Biochemistry

Free Version

Upgrade subject to access all content

Easy

Enzyme Assay Protocol

BIOCHM-D1Y4G5

A student was assigned the task of purifying an enzyme known to be secreted from yeast into the culture medium when grown in the presence of high glucose. The enzyme was described as a glycoprotein with a large net negative charge and a native molecular mass greater than 75 kDa. The following protocol was used, but in the end, no enzyme activity was recovered.

Protocol:

Step 1. Centrifugation of the crude yeast extract followed by dialysis (10 kDa molecular weight cutoff [MWCO]) of the recovered supernatant

Step 2. Ammonium sulfate precipitation, centrifugation, and dialysis (25 kDa MWCO) of the recovered supernatant

Step 3. Cation exchange chromatography followed by dialysis (10 kDa MWCO) of the collected eluate

Step 4. ConA (a carbohydrate binding lectin) affinity chromatography followed by dialysis (10 kDa MWCO) of the collected eluate.

Considering the above protocol and the known properties of the enzyme, which of the following is the MOST likely explanation for the negative result?

A

The enzyme could not bind to the cation exchange resin and was lost in Step 3. An anion exchange resin should have been used.

B

The dialysis tubing pore size in Step 2 allowed the enzyme to diffuse out of the dialysis bag where it was lost to the external buffer.

C

The enzyme was lost in the first centrifugation Step 1 when the yeast supernatant was retained rather than the pellet.

D

The enzyme was lost in the ConA affinity chromatography Step 4 because it did not contain carbohydrate residues thus preventing it from binding to the resin.