Reversible reaction of 5,5V-dithiobis(2-nitrobenzoate) with the hemoglobins of the domestic cat: Acetylation of NH3 + terminal group of the h 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 5,5V-dithiobis(2-nitrobenzoate) with the CysF9[93]h sulfhydryl group of domestic cat hemoglobins is a reversible process. In the major hemoglobin, in which the NH3 + terminal group of GlyNA1[1]h 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]h 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]h. Quantitative analyses of the complex profiles enabled us to estimate pKas of 7.47T0.3; 6.53T0.03 and 8.49T0.3 for GlyNA1[1]h, HisH21[143]h and other histidines within 2 nm of the sulfhydryl, and CysF9[93]h, respectively, of the major hemoglobin. Analyses of the simple profiles gave pKas of 6.33T0.17 and 8.54T0.5 for HisH21[143]h and other histidines within a distance of 2 nm of the sulfhydryl, and CysF9[93]h of the minor hemoglobin, respectively. D 2005 Elsevier B.V. All rights reserved.