Peroxidase

Horse radish peroxidase (HRP) EC1.11.1.7 is a commercially important enzyme used in techniques such as ELISA and immunohistochemistry. It catalyses the reduction of hydrogen peroxide to water in the presence of a suitable electron donor. The donor commonly used is 3,3’,5,5’-tetramethylbenzidine (TMB) which is colourless until oxidised when it becomes blue.

hydrogen peroxide H₂O₂	→	water + H⁺ H₂O + H⁺
hydrogen acceptor + H⁺ TMB	→	oxidised hydrogen acceptor oxidised TMB

[Catalase EC1.11.1.6 converts hydrogen peroxide to water and oxygen]

Safety

At the concentrations used here there are no hazards.

Good laboratory practices should be observed.

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Results

Comparison of results using different sources of enzyme

Effect of changing substrate concentration

Effect of changing enzyme concentration

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Methods

TMB

The TMB solution used to obtain the results shown here was made by dissolving 10mg of TMB in 2cm³ of methanol then diluting to 100cm³ with 0.05M phosphate/citrate buffer pH5.

Cuvettes contained 2cm³ of TMB solution, 0.1cm³ hydrogen peroxide solution and 0.1cm³ enzyme solution.

Enzyme extraction

The enzyme was extracted by grinding 0.5g of horse radish root (or alternative source) with 50cm³ of 0.5M phosphate/citrate buffer pH5. The extract was filtered through butter muslin.

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