Hemoglobins with multiple reactive sulphydryl groups: the reaction of dog hemoglobin with 5,5'-dithiobis (2-nitrobenzoate).

Dog hemoglobin has four sulphydryl groups per (tetramer) molecule located at the G18(111)alpha and F9(93)beta positions. The two sulphydryls at the G18(111)alpha positions are unreactive toward nonmercurial sulphydryl reagents, but those at the F9(93)beta positions are reactive toward these reagents. We have studied the kinetics of the reaction of dog hemoglobin with 5,5'-dithiobis (2-nitrobenzoic acid) as a function of pH. At all pH values studied, the reaction is kinetically monophasic. Quantitative analysis of the pH dependence of the apparent second-order rate constant shows that two ionizable groups are linked to the reaction of the sulphydryl group. Their pKa values are 5.57 and 9.0. These values are assigned to HisHC3(146)beta and to the CysF9(93)beta sulphydryl. We find that dog carbonmonoxyhemoglobin is significantly--almost an order of magnitude--less reactive than the aquomet, azidomet, and oxy derivatives. This result may be due to a greater tendency (at acid pH) for the salt bridge between HisHC3(146)beta and AspFG1(94)beta to form in the carbonmonoxy than in the other derivatives. Formation of this salt bridge is known to hinder access to the CysF9(93)beta sulphydryl [Perutz, M. F. (1970), Nature 228, 734-739].