Previous studies on the sedimentation of the beef plasma amine oxidase indicated that it might be an associating-dissociating system. This was confirmed by the studies reported here. A reinvestigation of the molecular weight of the native enzyme by the methods of gel filtration and sedimentation equilibrium gave a (minimum) molecular weight of 166,700 and 170,000, respectively, compared with the average value obtained previously of 260,000. In addition, the sedimentation velocity pattern of the enzyme was symmetrical at pH 7.0, but was sharpened considerably in acidic or alkaline solutions. Whether this is due to a pH-dependent association- dissociation process or charge effect was not ascertained definitively. The sedimentation velocity patterns of the enzyme were also dependent upon time of storage and upon concentration of the enzyme solution. After 8 weeks at 4°, multiple boundaries were observed in the ultracentrifuge pattern which exhibited S20,w values corresponding to monomer, dimer, and trimers. This may be due to the fact that with time and at high protein concentrations, the association of the monomer becomes slowly irreversible. The enzyme in its monomeric form of mol wt 170,000 was not easily dissociated into subunits. Acid, base, 8 M urea, and 6 M guanidine hydrochloride failed in this regard, indicating the presence of covalent bonds. Guanidine hydrochloride (6 M) in combination with 0.1M mercaptoethanol was found to be efficient for the dissociation process. A molecular weight of 87,000 was obtained from the studies of the reduced enzyme by combined sedimentation and diffusion measurements and by the sedimentation equilibrium studies, suggesting the monomer is composed of two polypeptide chains of equivalent size. The S-sulfo derivative was hydrolyzed by trypsin and the fingerprint pattern disclosed that there were about 38 peptides. From the known lysine and arginine content of the enzyme, it is concluded tha the enzyme consists of two identical subunits. © 1968, American Chemical Society. All rights reserved.
