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.
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?