HEMOGLOBINS WITH MULTIPLE REACTIVE SULFHYDRYL-GROUPS - THE REACTION OF PIGEON HEMOGLOBIN WITH 5,5'-DITHIOBIS (2-NITROBENZOIC ACID)

Pigeon hemoglobin has eight reactive sulphydryl groups per (tetramer) molecule, as determined by Boyer titration with p-chloromercuribenzoat e. However, only four of these are titratable with 5,5'-dithiobis(2-ni trobenzoate) under the same experimental conditions. The time course o f the reaction of pigeon hemoglobin with 5,5'-dithiobis(2-nitrobenzoat e) is biphasic. In the pH range 6-9, the fast phase is between one and two orders of magnitude faster than the slow phase. For the fast phas e, k(app), the apparent second-order rate constant, increases monotono usly with pH. Quantitative analysis reveals that the reaction of the s ulphydryl group responsible for this phase is coupled to the ionizatio n of two groups with pK(a) values of 6.15 +/- 0.1 and 8.5 +/- 0.1. The se pK(a) values are assigned to HisHC3(146)beta and to the CysF9(93)be ta sulphydryl group, respectively. For the slow phase the k(app), vs. pH profiles are bowl-shaped. Analysis reveals that the reaction of the sulphydryl group to which this phase may be attributed is coupled to the ionization of two groups with mean pK(a) values of 6.53 +/- 0.1 an d 8.25 +/- 0.1. Examination of the structure of hemoglobin allows us t o assign these values to HisG19(117)beta and CysB5(23)beta, respective ly. The CysB5(23)beta sulphydryl is in the region of the molecule wher e amino acid substitutions have been found to give rise to significant changes in the oxygen affinity of hemoglobin [Huang et al. (1990), Bi ochemistry 29, 7020-7023].