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  • Title: Spectrometric assay for horseradish peroxidase activity based on the linkage of conjugated system formed by oxidative decarboxylation.
    Author: Yamaguchi M, Sato S.
    Journal: Spectrochim Acta A Mol Biomol Spectrosc; 2017 Mar 05; 174():189-194. PubMed ID: 27912178.
    Abstract:
    Horseradish peroxidase (HRP)-catalyzed oxidation of 2,2-bis[3-acethylfilicinic acid-5-yl]acetic acid (BAFA, 4) produces Dehydro-3,3'-diacetyl-5,5'-methylenedifilicinic acid (DDMF, 3). A new photometric hydrogen donor (4) for peroxidase (POD)-catalyzed oxidation was demonstrated to be potentially useful for spectrocolorimetric and spectrofluorometric determination of HRP. Our developed colorimetric (absorption at 483nm) and fluorometric (emission at 529nm) systems both gave a linear calibration curve for HRP (y=0.0025×+0.0237; R2=0.9997, and y=0.241×+3.194; R2=0.9914, respectively) in the same concentration range of 9.1×10-8-1.1×10-6nmol/L. The calculated Km and Vmax values of 5.10×10-4M and 5.13×10-6M/min, respectively. The results indicate that the quantification of HRP using BAFA 4 as hydrogen donor is possible.
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