Single-step purification of peroxidase by 4-aminobenzohydrazide from Turkish blackradish and Turnip roots

Abstract

Peroxidases (PODs) were purified from the Turkish blackradish (Raphanus sativus L.) (TBR) and Turnip (Brassica rapa L.) using a simple and effective single-step method. An affinity resin was synthesised by coupling the 4-aminobenzohydrazide ligand and the l-tyrosine spacer-arm to CNBr-activated-Sepharose-4B. The purification factors for the TBR-POD and the Turnip-POD were 40.3-fold (with a yield of 10.6%) and 269.3-fold (with a yield of 9%), respectively. The molecular masses of the TBR-POD and Turnip-POD were approximately 67.3 and 65.8kDa, respectively. For guaiacol, the Km and Vmax values were calculated as 24.88mM and 3.23EU/mL, respectively for TBR-POD and as 4.09mM and 0.797EU/mL for the Turnip-POD. For H2O2, the Km and Vmax values were calculated as 3.247mM and 0.799EU/mL, respectively for TBR-POD, and as 12.49mM and 4.055EU/mL, respectively for the Turnip-POD. Furthermore, 4-aminobenzohydrazide was determined to be a non-competitive inhibitor of TBR-POD and Turnip-POD.

Keywords: Affinity chromatography; Inhibition; Kinetics; Peroxidase.