An investigation of a method for the separation of labile metalloenzymes.

Abstract

A method for the separation of metalloenzymes has been investigated. The activity and metal conbent of a metalloenzyme was measured before and after elution from aSephadex gel chromatography column to discern whether it was eluted with its metal ion still bound. A metal spiked buffer solution was employed to stabilize the metal-enzyme complex during elution. The results show that a stable zinc metalloenzyme (carboxypeptidase A) may be eluted without the use of a metal spiked buffer solution. However, a more labile zinc metalloenzyme (a-amylase) required the metal spiked buffer solution to prevent dissociation during elution. Ninety-eight percent or better of the metal content and enzyme activity was recovered upon elution of both enzymes. The a-amylase, the zinc content of which is less well established, was found to have a mole ratio of Zn:enzyme of 1:2.