Effect of pH and Acid Phosphatase

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The practical was carried out to investigate the effect of pH on the reaction of the enzyme acid phosphatase.

Of the many functions of proteins, catalysis is by far the most vital. When catalysis is not present, most reactions in the biological systems take place very slowly to produce at an adequate pace for metabolising organism. The catalysts that take this role are called enzymes. Enzymes are the most efficient catalysts; they can enhance rate of reaction by up to 1020 over uncatalysed reactions. (Campbell et al, 2012).

Enzyme catalysis is dependant upon factors such as concentration of enzyme and substrate, temperature and pH. These factors determine the rate of reaction, and an increase in temperature or pH above the optimum will
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After 30 seconds the same enzyme volume was added to the second flask. This was continued to the third, fourth and fifth flasks with interval of 30 seconds until the final addition occurred at 2.5 minutes.

3ml of sample was taken first flask at 4 minutes and added to the appropriate tube of sodium hydroxide, from the second flask at 4.5 minute and so on, each flask was sampled at 30 second intervals. The sampling was then repeated starting at 8,12,16 minutes. The final sample from the last flask was taken at 18.5 minutes. Once the sampling was completed, measurements of absorbance were obtained for solution in each tube at 405 nm.
Discussion

Tables 2,3,4,5 and 6 show that as duration increased the absorbance also increased for each pH. The solution in the conical flask became darker (yellow) in time this is because the substrate, p-nitrophenyl phosphate was catalysed by acid phosphatase, releasing Nitrophenolate anion. It was the Nitrophenolate anion giving off the yellow colour; the presence of this feature increases the absorbance rate. The addition of sodium hydroxide distorted the shape of the enzyme making it no longer effective in its function.

The addition of sodium hydroxide at different times means that the amount of Nitrophenolate anion released will be different, thus, will have different absorbance readings (generally lower). The more time the enzyme is exposed to the substrate without the addition of
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