One-step purification of lactoperoxidase from bovine milk by affinity chromatography

Sulphanilamide was determined to be a new inhibitor of lactoperoxidase (LPO) with an IC50 of 0.848.10(-5) M. The K-i for sulphanilamide was determined to be 3.57.10(-5) M and sulphanilamide showed competitive inhibition, which makes it a suitable ligand for constructing a Sepharose 4B-L-tyrosine affinity matrix. The affinity matrix was synthesised by coupling sulphanilamide as the ligand and L-tyrosine as the spacer arm to a cyanogen bromide (CNBr)-activated-Sepharose 4B matrix. Lactoperoxidase was purified 409-fold from the synthesized affinity matrix in a single step, with a yield of 62.3% and a specific activity of 40.9 EU/mg protein. The enzyme activity was measured using ABTS as a chromogenic substrate (pH 6.0). The degree of LPO purification was monitored by SDS-PAGE and its R-z (A(412)/A(280)) value. The R-z value for the purified LPO was found to be 0.7. Maximum binding was achieved and K-m and V-max values were determined. (C) 2012 Elsevier Ltd. All rights reserved.