The purpose of the experiment was to study the effect of P-BAPA on the hydrolytic enzyme trypsin and to look at the effect of the competitive inhibition from chicken egg white. To do so, the technique used for the effects of inhibitors on trypsin activity was the spectrophotometer used to measure absorbance of P-nitro aniline as a function of concentration in order to get a standard curve. Absorbance of p-nitrroaniline was used as a probe for all reaction.
The experiment was performed by firstly adding enzyme to the substrate; the solution was the product and buffer reading taking respectively. The spectrometer was turn on and set to and absorbance reading of 405nm. Also, five tubes were set up containing different amount of standard of 50μM P-nitro aniline solution, a mix of3ml (0.05M, Tris, PH 9.2) and 300μl BAPA (4.6mg/ml in DMF) was put in a solvent resistant cuvette and placed in the spectrophotometer to take 5 (five) different reading of the standard curve for P-nitro aniline using it as a blank.
The next step was to determine the initial reaction rate in the presence of inhibitor, which shows the activity of the trypsin in the presence of varying amount of inhibitors, by doing so, 200μl of trypsin was added (1mg/ml in buffer) to the three cuvette given to perform the experiment which contained buffer and BAPA and mix. The cuvette was placed into the spectrophotometer by starting the stop watch and recording the OD reading every 30 seconds for 5 (five) minutes. Whiles the cuvette remains in the machine for the entire 5 minutes and noting the value every 30 seconds as given in the instruction. The process was repeated by using a clean cuvette each time but in the presence of different amount of trypsin inhibitor (stock solution of 0.1mg/ml in buffer).and by putting the mix in a beaker separately to avoid melting/dissolve any plastic. And in each case of the mix, the BAPA was the last solution that was added to the cuvette. The...