Affinity chromatography of frog epidermis dopa-oxidase
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Benzoic acid is a competitive inhibitor of the enzyme dopa-oxidase. p-Aminobenzoic acid was coupled by a single-step reaction of the amino group to: (1) CNBr-activated Sepharose 4B: (2) Enzacryl AA, by reaction with thiophosgene (pH 10.0); and (3) CM-Sephadex G-50, modified to azide by the Curtis procedure. These three solid supports were used as affinity adsorbents in the dopa-oxidase purification. The enzyme was obtained from frog epidermis, and was retained at pH 4.7 with 0.1 M acetate buffer. The enzyme elution was carried out using a linear pH gradient with 0.1 M phosphate buffer pH 8.0. We have examined the interaction of the enzyme with the immobilized PABA in relation to the nature of the support, the lengthening of the “arm” of the ligand, bathwise adsorption, and the enzyme activation by immobilized trypsin.
KeywordsAffinity Chromatography PABA Enzyme Form Elution Pattern Mushroom Tyrosinase
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