Haemoglobins with multiple reactive sulfhydryl groups: reactions of 5,5'-dithiobis(2-nitrobenzoate) with CysF9[93] and CysH3[125] of guinea pig haemoglobin

Abstract

Guinea pig haemoglobin has six sulfhydryl groups, one at each of the positions F9[93], G11[104] and H3[125], which appear in pairs. Titration with p-hydroxymercuri(II)- benzoic acid and with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) indicate that four of the six sulfhydryls are reactive. The time course of the DTNB reaction is triphasic at pH > 7. The fast phase is about 2 orders of magnitude faster than the intermediate phase and can be clearly separated from it. The intermediate phase, where it exists, is about 3-4 times faster than the slow phase. However, the amplitude of the intermediate phase, which has a maximum of about 15% of the total signal amplitude, becomes negligible as it approaches pH 7, so that the kinetics eventually becomes biphasic.

The pH dependence profile of kapp, the apparent second-order rate constant, for the fast phase resembles the titration curve of a diprotic acid. Quantitative analysis indicates that the reactivity of the sulfhydryl group to which this phase may be attributed is linked to two ionizable groups with pKas of 6.4 0.1 and 7.8 0.2. These values are assigned to HisHC3[146] and CysF9[93], respectively. The pH dependence profile of kapp for the slow phase resembles the titration curve of a monoprotic acid. Quantitative analysis indicates that the sulfhydryl group to which this phase may be attributed is linked to a single ionizable group with a pKa of 6.1 0.2. Examination of the structure of guinea pig haemoglobin near the H3[125] position shows that there is a lysine residue, LysA5[8], which is only 4 (0.4 nm) away from CysH3[125]. The presence of this lysine lowers the pKa of CysH3[125] from the normal value for sulfhydryl groups (between 8 and 8.5) to 6.1.



(Received July 31, 2001; revised May 1, 2002)


(Bulletin of The Chemical Society of Ethiopia: 2002 16 (2): 175-186)