title: Spectrometric assay for horseradish peroxidase activity based on the linkage of conjugated system formed by oxidative decarboxylation.
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 Kmand Vmaxvalues 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.
authors.affiliation: Department of Biochemical Engineering, Graduate School of Science and Engineering, Yamagata University, Jonan 4-3-16, Yonezawa-shi, Yamagata 992-8510, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Yamaguchi
authors.affiliation: Department of Biochemical Engineering, Graduate School of Science and Engineering, Yamagata University, Jonan 4-3-16, Yonezawa-shi, Yamagata 992-8510, Japan. Electronic address: shingo-s@yz.yamagata-u.ac.jp.
authors.forename: Shingo
authors.initials: S
authors.lastname: Sato
mesh_terms: D055162:Biocatalysis; D002138:Calibration; D003653:Decarboxylation; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D012997:Solvents; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors
chemical_list: D012997:Solvents; D006735:Horseradish Peroxidase
keywords: Carthamin-precursor model; Horseradish peroxidase; Oxidative decarboxylation; Spectrocolorimetry; Spectrofluorometry