25. Purification tables are often used to keep track of the yield and pu- rifica

Question

25. Purification tables are often used to keep track of the yield and pu- rification of a protein. The specific activity is a ratio of the amount of the protein of interest, in this case Mb, obtained at a given step (umol or enzyme units) divided by the amount (mg) of total protein. The yield is the ratio of the amount of the protein of interest obtained at a given step (umol or enzyme units) divided by the original amount present in the crude extract, often converted to percent yield by multiplying by 100. The fold purification is the ratio of the specific activity of the purified protein to that of the crude preparation. (a) For the purification table below, calculate the specific activity, % yield, and fold purification for the empty cells

Explanation / Answer

a)

Specific Activity = amount of protein of interest (mol or enzyme units) / amount of total protein

% Yield = amount of protein of interest obtained / original protein present in the crude extract

Fold Purification = specific activity of purified protein / crude amount

b)DEAE is a weak anion exchanger.This exchange is used to separate proteins that have faintly differing charges.The resin carries positive charge and hence interacts favourably with negative charges.Positive charge of the resin is due to the protonated amine group.This kind of electrostatic interaction is dependent on the pH range and should be less than the pKa of amine group.Also such a negative charge should also be found in the protein to be extracted from a crude preparation.Hence using DEAE would cause greater loss of Mb compared to using the affinity chromatography method because affinity chromatography is based on the specific binding of the protein to the stationary phase and hence the product will be eluted out last.

c)Affinity Chromatography

d)Affinity Chromatography

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