Reactions Of 5,5\'-Dithiobis(2 -Nitrobenzoate) with CysB5(23)β and CysF9(93)β 0f Duck Major Hemoglobin

Abstract

Duck major hemoglobin contains ten sulphydryl groups per tetramer molecule as revealed by its amino acid sequence. Boyer titration of this hemoglobin with p-hydroxymercuri(II)benzoic acid (pMB) indicates that there are only six reactive sulphydryl groups per (tetramer) molecule, while titration with 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) indicates only four. The kinetics of the reaction of DTNB with the oxy-, carbonmonoxy-, and aquomet derivatives of this hemoglobin were studied at 20oC as a function of pH (pH 7.0 9.0) under pseudo-first order conditions. Biphasic time courses were obtained under all experimental conditions and the two kinetic phases were well separated along the time axis. The apparent second-order rate constants, kapp, were determined from the observed pseudo- first-order rate constants, kobs. The dependence of kapp on pH for each of the two kinetic phases is simple, resembling the titration curve of a diprotic acid. For the fast kinetic phase, quantitative analyses of the simple pH dependence profiles gave pKas of 5.5 ± 0.5 and 8.6 ± 0.2. The pKa of 5.5 ± 0.5 is assigned to HisHC3(146)β, the pKa of 8.6 0.2 is assigned to CysF9(93)β. For the slow kinetic phase, quantitative analyses of the simple pH dependence profiles gave pKas of 6.2 ± 0.6 and 8.8 ± 0.1. The pKa of 6.2 ± 0.6 is assigned to HisG19(117) β; the pKa of 8.8 is assigned to CysB5(23) β. It is significant to note that the rates of both CysF9(93) β and CysB5(23) β of the carbonmonoxy derivative were found to be much greater than those of oxy and aquomet derivatives. KEY WORDS:- Hemoglobin, sulphydryl groups, duck, 5,5'-dithiobis(2-nitrobenzoic acid). Global Journal of Pure and Applied Sciences Vol.11(2) 2005: 209-215