A colorimetric assay for steady-state analyses of iodo- and bromoperoxidase activities

作者：Verhaeghe Elodie; Buisson David; Zekri Elisabeth; Leblanc Catherine; Potin Philippe; Ambroise Yves^*

来源：Analytical Biochemistry, 2008, 379(1): 60-65.

DOI：10.1016/j.ab.2008.04.041

• 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.

• 出版日期2008-8-1
• 单位中国地震局