Horseradish peroxidase substrate peroxidation

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

B. Chance, Enzyme-substrate compounds of horseradish peroxidase and peroxides. II. Kinetics of formation and decomposition of the primary and secondary complexes, Arch. Biochem. Biophys. 22 (1949) 224. 

Extensive studies have established that the catalytic cycle for the reduction of hydroperoxides by horseradish peroxidase is the one depicted in Figure 6 (38). The resting enzyme interacts with the peroxide to form an enzyme-substrate complex that decomposes to alcohol and an iron-oxo complex that is two oxidizing equivalents above the resting state of the enzyme. For catalytic turnover to occur the iron-oxo complex must be reduced. The two electrons are furnished by reducing substrates either by electron transfer from substrate to enzyme or by oxygen transfer from enzyme to substrate. Substrate oxidation by the iron-oxo complex supports continuous hydroperoxide reduction. When either reducing substrate or hydroperoxide is exhausted, the catalytic cycle stops. 

A quite different approach came from Chance and others using heme enzymes (1947). Purified horseradish peroxidase has a characteristic absorption spectrum which was visibly altered in the presence of hydrogen peroxide. When an appropriate substrate was added it was oxidized by the hydrogen peroxide and the spectrum reverted to that of the original state of the enzyme. Similar studies were performed with catalase, showing that prosthetic groups in enzymes underwent reversible changes in the course of their reactions. 

Mahmoudi A, Nazari K, Khosraneh M et al (2008) Can amino acids protect horseradish peroxidase against its suicide-peroxide substrate Enzyme Microb Technol 43 329-335... 

Smith AT, Sanders SA, Thomeley RNF et al (1992) Characterisation of a haem active-site mutant of horseradish peroxidase, Phe41 Val, with altered reactivity towards hydrogen peroxide and reducing substrates. Eur J Biochem 207 507-519... 

Certain fluorescent compounds, such as the coumarin derivative, scopoletin, can act as hydrogen donors in the oxidative reaction catalysed by horseradish peroxidase (Udenfriend, 1969), an enzyme that exhibits substrate specificity for hydrogen peroxide... 

Calibration of the system The scopoletin concentration is adjusted in such a way that its fluorescence exceeds the substrate-induced fluorescence at least tenfold. Any marked decrease in fluorescence intensity therefore demonstrates an oxidation of scopoletin by H202 via horseradish peroxidase. Hence, after suitable calibration of the system, the rate of loss of fluoresence can be used to measure the rate of hydrogen peroxide production. 

---