ラット骨格筋ミオグロビンの単離と諸特性

Abstract

Myoglobin was isolated and purified by heat denaturation-gel filtrationchromatofocusing procedure from water extract of skeletal muscles of the rat. One major (fraction Ⅰ) and two minor (fractions Ⅱ and Ⅲ) omponents were finally obtained. Amino acid sequence of the major component was determined by an automatic Edman degradation procedure. As compared with murine myoglobin, we found amino acid replacements at 9 positions. Cysteine residue, with sulfhydryl group highly reactive to p-chloromercuribenzoate (PMB), was present at position 66 in both rat and murine myoglobins. The two minor components with higher anodic electrophoretic mobilities and with sulfhydryl group unreactive to PMB were found to be derived through mixed disulfide formation with cysteine (component Ⅱ) or glutathione (component Ⅲ) at the 66th cysteine residue of the major component. In view of the hydropathy profile, secondary structures of rat and murine myoglobins were little different from each other and a prominent hydrophobic lobe around position 70, corresponding to the E-helix containing the distal histidine residue at position 64, was noted. The three components showed essentially identical oxygen equilibrium properties with neither homotropic nor heterotropic allosteric interactions. Halfoxygenation pressure (P₅₀) of the proteins calculated from the kinetic measurements, assuming a simple bimolecular reaction model, agreed very well with those determined by oxygen equilibria.