PURIFICATION OF -GALACTOSIDASE AND ENZYME ASSAY 2. Create a plot of AAbs/min vs
ID: 149633 • Letter: P
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PURIFICATION OF -GALACTOSIDASE AND ENZYME ASSAY 2. Create a plot of AAbs/min vs enzyme aliquot Sample type Volume of sample AAbs/min (ul) Crude Crude Crude Purified Purified Purified 0.002 0.004 0.007 0.001 0.002 0.003 2 25 AAbs/min vs enzyme aliquot 30 y 38143x-5.9857 R2 0.8573 25 o 10 0.002 0.004 0.006 0.008 AAbs/min Are these curves what you would expect to see and why? Explain what AAbs/min means in real life, what are you actually measuring? 3. Discuss the following in your report A. Are your purification results what you would expect and why? (Specifically address: If specific activity is higher or lower after purification? If your protein concentration is different after purification, why?) B. List one thing that could be done to improve your experimental results 4. Additional question: After purification of an enzyme you do an activity assay to compare crude and pure activity. You add the same amount of protein on a mass basis to each activity assay and you see activity in the assay with the crude sample but not in the assay with the purified sample. What does this mean?Explanation / Answer
2. No, we do not expect this kind of a result. Delta Abs/min actually measures the rate of enzyme activity. The graph is not properly plotted. The volume of samples plotted in the graph do not match with the table. Moreover abs.below 0.05 AOD/min is generally considered no activity.From the table it can be seen that the rate of enzyme activity is more in the crude enzyme than the purified. We expect more activity in the purified enzyme.
Delta Absorbance actually means either the increase or decrease in enzyme activity and measures the rate of enzyme activity.
3. A The purification results are not what is expected. The purified enzyme should have a higher specific activity than the crude extract. Specific enzyme activity is a measure of enzyme purity and quoted as units/mg. The value becomes larger as an enzyme preparation becomes purer since the amount of protein (mg) is typically less, but the rate of reaction stays the same (or may increase due to reduced interference or removal of inhibitors).However this is not the case here. 5ul of the purified enzyme yielded delta abs. of 0.0001 in contrast to 0.0007 for the crude sample.
The protein concentration is definitely different after purification. The crude extract contains the protein of interest along with the others. So, If one measures the protein conc. of the crude ,one will get a higher concentration than once the protein of interest is purified.
B. In order to improve the results , one needs to start with a lot of starting material that is if one is isolating beta galactosidase from bacteria, one needs to culture the bacteria in large volume. This is required as the delta absorbance of the crude lysate lies in the lower range from 0.0001 to 0.0007. If the crude is so low, we cannot expect a good result after purification of the protein.
4. This means that the concentration of the purified protein is very low and also the enzyme ativity is lost in the purification process may be due to exposure to high temperature or mis handling etc.
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