Abstract
Peroxidase tagged proteins are being used successfully as immune-histological probes for the demonstration of tissue antigens, and in enzyme amplified immunoassay systems for the quantitative determination of soluble and insoluble antigens. The glycoprotein nature of peroxidases can be exploited for conjugation to proteins of interest. Peroxidase extracted from the bulbs of Brassica oleracea gongylodes was salted out at 40-80% ammonium sulfate saturation and activated by treatment with 1-Fluoro-2,4-dinitro benzene (FDNB) and periodate. Treatment with 0.08% FDNB and 12.5mM periodate was optimized for activation of the enzyme. The treated enzyme was found to conjugate successfully to immunoglobulin fractions harvested from egg yolk (IgY), human plasma and goat serum. Enzyme conjugated to IgY fraction showed improvement in its pH stability and temperature stability. The affinity of the enzyme for its substrate phenol did not alter to a significant extent upon activation and conjugation. The conjugates exhibited high affinity towards phenol, bromocresol purple and bromothymol blue in comparison to HRP conjugates prepared using the same protocol. Int. J. Appl. Sci. Biotechnol. Vol 5(1): 59-65
Highlights
Peroxidases (EC 1.11.1.7/ phenolic donor:hydrogenperoxide oxidoreductase) are heme containing enzymes distributed widely in plant kingdom in a variety of tissues (Duarte-Vazquez et al, 2007)
Peroxidase extracted from horse radish (HRP)) roots in conjugation with antibodies and other protein tools is being used extensively in immunoassay probes for the detection of tissue antigens and measurement of antigens in enzyme amplified immunoassays
The bulb of Brassica oleracea gongylodes which is used as a vegetable in some parts of India is a reservoir of peroxidase
Summary
Peroxidases (EC 1.11.1.7/ phenolic donor:hydrogenperoxide oxidoreductase) are heme containing enzymes distributed widely in plant kingdom in a variety of tissues (Duarte-Vazquez et al, 2007). From the commercial point of view, peroxidases are enzymes of great significance as they have varied applications (Yu et al, 1994; Bholay et al, 2012; Dorantes et al, 2012, Chandrasekaran et al, 2014). These enzymes catalyze the oxidation by hydrogen peroxide of a broad range of hydrogen donor substrates such as phenols, ascorbic acid, aromatic amines and their derivatives (Ye et al, 2006; Vernawal et al, 2006). The storage stability of the enzyme was reportedly satisfactory
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