Reversible reaction of 5,5′-dithiobis(2-nitrobenzoate) with the hemoglobins of the domestic cat: Acetylation of NH 3+ terminal group of the β chain transforms the complex pH dependence of the forward apparent second order rate constant to a simple form

Abstract

We demonstrate kinetically that the reaction of 5,5′-dithiobis(2-nitrobenzoate) with the CysF9[93]β sulfhydryl group of domestic cat hemoglobins is a reversible process. In the major hemoglobin, in which the NH 3 + terminal group of GlyNA1[1]β is free, k f, the apparent forward second order rate constant, has a complex pH dependence profile. In the minor hemoglobin, the NH 3 + terminal group of SerNA1[1]β is acetylated, and the pH dependence profile of k f is simple. These results support the proposal that the positively charged groups at the organic phosphate binding site are electrostatically linked to CysF9[93]β. Quantitative analyses of the complex profiles enabled us to estimate pK as of 7.47 ± 0.3; 6.53 ± 0.03 and 8.49 ± 0.3 for GlyNA1[1]β, HisH21[143]β and other histidines within 2 nm of the sulfhydryl, and CysF9[93]β, respectively, of the major hemoglobin. Analyses of the simple profiles gave pK as of 6.33 ± 0.17 and 8.54 ± 0.5 for HisH21[143]β and other histidines within a distance of 2 nm of the sulfhydryl, and CysF9[93]β of the minor hemoglobin, respectively.