Ligand-dependent reactivity of the CysB5[23] β sulfhydryl group of the major haemoglobin of chicken

Abstract

Chicken haemoglobin contains eight reactive sulfhydryl groups per (tetramer) molecule, as determined by Boyer titration with p-chloromercury(II)benzoic acid. However, only four of these sulfhydryls are reactive towards 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). They are at the F9[93] and B5[23] positions of each of the two β subunits in the molecule. The time course of the DTNB reaction is biphasic. With oxyhaemoglobin, k _app , the apparent second-order rate constant of the fast phase, increases monotonically with pH, the simple profile resembling the titration curve of a diprotic acid; the pH-dependence of k _app for the slow phase is bowl-shaped. With carbonmonoxyhaemoglobin and aquomethaemoglobin, k _app for the fast phase is bowl-shaped whilst k _app for the slow phase increases monotonically with pH. Quantitative analyses of the simple profiles show that the reactivity of the sulfhydryl group to which they may be attributed is subject to the influence of two ionizable groups on the molecule, with mean pK _a values of 6.4±0.1 and ca. 8.4±0.3. These pK _a values are assigned to HisHC3[146]β and CysF9[93]β, respectively. Quantitative analyses of the bowl-shaped profiles show that the reactivity of the sulfhydryl group to which they may be attributed is subject to the influence of two ionizable groups on the protein, with mean pK _a s of 6.85±0.05 and 8.3±0.2. These values are assigned to HisG19[117]β and CysB5[23]β, respectively. It is highly significant that the CysB5[23]β sulfhydryl groups of carbonmonoxy- and aquomet-haemoglobin react ca. 100 times faster than that of oxyhaemoglobin. By contrast, the difference in the reactivities of the CysF9[93]β sulfhydryls of the three haemoglobin derivatives is no more than four-fold. This indicates that, in chicken haemoglobin, changes in the haem ligand give rise to structural changes in the neighbourhood of the CysB5[23]β sulfhydryl which are far more significant than those in the neighbourhood of the CysF9[93]β sulfhydryl.