Q.1. Protein (10 mL of crude extract with activity of 600 U/mL, when diluted 15fold it has OD280 of 0.8) was purified using affinity chromatography resulting in
2.5 mL pooled fraction with protein content of 3.5 mg/mL and activity of 1800
U/mL. Calculate specific activity and percent recovery. (6)
Note: one unit of optical density at 280 nm corresponds approximately to 1 mg/mL
protein.
Q.2. A crude cell free extract contain 32mg protein per ml. 10 microliter of the
extract catalysed a reaction at a rate of 0.14 μmol/min under standard optimum
assay conditions. 50 milliliters of the extract were fractionated by ammonium
sulfate precipitation. The fraction precipitated between 20% and 40% saturation
was re-dissolved in 10ml. This solution was found to contain 50mg protein/ml. 10
microliter of this purified fraction catalyzed the reaction at a rate of 0.65μmol/min.
Calculate
a) The percent recovery of the enzyme in the purified fraction and b) The degree of
purification obtained by the fractionation (the purification factor)
(7)