Loading…

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

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 spectrophotometr...

Full description

Saved in:
Bibliographic Details
Published in:Analytical biochemistry 2008-08, Vol.379 (1), p.60-65
Main Authors: Verhaeghe, Elodie, Buisson, David, Zekri, Elisabeth, Leblanc, Catherine, Potin, Philippe, Ambroise, Yves
Format: Article
Language:English
Subjects:
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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 2O 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 haloperoxidases.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2008.04.041