Purification of lactoperoxidase from bovine milk and investigation of the kinetic properties

Lactoperoxidase (LPO) was purified from bovine milk using Amberlite CG 50 H + resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 g el filtration chromatography. During the purification steps, the activity o f enzyme was measured using 2,2 ' -azino-bis (3-ethylbenzthiazoline-6 sulfo nic acid) diamonium salt (ABTS) as a chromogenic substrate at pH 6. Optimum pH and optimum temperature values for LPO were determined for ABTS, p-phen ylendiamine, catechol, epinephrine, and pyrogallol as substrates, and then K-m and V-max values for the same substrate were obtained by means of Linew eaver-Burk graphics. The purification degree of the enzyme was controlled b y SDS-PAGE and R-z (A(412)/A(280)) values. K-m values, at optimum pH and 20 degreesC, were 0.197 mM, 0.063 mM, 0.64 mM , 25.2 mM, and 63.95 mM for p-phenylendiamine, ABTS, epinephrine, pyrogallo l, and catechol, respectively. V-max values, at optimum pH and 20 degreesC, were 3.5x10(-5) EU/mL, 4.0x10(-5) EU/mL, 5.8x10(-4) EU/mL, 8.4x10(-4) EU/m L, and 1.01x10(-3) EU/mL for the same substrates, respectively. p-Phenylend iamine was first found as a new substrate for LPO.