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 lead to the denaturation of the enzyme and a decrease in the rate of reaction. There are many phosphatase enzymes but they are classified as those with alkaline and acid pH optimum. Both catalyse general reaction: ROPO3H2+ H2O –ROH + HPO42-+2H+.
Each enzymes works with a small range of pH, there is a pH at which its activity is greatest called optimal pH. This is due to the changes in pH can make a break intra and intermolecular bonds, distorting the shape of the enzyme, and its effectiveness. Generally, enzymes have an optimum pH this doesn’t go to say that the optimum is the same for each enzyme. For example the optimum pH for enzyme pepsin found acidic lumen in the stomach is lower than that of the enzyme carbonic anhydrase that works in the cytosol at neural pH.
Predominantly, enzymes show specificity for phosphatase monoesters but are relatively non-specific to (RO-). This can be used to construct an assay that uses non-physiological substrate, p-nitrophenyl phosphate, and one that has a product, which can be determined spectrometrically.
Nitrophenolate anion gives off a yellow colour this shows the maximum absorbance at 405 nm and providing the pH is more than 9 is basically the product alone.
Phosphatases are enzymes that take groups from substrates. Phosphorylated compounds are widely in living systems. They serve as storage forms for energy (e.g. ATP), as a part of informational macromolecules (i.e. nucleotides and deoxynucleotides) as effectors of some enzymes (e.g. fructose 1-6 biphosphate), and as second messengers (i.e. inositol phosphate and cAMP). Phosphorylation and de-phosphorylation reactions of proteins mediated by protein kinases and protein phosphatase, balance a lot of enzyme activities.
The first segment of the experiment was a trial run whereby the amount of enzymes was determined for the main experiment. Firstly, five test tubes were labelled 1-5, then 3 ml of 0.1 M sodium hydroxide was pipetted into each test tube. 20 ml of 0.05 M citrate buffer pH 5.0 containing 4 mM p-nitrophenyl phosphate was added to a 50 ml conical flask. 3 ml of the sample was then removed from conical flask and added to tube 1. This was then mixed and left to stand. An addition of 0.6 ml of acid...