Cat Hemoglobin: pH DEPENDENCE OF COOPERATIVITY AND LIGAND BINDING

Abstract

Abstract Cat hemoglobin has been found to exist in three forms which differ in the cooperativity of oxygen binding at low and high pH values. Type I cat hemoglobin has a high cooperativity (n = 2.7 to 3.1) at both high (pH = 7.8 to 9.0) and low (pH = 6.5 to 6.9) pH values. Type II hemoglobin exhibits low cooperativity (n = 1.8 to 2.3) in the low pH range but high cooperativity in the high pH range. Types I and II have identical p(O2)hb50 values. Cat hemoglobin type III has a low cooperativity in both pH ranges. Results presented indicate that changes in the reactivity of sulfhydryl groups coincide with the difference in cooperativity among the three cat hemoglobin forms. Stored at 4° as a hemolysate, in the oxy form, type I hemoglobin gradually converts to type II and then type III cat hemoglobins. The conversion occurs much more slowly if purified cat hemoglobin is stored in the carbon monoxide form. Type III hemoglobin can be converted to type II hemoglobin by incubation with dithiothreitol. Molar equivalents of p-hydroxymercuribenzoate, 1.4 and 2.4, convert type I to type II and type III, respectively. Ultracentrifuge experiments indicate that the tetramer-dimer subunit dissociation constants of the liganded forms of types I and II of cat hemoglobin are pH-dependent. In addition, liganded cat hemoglobin dissociates about 10 times more readily than any other mammalian hemoglobin investigated. The results are discussed in light of known facts about the structure of cat hemoglobin and applied to an evaluation of the validity of the allosteric model for cooperativity of multi-subunit proteins.