Reactions of 5,5\'-dithiobis(2-nitrobenzoate) with CysB5(23)β and CysF9(93)β Of Duck Minor Hemoglobin
AbstractThe reactivities of the sulphydryl groups of duck minor hemoglobin have been studied for a comparison with those of duck major hemoglobin. Boyer titration with p-hydroxymercuri(II)benzoic acid (pMB) gave four reactive sulphydryl groups per (tetramer) molecule. Titration with 5,5'- dithiobis(2-nitrobenzoic acid) (DTNB) also gave four reactive sulphydryl groups per molecule.
The kinetics of the reaction of DTNB with the oxy-, carbonmonoxy- and aquomet derivatives was studied at 200C as a function of 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 calculated from the observed pseudo-first-order rate constant, 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 8.4 ± 0.3. The pKa of 5.5 is assigned to HisHC3(146) β; the pKa of 8.4 is assigned to CysF9(93)β. For the slow kinetic phase, quantitative analyses of the simple pH dependence profiles gave pKas of 5.3 ± 0.3 and 8.7 ± 0.1. The pKa of 5.3 is assigned to HisG19(117) β; the pKa of 8.7 is assigned to CysB5(23) β. CysF9(93) β is responsible for the fast phase while CysB5(23) β is responsible for the slow phase. The result obtained showed that the reactivity of the CysF9(93) β sulphydryl of the minor hemoglobin differs from that of the major hemoglobin. It was observed that for the CysF9(93) β reaction, the oxy and aquomet derivatives of the major hemoglobin reacted about 3-fold faster than the corresponding derivatives of the minor hemoglobin. The carbonmonoxy derivative of the major hemoglobin reacted about 20-fold faster than the corresponding derivative of the minor hemoglobin. For the CysB5(23) β reaction, the oxy, carbonmonoxy and aquomet derivatives of the major hemoglobin reacted at about the same rate as the corresponding derivatives of the minor hemoglobin.
KEY WORDS: Hemoglobin, sulphydryl groups, duck, p-hydroxymercuri (II) benzoate, 5,5'-dithiobis(2-nitrobenzoic acid).
Global Journal of Pure and Applied Sciences Vol.11(2) 2005: 225-231