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Horseradish peroxidase assay - Lab Report Example

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They speed up chemical reactions in the body by providing an alternate pathway for a reaction to follow with a lower activation energy. The enzymes do not take part in the actual chemical reactions and are therefore unchanged when…
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Horseradish peroxidase assay
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Download file to see previous pages For each enzyme there is a small of range of pH within which it works optimally. Enzymes have active sites in their structures. The active site is the part of the enzyme that has the correct shape and the functional groups required to bind to the substrate (Dunford, 1999).
Enzyme activity can be measured in any one of these two ways: observing the rate at which the substrate disappears during a reaction or measuring the rate at which the product is formed. Enzyme assays are used in such measurements. There are two methods that have been developed for use in measuring the amount of substrates or products in a chemical reaction: continuous and fixed-timed assays. Continuous assay make use of a spectrophotometer to measure the rates at which the substrate disappears and products form in real-time (Leskovac, 2003).
To measure the peroxidase activity a change in the amount of product formed will be evaluated over time. For the breakdown of peroxide by peroxidase, the simplest molecule that can be measured is O2 gas, the product of the decomposition of peroxide. To accomplish this the real volume of O2 gas produced is measured by use of an indicator. For this experiment an indicator (pyrogallol) that shows the presence of O2 gas will be used (Dunford, 2010).
2.50 cm3, 0.35 cm3, 0.10 cm3, and 0.35 cm3 of deionized water, buffer solution (at a pH of 6.0), hydrogen peroxide, and pyrogallol respectively were pipetted into two separate cuvettes labelled Cuvette 1 and Cuvette 2. The contents of the cuvettes were then mixed well using a small glass rod. The spectrophotometer was set to 420 nm after which Cuvette 1 was placed into it. 0.1 ml of the buffer solution was added to the cuvette and then stirred using a small glass rod. The readings of the spectrophotometer were recorded every 10 seconds for 5 minutes.
Cuvette 2 (blank) was placed into the spectrophotometer. 0.1 ml of ...Download file to see next pagesRead More
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