Ionizable groups linked to the reaction of 2,2'-dithiobispyridine with hemoglobin

The pH-dependence of the second-order rate-constant for the reaction of 2,2'-dithiobispyridine with the CysF9(93) beta sulphydryl group of hemoglobin in the R quaternary structure is analyzed in terms of a tentative model based on the observation that this sulphydryl exists as a mixture of two tertiary conformations in dynamic equilibrium. For the four aquomethemoglobins studied (human A and S, dog and rabbit), the equation derived from this model gives a better fit than a simpler equation based on the assumption of only one tertiary conformation. For the corresponding carbonmonoxyhemoglobins the simpler equation gives a better fit. The dog and rabbit oxy and azidomet data are better fitted by the model equation, whereas the data for the corresponding human A and S derivatives are better fitted by the simpler equation. From the analysis pKa values of 6.1 and 8.7 are obtained for the ionization of groups coupled to the presumed conformational transition. The pKa of 6.1 is assigned to HisHC3(146) beta; the pKa of 8.7 is assigned to the CysF9(93) beta sulphydryl group in its external conformation. It is estimated that the pKa of this sulphydryl may be as high as 12.9 in its internal conformation.