期刊
ANALYTICAL BIOCHEMISTRY
卷 379, 期 1, 页码 60-65出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2008.04.041
关键词
iodoperoxidase; bromoperoxidase; thymol blue; vanadium
The standard assay for iodoperoxidase activity is based on the spectrophotometric detection of triiodide formed during the enzymatic reaction. However, chemical instability of I(3)(-) has limited the method to high iodide concentrations and acidic conditions. Here we describe a simple spectrophotometric assay for the determination of iodoperoxidase activities of vanadium haloperoxidases based on the halogenation of thymol blue. The relation between color and chemical entities produced by the vHPO/H(2)O(2)/I(-) catalytic system was characterized. The method was extended to bromine and, for the first time, allowed measurement of both iodo- and bromoperoxidase activities using the same assay. The kinetic parameters (K(m) and k(cat)) of bromide and iodide for vanadium bromoperoxidase from Ascophyllum nodosum were determined at pH 8.0 from steady-state kinetic analyses. The results are concordant with an ordered two-substrate mechanism. It is proposed that halide selectivity is guided by the chemical reactivity of peroxovanadium intermediate rather than substrate binding. This method is superior to the standard I(3)(-) assay, and we believe that it will find applications for the characterization of other vanadium as well as heme haloperoxiclases. (C) 2008 Elsevier Inc. All rights reserved.
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