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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...
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Published in: | Analytical biochemistry 2008-08, Vol.379 (1), p.60-65 |
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Main Authors: | , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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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. |
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ISSN: | 0003-2697 1096-0309 |
DOI: | 10.1016/j.ab.2008.04.041 |