Isolation of 3,4-dihydroxyphenylalanine-containing proteins using boronate affinity chromatography

A rapid procedure for the isolation of 3,4-dihydroxyphenylalanine-containing proteins has been developed in which the protein is selectively bound to a m-phenylboronate agarose column, and eluted with 1.0 M ammonium acetate, pH 3.0. The method is based on the affinity of boronates for diols including catechol. The chromatography is carried out in the absence of oxygen to prevent oxidation of the catechol. Other proteins are eluted beforehand with 0.25 M ammonium acetate, pH 8.5, or for glycoproteins with a Tris buffer containing 0.2 M sorbitol, pH 8.5.