Reversible reaction of 5,5′-dithiobis(2-nitrobenzoate) with the CysF9[93]β sulfhydryl groups of the hemoglobins of the domestic cat: Variation of the equilibrium and reverse rate constants with pH

We have determined for the first time the equilibrium constant, Keq, for the reaction of Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoate), with the CysF9[93]β sulfhydryl groups of the hemoglobins of the domestic cat. In the pH range 5.6 to 9.0 Kequ varies over four orders of magnitude - between ca 10 and 10− 3 - for all hemoglobin derivatives. Using these Kequ values and published data on the dependence of the apparent second order forward rate constant, kf, on pH we have calculated the apparent second order reverse rate constant, kr, as a function of pH. This parameter increases strongly with pH, particularly above pH 7.5. Quantitative analyses of the pH dependence profiles of log10kr indicate that the reverse reaction is coupled to the ionization of two groups on the protein with pKas of 7.2 ± 0.2 and 9.4 ± 0.1 in the major hemoglobin and 6.7 ± 0.3 and 8.4 ± 0.1 in the minor hemoglobin.