Date of Award
1992
Publication Type
Master Thesis
Degree Name
M.Sc.
Department
Chemistry and Biochemistry
Keywords
Chemistry, Biochemistry.
Rights
info:eu-repo/semantics/openAccess
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Abstract
Verification of the purity of a commercial Horseradish peroxidase (HRP) preparation, kinetic analyses of two chromogenic assays and an investigation of the mechanisms of enzyme inactivation by two of its substrates, hydrogen peroxide ($\rm{H\sp2 O\sb2}$) and phenol, are described. Isoelectricfocusing of Boehringer Mannheim Grad II HRP preparation revealed that it is composed of neutral isozymes B and C, as reported by the Manufacturer. No other contaminating isoenzymes were detected. Kinetic analysis (T = 25$\sp\circ$C, pH 7.4) of the 4-amino-antipyrine (AAP)/3,5-dichloro-2-hydroxybenzenesulfonic acid (HDCBS) chromogen system yielded K$\sb{\rm mapp}$ values for $\rm{H\sb2 O\sb2}$, AAP and HDCBS of 41.0$\mu$M, 3.94mM and 1.4mM, respectively. Hydrogen peroxide, in the absence of donor substrates and at concentrations above 100$\mu$M, inactivates HRP in a time-dependent and irreversible mechanism-based suicide inactivation that does not require a pre-association of $\rm{H\sb2 O\sb2}$ with HRP before substrate turnover. Protection against inactivation is afforded in the presence of donor substrates. Phenoxy radicals generated during the oxidation of phenol by HRP/$\rm{H\sb2 O\sb2}$ also inactivate the enzyme in an irreversible, mechanism-based time-dependent inactivation that follows a single-exponential decay. (Abstract shortened by UMI.)Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1992 .B395. Source: Masters Abstracts International, Volume: 31-04, page: 1793. Thesis (M.Sc.)--University of Windsor (Canada), 1992.
Recommended Citation
Baynton, Kathy J., "Investigation of the pathways leading to reversible and irreversible inactivation of horseradish peroxidase." (1992). Electronic Theses and Dissertations. 2510.
https://scholar.uwindsor.ca/etd/2510
