STRUCTURE AND FUNCTION OF HEMOGLOBIN PHILLYRIN (TYR C1 (35) BETA-]PHE)

Hb Philly was purified by isoelectric focusing on polyacrylamide gel, O2 equilibrium, proton NMR spectra, mechanical stability and pH-dependent UV difference spectrum were studied. Stripped Hb Philly bound O2 non-co-operatively with high affinity. Inorganic phosphate and 2,3-diphosphoglycerate had little effect on the equilibrium curve, but inositol hexaphosphate lowered the affinity and induced co-operativity. These properties were explained by the NMR spectra which showed that stripped deoxyhemoglobin Philly had the quaternary oxy structure and that inositol hexaphosphate converted it to the deoxy structure. An exchangeable proton resonance at -8.3 ppm from water, which was present in oxy- and deoxyhemoglobin A, was absent in both these derivatives of Hb Philly and can be assigned to one of the H bonds made by tyrosine Cl-(35).beta., probably the one to aspartate H8(126).alpha. at the .alpha.1.beta.1 contact. Hb Philly showed the same pH-dependent UV difference spectrum as Hb A, only weaker, so that a tyrosine other than 35.beta. must be mainly responsible for this.