Purification and Partial Characterization of Three Turnip (Brassica n apus L. var. esculenta D.C.) Peroxidases

• Published in: Journal of agricultural and food chemistry Vol. 48; no. 5; pp. 1574 - 1579
• Main Authors: Duarte-Vázquez, Miguel A, García-Almendárez, Blanca, Regalado, Carlos, Whitaker, John R
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.05.2000
• Subjects: Biological and medical sciences; Biotechnology; Brassica - enzymology; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Enzyme engineering; Fundamental and applied biological sciences. Psychology; Improved methods for extraction and purification of enzymes; Isoenzymes - chemistry; Isoenzymes - isolation & purification; Kinetics; Methods. Procedures. Technologies; Peroxidases - antagonists & inhibitors; Peroxidases - chemistry; Peroxidases - isolation & purification; Purification; Root; Enzyme; Isozyme; Peroxidases; Enzymatic activity; Cruciferae; Dicotyledones; Angiospermae; Plant origin; Brassica napus; Spermatophyta; Peroxidase; Oxidoreductases; Physicochemical properties
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf990454j

Three turnip peroxidases (fractions C1, C2, and C3) were partially purified and characterized, to permit study of their feasibility for use in clinical and enzyme immunoassays. These fractions represented 20% of the initial activity, and fractions C1 and C2 were purified to homogeneity. The optimum pH was between 5.0 and 5.5, while optimum temperature ranged from 40 to 55 °C. The ABTS K m values for the two acidic fractions (C2 and C3) were 0.70 and 0.42 mM, respectively; about 5 times lower than that reported for the acidic commercial horseradish peroxidase (HRP). Fraction C3 had 4 times higher K m value than commercial cationic HRP. The molecular weights determined by SDS−PAGE ranged from 39.2 to 42.5 kDa. Activation energies for inactivation were 113 (C1), 130 (C2), and 172 kJ/mol (C3) which are higher or comparable to other peroxidase isoenzymes reported. Fractions C1 and C3 represent an alternative source of peroxidase because of their higher purification yield and specific activity, when compared to fraction C2. Keywords: Peroxidase isoenzymes; turnip; protein purification, Brassica napus L.