High-performance liquid chromatography of β-lactamases

Abstract

Wide-pore, chemically bonded, reversed-phase HPLC packings have been used for the purification of β-lactamases. Several chromatographic systems have been investigated and although the recovery of enzyme activity was only between 10 and 28%, depending on the mobile phase constituents, a maximum protein recovery of 87% was achieved with a mobile phase containing trifluoroacetic acid. The analytical method was assessed in terms of reproducibility and linearity and the technique was applied to the isolation of β-lactamase from culture media. Protein purity was determined by polyacrylamide gel electrophoresis.