Alkaline Phosphatase Lab Report
Aim
The aim of the experiments to be carried out is to determine the kinetic parameters, Km and Vmax, of Alkaline Phosphatase.
Theory, Principles and Application of Principles
Enzymes are a huge varying group of proteins which are needed to carry out essential metabolic functions in cells. Substrate-specific enzymes, like Alkaline Phosphatase, act as catalysts lowering the needed activation energy to convert the substrate to product.
Enzymes are made up of amino-acids and amino-groups have side chains referred to as R-groups. These R-groups have different degrees of protonation at different pH levels – meaning they can carry different charges at different pH levels, these charges together make up the overall charge of the enzyme. The charge
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The SPM was set to 34oC. Seven test tubes were used in determining the optimal temperature – in the first test tube a solution of 5mM p-Nitrophenol Phosphate with a buffer of pH 8 was used, when the 10 units of enzyme AP was added into the test tube (final volume of 3ml) then the experiment was immediately under way. The test tubes contents were hastily transferred to the cuvette and the put into the SPM, the absorbency readings were recorded every minute for a total of 10 minutes. This haste ensured the enzyme would not have ample time to form product that would have been unaccounted for, thus resulting in skewed readings and a faulty report. When the results for the first test tube were recorded, then the next solution/mixture was prepared. The second test tube was exactly the same as the first, the only difference being that the SPM was this time set to 35oC. The temperature of the SPM gets increased by 1oC for every test tube solution, until test tube 7 with an SPM temperature of 40oC.
After all the absorbencies for the varying temperatures had been recorded – the product concentration of each test tube solution was calculated using the absorbency readings at 10 minutes for each respective test tube mixture. The product concentration was calculated using Beer-Lamberts’ law of A = ECL. A graph plotting the product concentration against their corresponding temperatures was then drawn up, the peak of the graph being the optimal
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