Phospholipase A. is an enzyme used by Leukocytes to destroy bacteria, by causing membrane permeability. In multiple purification steps, we must isolate the enzyme to be able to conclude it can be used for antibiotic use. Using homogenate, acid precipitation, ion exchange chromatography, and 2 from of gel filtration, the purification will be tested. I hypothesize these purification steps will isolate and purify the enzyme desired.
Results
Figure 1 shows Sephadex carboxymethyl ion exchange chromatography of a leukocyte’s cytoplasm. The 3 things being measured in this graph are: overall protein concentration, permeability-increasing activity(PI) and phospholipid A(PLA) activity. PLA activity is measured by the destruction of the …show more content…
Figure 2 shows the SDS-PAGE of the fraction number, which is the X-axis of Figure 1. In figure 2, a constant level of protein must be used through each lane of the SDS, in each step. The sample enzyme is placed in the gel, between every lane. A current is then run through the gel. The number of bands, in each lane, decreases with increasing purification, while the amount of the protein of interest increases as the total amount of protein decreases. Thus with greater purification of the protein, less total protein is left to work with. In contrast low purification leaves many other proteins to contaminate the sample. Referring to Figure 1, E has the highest peak, although it's mainly protein that is not of interest to us. There are 4 bands of protein and 2 bands are of considerable thickness, meaning there are multiple proteins of similar sizes present. The peak F is considerably smaller, but there is more purification. There are only 3 bands of protein and one is thick. PI is also coming into play at this