Purification, crystallization and preliminary X-ray crystallographic analysis of lactoperoxidase from buffalo milk.

The lactoperoxidase was prepared from buffalo milk and purified using CM-Sephadex C-50 and Sephadex G-100. The activity of the enzyme was measured using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt as a chromogenic substrate at pH 6.0. The purified protein was crystallized from 0.01 M sodium phosphate buffer (pH 8.0) with 10%(v/v) ethanol by the sitting-drop vapour-diffusion method. The green-coloured plate-like crystals are orthorhombic in space group P2(1)2(1)2(1) with unit-cell dimensions a = 116.9, b = 103.2 and c = 62.3 A. The asymmetric unit contains one molecule with a solvent content of 52%. The crystals were stable in the X-ray beam and diffract beyond 3.2 A. The native data to 3.5 A have been collected and the structure determination is in progress.