title: One-step purification of lactoperoxidase from bovine milk by affinity chromatography.
abstract: Sulphanilamide was determined to be a new inhibitor of lactoperoxidase (LPO) with an IC(50) 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.
authors.affiliation: Ataturk University, Ispir Hamza Polat Vocational Training School, 25900-Erzurum, Turkey.
authors.forename: Ali
authors.initials: A
authors.lastname: Atasever
authors.forename: Hasan
authors.initials: H
authors.lastname: Ozdemir
authors.forename: Ilhami
authors.initials: I
authors.lastname: Gulcin
authors.forename: O
authors.initials: O
authors.lastname: Irfan Kufrevioglu
mesh_terms: D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D007700:Kinetics; D007784:Lactoperoxidase / Q000737:chemistry / Q000302:isolation & purification*; D008892:Milk / Q000737:chemistry* / Q000201:enzymology*
chemical_list: D007784:Lactoperoxidase