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 gel filtration chromatography. During the purification steps, the activity of enzyme was measured using 2,2'-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) diamonium salt (ABTS) as a chromogenic substrate at pH 6. Optimum pH and optimum temperature values for LPO were determined for ABTS, p-phenylendiamine, catechol, epinephrine, and pyrogallol as substrates, and then Km and Vmax values for the same substrate were obtained by means of Lineweaver-Burk graphics. The purification degree of the enzyme was controlled by SDS-PAGE and Rz (A412/A280) values. Km values, at optimum pH and 20 degrees C, were 0.197 mM, 0.063 mM, 0.64 mM, 25.2 mM, and 63.95 mM for p-phenylendiamine, ABTS, epinephrine, pyrogallol, and catechol, respectively. Vmax values, at optimum pH and 20 degrees C, were 3.5x10(-5) EU/mL, 4.0x10(-5) EU/mL, 5.8x10(-4) EU/mL, 8.4x10(-4) EU/mL, and 1.01x10(-3) EU/mL for the same substrates, respectively. p-Phenylendiamine was first found as a new substrate for LPO.
Ozdemir
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Department of Biochemistry, Faculty of Science and Arts, Ataturk University, Erzurum, Turkey.
Aygul
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Küfrevioglu
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OI
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003911:Dextrans; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents / Q000737:chemistry; D007700:Kinetics; D007784:Lactoperoxidase / Q000302:isolation & purification*; D008892:Milk / Q000201:enzymology*; D012117:Resins, Synthetic; D013451:Sulfonic Acids / Q000737:chemistry
D000596:Amino Acids; D052160:Benzothiazoles; D003911:Dextrans; D007202:Indicators and Reagents; D012117:Resins, Synthetic; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; C025614:sephadex; C046797:amberlite; D007784:Lactoperoxidase