The association-dissociation and some physical-chemical studies of plasma amine oxidase

Abstract

Beef plasma amine oxidase has been isolated and crystallized by Yamada and Yasunobu (J. Biol. Chem., 237, 1511, 1962). The high molecular weight reported for the enzyme and other preliminary studies (Gee, Master's Thesis, U. of Hawaii, 1963) indicated that the enzyme might be composed of subunits. An investigation was made of the 3ssociationdissociation properties of plasma amine oxidase. The usual methods employed to break down non-covalent linkages in proteins, such as raising or lowering the pH, detergent treatment, succinylation, and urea denaturation, proved to be unsuccessful in dissociating the enzyme as revealed by ultracentrifugal analysis of the treated protein. Dissociation was achieved only in 6 M guanidine-hydrochloride in the presence of ~ reducing agent. Determination of Sulfhydryl groups and the total cystine and cysteine content of the enzyme revealed that there are two free thiol groups and nine disulfide bonds present in the molecule. The number of -S-S- bridges gave support to the hypothesis that the polypeptide chains of PAO are covalently bonded. A re-evaluation of the molecular weight of the native enzyme led to the realization that PAO is a chemically interacting system and exists in solution as monomeric species in rapid equilibrium with higher polymers. The techniques of sedimentation equilibrium and Sephadex gel filtration gave a molecular weight of 170,000 for native PAD. The high molecular weight of 261,000 previously reported (Yamada et al., Biochim. Biophys. Acta, 8l, 165, 1964) was explained as probably being an average of the monomer and higher polymers. Molecular weight determinations of the reduced enzyme in 6 M guanidine- hydrochloride by sedimentation equilibrium and sedimentation-diffusion methods yielded values of 86,600 and 87,000, respectively. The data suggested that plasma amine oxidase is composed of two polypeptide chains which are covalently linked by disulfide bonds. The enzyme was found to undergo a loss of activity upon standing at low temperatures which was characterized by the presence of faster moving components along with the original component in the ultracentrifuge. The sedimentation coefficients of these components were indicative of the presence of monomers, dimers, and trimers. This association phenomenon appeared to be a mass action effect. A 1% enzyme solution in 0.06 M phosphate buffer, pH 7.0 gave rise to the presence of more polymer and greater loss of activity than did a 0.5% solution prepared and kept under identical conditions.