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 I3- 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 I3- assay, and we believe that it will find applications for the characterization of other vanadium as well as heme haloperoxidases.
Verhaeghe
Elodie
E
CEA, iBiTecS, Service de Chimie Bioorganique et de Marquage, Gif sur Yvette F-91191, France.
Buisson
David
D
Zekri
Elisabeth
E
Leblanc
Catherine
C
Potin
Philippe
P
Ambroise
Yves
Y
D020418:Algal Proteins / Q000378:metabolism; D044646:Ascophyllum / Q000201:enzymology; D001681:Biological Assay / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007453:Iodide Peroxidase / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D010544:Peroxidases / Q000378:metabolism*; D013944:Thymolphthalein / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D014639:Vanadium / Q000737:chemistry / Q000378:metabolism
D020418:Algal Proteins; D014639:Vanadium; C013713:thymol blue; D010544:Peroxidases; C026195:bromide peroxidase; D007453:Iodide Peroxidase; D013944:Thymolphthalein