An investigation of the functioning of the two major haemoglobins of the Sphenodon using fast reaction kinetic methods.

The blood of the Sphenodon (Sphenodon punctatus) has been fractionated into two major and one minor haemoglobin components by ion-exchange chromatography. The two major haemoglobins have been studied in terms of their kinetic reactions with both O2 and CO. The combination of flash photolysis and stopped-flow indicates kinetic differences between two states of the proteins identified with the allosteric T and R forms. The major kinetic findings show that (i) in these haemoglobins the T state is retained to a higher level of ligation than that commonly found in mammals, (ii) the rate of conversion from the R to the T state (k0RT) is some three orders of magnitude lower in the Sphenodon haemoglobins than in mammalian systems. A comparison between the kinetic and equilibrium data for these haemoglobins indicates that the very weak cooperativity exhibited by these proteins arises from the close similarity of the affinities of the two allosteric states together with a very low value for the allosteric equilibrium constant, although very significant kinetic differences exist between the two states.