Purification and properties of carboxypeptidase G2 from Pseudomonas sp. strain RS‐16: Use of a novel triazine dye affinity method

Abstract

A folate‐degrading enzyme, carboxypeptidase G2, has been purified on a large scale from Pseudomonas sp. strain RS‐16. Homogeneous enzyme was obtained by a three‐step procedure involving ion‐exchange chromatography and a novel triazine dye (affinity) chromatography step which utilizes Zn2+ to promote adsorption of the enzyme. Enzyme was selectively eluted by the use of a chelating agent (EDTA) and a step change in pH. The enzyme is a dimeric protein (Mr 83000) with two identical subunits of 41800 and contains four atoms of zinc per enzyme molecule, which are required for full activity. The enzyme follows Michaelis‐Menten kinetics with Km values of 4.0 μM for folate, 8.0 üM for methotrexate and 34.0 μM for 5‐methyltetrahydrofolate, the predominant form of reduced folate found in plasma. Copyright © 1985, Wiley Blackwell. All rights reserved