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

NA Adesola Babarinde
J Oyebamiji Babalola
I Ugochukwu Uko


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