A New Approach for Affinity-Based Purification of Horseradish Peroxidase.

We have developed efficient procedure for isolation of horseradish peroxidase (HRP) using aminobenzohydrazide based affinity chromatography. Sepharose 4B-bounded aminobenzohydrazides are suitable for long term use and large scale purification. In this study, 26 aminobenzohydrazide derivatives were synthesized, characterized and defined as new HRP inhibitors. In addition, detailed inhibition effect of these molecules on HRP enzyme were investigated. Affinity matrix was formed by bonding aminobenzohydrazides which exhibited inhibitory activity to Sepharose-4B-L-tyrosine. HRP was isolated from crude homogenate in single-step and purification factors were recorded as 1151 fold (recovery of 8.5 %) with 4-amino 3-bromo benzohydrazide, and purification factors were recorded as 166.16 fold (recovery of 16.67 %) with 3-amino 4-chloro benzohydrazide. This article is protected by copyright. All rights reserved.