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 effects 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 1,151-fold (recovery of 8.5%) with 4-amino 3-bromo benzohydrazide and as 166.16-fold (recovery of 16.67 %) with 3-amino 4-chloro benzohydrazide.

Purification of peroxidase enzyme from radish species in fast and high yield with affinity chromatography technique.

In this study, an effective single step affinity method is presented for purifying plant peroxidase (POD) enzymes from radish species. This method make possible to purify the enzymes in high yield and purity. Briefly, 10 different 4-amino benzohydrazide derivatives were synthesized and identified as new competitive POD inhibitors. Then, these derivatives were coupled to Sepharose 4B-L-Tyrosine support matrix by diazotization to form the affinity gels. Purification factors were recorded as 54.8% yield - 665-fold, 33.8% yield - 613-fold, 22.7% yield - 595-fold, 34.4% yield - 781-fold, 40.9% yield - 282-fold for turnip (T-POD), black radish (BR-POD), daikon (D-POD), sweet radish (SR-POD) and kohlrabi radish, (KR-POD), respectively. It has also been shown that the affinity gels, which prepared using the 4-amino 3-bromo benzohydrazide and 4-amino 2-nitro benzohydrazide molecules, capable to purify all radish species POD enzymes in high purity and yield.