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 CysF993]beta sulfhydryl group of domestic cat hemoglobins is a reversible process. In the major hemoglobin, in which the NH3+ terminal group of GlyNA11]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 SerNA11]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 CysF993]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 GlyNA11]beta, HisH21143]beta and other histidines within 2 nm of the sulfhydryl, and CysF993]beta, respectively, of the major hemoglobin. Analyses of the simple profiles gave pKas of 6.33 +/- 0.17 and 8.54 +/- 0.5 for HisH21143]beta and other histidines within a distance of 2 nm of the sulfhydryl, and CysF993]beta of the minor hemoglobin, respectively.