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 (A₄₁₂/A₂₈₀) 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.