P-Nitrophenol Lab Report
A standard curve of p-nitrophenol was drawn at the start of the project, before any enzyme assay of the fractions. The standard curve is set up so the equation of the line can be used to measure the enzyme activity in each sample fraction. P-nitrophenol shows a linear relationship with acid phosphatase.
An Enzyme assay gives the activity levels of acid phosphatase in each fraction. The results from each fraction were placed into the equation of the line from the standard curve of p-nitrophenol. A standard curve of p-nitrophenol is used because this is the product created after acid phosphatase is reacted with the specific buffer.
The activity in each homogenate changed the more the samples were purified i.e. for each …show more content…
From the assay used protein can also be detected. This met that the acid phosphatase activity results were not reliable till the protein was taken out. This involved the samples being tested at two absorbances, 208nm and 260nm. These absorbances absorb light in the UV region. Protein absorbs in the UV region because they contain aromatic side chains. The two absorbances were taken from each other and the total protein found. The total protein was low in the samples. Reasons for this would be that they were vegetables. Vegetables are not usually used as protein supplements. Direct UV-light absorbances do have disadvantages associated with them. These disadvantages can make them impractical for use with typical protein samples. Broccoli is shown to have the most protein concentration in Fraction 1 while it decreases as the fractions are purified. The lowest protein concentration is found in frozen cauliflower. This sample starts off at a very low level of protein until it finally goes to zero. The protein level is slightly higher in fresh bought cauliflower but by the last fraction, fraction 3, the protein levels have also reached zero. This shows that protein in the samples had been extracted. Enzyme activity was still present showing acid phosphatase.
Although unexpected broccoli Fraction 2 (F2) has the highest percentage recovery of acid phosphatase. This shows that acid phosphatase was present at a higher level in broccoli Fraction 2. While the other samples show a decrease in acid phosphatase recovery. This could show that acid phosphatase was precipitated out into the pellet and discarded after