1. In the just concluded experiment of gel filtration, Identify and record a tes
ID: 258146 • Letter: 1
Question
1. In the just concluded experiment of gel filtration, Identify and record a test tube/Fraction with the highest absorbance yet WITHOUT our enzyme of interest?
2. Why does this said fraction have a High absorbance but no enzyme of interest?
3. List the possible proteins accounting for this high absorbance and how would you separate / Purify alpha-mannosidase from those enzymes?
Absorbance at 280 nm Absorbance at 400 nm 0.7 0.3 0.2 30 32 36 38 40 42 Qualitative Protein Assay vs. Enzyme Assay Qualitative 0.8 05 02 0.2 -0.4 -0.6 022530S 4 -4-Absorbance at280 m, ?Abarbace at400 mmExplanation / Answer
1. We are getting the highest absorbance 1 at 280 nm.
2. The 400nm absorbance is specially for checking whether the sample contains other proteins or not. If yes, then we will get the absorbance. These other proteins do not contain aromatic amino acids, which is the basic requirements for the absorbance under 280 nm.
3. Here we are getting high absorbance due to presence of aromatic rign structures like FAD, FMN, Porphyrin and NAD. Also, carbohydrates impurities can affect the absorbance. The best process for the purification of ?-mannosidase is the dialysis process by potassium phosphate buffer of various pHs.
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