NH3+ terminal group of SerNA1[1]beta is acetylated, and the pH dependence profile of kf is simple. These results support the proposal that the positively charged groups at the organic phosphate binding site are electrostatically linked to CysF9[93]beta. Quantitative analyses of the complex profiles enabled us to estimate pKas of 7.47 +/- 0.3; 6.53 +/- 0.03 and 8.49 +/- 0.3 for GlyNA1[1]beta, HisH21[143]beta and other histidines within 2 nm of the sulfhydryl, and CysF9[93]beta, respectively, of the major hemoglobin. Analyses of the simple profiles gave pKas of 6.33 +/- 0.17 and 8.54 +/- 0.5 for HisH21[143]beta and other histidines within a distance of 2 nm of the sulfhydryl, and CysF9[93]beta of the minor hemoglobin, respectively.
Okonjo
Kehinde O
KO
Department of Chemistry, University of Ibadan, Ibadan, Nigeria. kehindeokonjo@yahoo.com
Fodeke
Adedayo A
AA
D000107:Acetylation; D000641:Ammonia / Q000737:chemistry*; D000818:Animals; D002415:Cats; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003545:Cysteine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013997:Time Factors
D006454:Hemoglobins; D013438:Sulfhydryl Compounds; D000641:Ammonia; D004228:Dithionitrobenzoic Acid; D003545:Cysteine