Molecular Sieve Studies of Interacting Protein Systems

Highly purified preparations of D-aIIIiII0 acid oxidase were obtained by removal of high molecular weight components after purification by established procedures.Molecular sieve studies were carried out on the size characterization of the highly purified apoenzyme molecule.In the microgram per ml concentration range the apoenzyme was shown to dissociate to a subunit with a molecular Stokes radius of 25.1 f 1 A and a molecular weight of 35,000 to 40,000.This molecular weight corresponds to that of a single flavin adenine dinucleotide-binding unit and presents the possibility that the monomer of the holoenzyme may be the active species.The enzyme n-amino acid oxidase (n-amino acid: oxygen oxidoreductase (deaminating), EC 1.4.3.3) has been shown to be an associating system in which the physical properties of the protein are affected by the presence of coenzyme and substrate (2, 3).However, the relative significance of association and conformation changes to the physical properties remains unclear, and the minimum molecular weight of apoenzyme, holoenzyme, and benzoate-complex is in dispute.The physical studies reported to date on n-amino acid oxidase have been carried out at protein concentrations greater than 0.5 mg per ml and, as shown below, are complicated by the presence of high molecular weight contaminants.In this paper we report (a) a further purification of the enzyme in which high molecular weight components are removed after purification by established procedures and (b) molecular sieve studies on the size characterization of the highly purified apo-