Acid phosphate reaction:
P-nitrophenyl phosphate + H2O -------------- p-nitrophenol + phosphate
(Substrate)
P-nitrophenol + OH- ---------------- p-nitrophenolate ion + H2O
(Product pale yellow) (Bright yellow at 405nm)
The pale yellow product formed gives a bright yellow colour in alkaline solutions; this also stops the enzymic reaction.
Method:
Before I begin with the experimental methods, I am going to quickly run through the health and safety issues relating to this experiment and the equipments used and the dangers they pose. As in most practical’s, gloves and goggles must be worn. NaOH is corrosive and harmful, and has the potential to irritate the skin. Another point of consideration is the handling of glassware including pipette, breakage can often occur due to the mishandling.
This experiment involves two stages, part A and B, part A involves the determination of a standard curve for p-nitrophenol formation, whilst part B will determine the optimum pH. The most crucial aspect to the experimentation is timing, in enzyme catalytic reactions a delay in the addition of substance can significantly alter results, and render the data inaccurate.
Switch on the spectrometer and label six test tubes 1-6. Using a 10ml pipette measure out different amount of standard 200µM p-nitrophenol solution. It is important that all volumes are made up to 6mls with 0.1M of NaOH. The content of the test tubes should mix gently and after mixing, place it in the spectrometer and read at 405nm. After taking all the reading, a table should be drawn and a standard curve. In part B of the experiment, a total of sixteen test tubes is needed. A total of 8 tubes containing 5.5 ml of 0.1M NaOH should be set aside for a later stage of the experiment. Label the remaining 8 test tubes pH 3 to pH 8. Then Add 2.0ml of 2.5 mM p-nitrophenyl phosphate to each tube. At this stage the test tubes should be placed in a water bath for 4 minutes, and to allow it to equilibrate at 37°C. After the stipulated time add 1.0 ml of distilled water to test tube one labelled pH 3 blank, this tube will used as the blank. Then add 1.0ml exactly of acid phosphatase, to test tube two labelled pH 3 and start the stop clock. After 2 minutes add 1.0ml of enzyme to test three, labelled pH 4. Continue this process for the remaining tubes at a 2 minutes interval.
After precisely 30 minutes in the water bath remove a 0.5ml sample from test tube 2 and add this to the first test tube of NaOH which was prepare and put aside. Continue this process for the remaining tubes at a 2 minutes interval and add it to the remaining tubes containing NaOH. Record a reading for the eight test tubes absorbance containing 0.5ml sample and 5.5ml of 0.1M NaOH at a wavelength of 405nm. Calibrate the spectrophotometer to zero using NaOH.
Results:
Table1.Standard curve for p-nitrophenol:
Table 2.Activty of acid phosphatase at varying pH:
Equation: y = 0.0138x + 0.1473, by using this equation I so able to substitute y which is the absorbance and find the p-nitrophenol concentration which is x.
For example: y = 0.0138x + 0.1473
0.15 = 0.0138x + 0.1473
Therefore: x = 0.15 – 0.1473 / 0.0138
x = 0.196 µM
Optimum pH = 5.0 (41.21µM)
Discussion and Analysis:
The experiment went according to the pre-set method, and the overall objectives have been achieved. In term results and data, when compared with other in class data, it seems that the general aims of the experiment have been achieved. A structural change in the enzyme was inevitable as the pH would alter the enzyme shape as well as the substrate. There was not much complication with the experimental methods; the only source of error identified is timing. A delay in the second part of the experiment could affect the results. The optimum pH was the main indicator of success or completion of the experiment; it is observable from the smooth curve graph that the optimum activity has been achieved.
