Fractionation of Horseradish Peroxidase by Preparative Isoelectric Focusing, Gel Chromatography and Ion‐Exchange Chromatography

Abstract

Horseradish peroxidase has been fractionated by preparative isoelectric focusing in a density gradient and in a layer of granulated gel using pH‐3–10 and narrow‐pH‐range carrier ampholytes at different total enzyme loads. The resolution of peroxidase isoenzymes in preparative‐layer isoelectric focusing was comparable to that obtained by analytical thin‐layer isoelectric focusing. Isoelectrically homogeneous isoenzymes could be isolated with good recovery in a single fractionation step. Despite the excellent separation of the individual isoenzymes by isoelectric focusing in gel layers, an effective purification, indicated by the absorbance ratio A403 nm/A278 nm, could not be achieved by focusing applied as a single step. By different fractionation sequences combining gel chromatography, ion‐exchange chromatography, and isoelectric focusing, individual isoenzymes with a high purity and homogeneous with respect to their size and charge properties have been isolated. Copyright © 1975, Wiley Blackwell. All rights reserved