Purification of Cowpea (Vigna unguiculata) Peroxidase with Single-Step Affinity Chromatography and, Characterization Study

Abstract

In this report, cowpea (Vigna unguiculata) peroxidase (POD) enzyme was isolated and characterized. Four different amino benzohydrazide derivatives were used as ligand in affinity chromatography and with 4-amino 3-bromo 2-methylbenzohidrazide, 53.6% purification yield (91.6 fold) was achieved in a single-step. The molecular weight of the purified cowpea POD showing single band at SDS-PAGE was calculated as 39 kDa. For evaluating the cowpea PODs affinity to ligands, IC50 and Ki values were determined. The molecule that caused the highest inhibition at the lowest concentration was found to be a 4-amino 3-bromo 5-chlorobenzohydrazide with the IC50 value of 78 µM. In the characterization assays, optimum parameters were determined for purified cowpea POD as pH= 6.0, ionic strength= 0.3 M and temperature= 40 ºC. In addition, substrate specificity tests were carried out with guaiacol, H2O2, 4-methylcatechol and ABTS substrates. According to the obtained data, the KM values of these substrates were calculated as 7.21 mM, 17.03 mM, 7.28 mM and 49.21 mM, respectively.

Keywords

• Peroxidase
• Cowpea
• Purification
• Affinity chromatography
• Characterization

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