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Biochemistry

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Enzyme Purification Chart: Calculation and Interpretation

BIOCHM-RLJHXF

Two students were assigned the task of purifying the enzyme alpha-galactosidase from a yeast extract.

After an initial centrifugation and dialysis of the crude extract, they employed batch (large-scale) and then column DEAE (diethyl-aminoethyl)-ion exchange chromatography. Following dialysis, the students then employed affinity chromatography.

After each step, the samples/fractions were assayed for enzyme activity and protein concentration. Active fractions were pooled and further processed.

A chart showing the results of the purification process is shown below. Note that the percent yield of enzyme activity (Units) and fold purification of the enzyme specific activity are only given for the starting material.

You should calculate these values for the remainder of the table and then choose the one statement below which correctly interprets the results of the purification scheme.

Purification Table of Alpha-Galactosidase from Yeast

Step Vol. (mL) Total Activity (Units) Total Protein (mg) Specific Activity (U/mg protein) Yield (%) Purification Factor
Crude Extract 100 28.3 1555 0.018 100 1
Batch-DEAE Ion Exchange 42 22.8 1011 0.023 - -
Column-DEAE Ion Exchange 15 12.2 480 0.025 - -
Affinity Chromatography 1.1 0.22 0.11 2.0 - -
A

Overall, the purification scheme was not very successful because less than 1% of the original protein concentration was recovered.

B

The very small total yield of enzyme following affinity chromatography makes this purification scheme impractical for obtaining a pure enzyme extract.

C

The column DEAE-ion exchange chromatography step could be skipped based on little improvement in the purification factor and a large loss in total enzyme compared to the batch-DEAE ion exchange chromatography step.

D

Alpha-galactosidase protein was lost at a higher rate than overall protein in the purification scheme. This suggests that this scheme was not very successful in obtaining relatively pure enzyme.