On the mechanism of the dissociation of haemoglobin

The reaction between p-chloromercuribenzoate (PCMB) and haemoglobin has been investigated to define the optimum conditions for dissociation to the subunits. The production of various molecular species has been followed by electrophoresis, gel filtration and ultracentrifugation. The reagent was applied to human haemoglobins A, S, C, Ja (Glasgow), A₂, H and F, and also to the haemoglobins of dog, rabbit, horse and pig. Evidence is presented for the occurrence of haemoglobins without α-chains containing dissimilar β-chains, i.e. β₂^Yβ₂^X The behaviour of haemoglobins in the presence of PCMB was found to correlate qualitatively and quantitatively with the nature and number of their sulphydryl groups. It is shown that attachment of reagent at position β-93 causes initial dissociation of the tetramer to the dimer: α₂β₂ ag 2αβ. From the location of these reactive sulphydryls it is concluded that the initial dissociation is along the bc plane of the haemoglobin molecule with the formation of two α¹β¹ dimers. Further dissociation by PCMB at pH 6.0 only occurred with haemoglobins containing a second sulphydryl group on the β-chain (β-112). From the data it is concluded that substitution at both unreactive thiols β-112 and α-104 causes final dissociation to the monomeric species. The contribution of the various sulphydryl groups to dissociation was also indicated by the behaviour of alkylated haemoglobins when treated with PCMB. Evidence is presented that dissociation of haemoglobin to half-molecules in several media occurs across the same plane as that initially affected by PCMB. The connection between the sigmoid oxygen tension curve and dissociation is discussed.