Spectrophotometric assay for horseradish peroxidase activity based on pyrocatechol-aniline coupling hydrogen donor.
The hydrogen donor couples pyrocatechol-aniline and phenol-aminoantipyrine in the presence of hydrogen peroxide were compared as chromogens for horseradish peroxidase (HRP) assay. UV-Visible spectroscopy and high-performance liquid chromatography analysis indicated that during the HRP biocatalytic process, pyrocatechol-aniline was converted to a pink-colored reagent with a lambda(max) of 510 nm, which was used in the assay of HRP activity. Electrochemical studies revealed adequate electron transfer ability for this color reagent to serve as a proper mediator for HRP also. Using pyrocatechol-aniline a higher sensitivity and lower detection limit was obtained relative to those of the phenol-aminoantipyrine couple, which is commonly used for HRP assay. A relative standard deviation of 2.9% was obtained for 20 HRP activity measurements, indicating a satisfactory reproducibility for this method. In addition, kinetic parameters of K(m) (12.5mM) and V(max) (12.2 mM min(-1)mg(-1)) were calculated for pyrocatechol-aniline. Regarding the superiority of pyrocatechol-aniline, this couple is suggested to be a better hydrogen donor for the HRP spectrophotometric assay.
Molaei Rad
A
A
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
Ghourchian
H
H
Moosavi-Movahedi
A A
AA
Hong
J
J
Nazari
K
K
D000814:Aniline Compounds / Q000737:chemistry* / Q000378:metabolism; D002396:Catechols / Q000737:chemistry* / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D011522:Protons; D015203:Reproducibility of Results; D013053:Spectrophotometry / Q000379:methods*
D000814:Aniline Compounds; D002396:Catechols; D011522:Protons; D006735:Horseradish Peroxidase; C034221:catechol; C023650:aniline