Author
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Jacks, Thomas |
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Submitted to: Analytical Letters
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 1/10/2005 Publication Date: 3/1/2005 Citation: Jacks, T.J. 2005. Evaluation of kojic acid for determining heme and nonheme haloperoxidase activities spectrofluorometrically. Analytical Letters. 38:921-927. Interpretive Summary: It is important to be able to measure enzymes present in crop plants because they can effect the quality and quantity of agricultural products. This paper describes a new method for measuring enzymes that cause discoloration of plant products and also protect plants against infection. The method is based on the reaction of the enzyme with a chemical to yield a product that fluoresces when illuminated with ultraviolet light. The intensity of the fluorescence is directly proportional to both the amount and the strength of the enzyme and is easily measured. In this manner, scientists, who are the principal users of this technological advance, are able to readily and reliably measure the amounts and strengths of the enzymes. Technical Abstract: Heme-containing haloperoxidases (heme haloperoxidases) catalyze peroxide-dependent oxidation of halide ions to hypohalite ions. Nonheme haloperoxidases catalyze peroxidation of alkyl acids to peroxyacids that subsequently oxidize halide ions to hypohalite ions. Hypohalite ions react with kojic acid spontaneously with concomitant production of fluorescence. This reaction was examined for suitability in assaying heme and nonheme haloperoxidase activities spectrofluorometrically. Heme haloperoxidases were assayed directly from hypohalite generation, whereas nonheme enzymes were assayed by coupling peroxyacid formation to bromide ion oxidation. Peroxidation of kojic acid by chloroperoxidase and myeloper-oxidase did not require halide ions and was not increased upon their addition, demonstrating that haloperoxidase activity was not distinguished from the classical peroxidase-like activity of these enzymes. Addition of bromide ions to specimens of nonheme haloperoxidase, however, resulted in increased rates of fluorescence generation, indicative of the presence of distinct peroxidase and haloperoxidase enzymes. The heme poison sodium azide inhibited chloroperoxidase, myeloperoxidase, and horeradish peroxidase activities totally, but inhibited only a portion of nonheme haloperoxidase activity, verifying that the last specimen was contamined with heme-containing peroxidase. Results show the utility of kojic acid for fluorometrically measuring haloperoxidase activity, particularly that of the nonheme enzyme. |
