Chapter 5, Problem 19P

You are a summer intern in a clinical hematology lab. The lab director gives you a sample of a patient's blood proteins and asks you to characterize the thrombin in the sample. She also tells you that thrombin is a serine protease important in blood clotting (see Table 5.3), and this patient is a newborn with uncontrolled bleeding.
a. To characterize the thrombin in the sample, you must remove two proteins that interfere with the thrombin activity assay: cytochrome c and lactoglobin. You find some CM-cellulose (see Figure 5A.5) and a phosphate buffer (pH 6.4) on the shelf in your lab. You decide to load the protein sample onto a column of CM-cellulose equilibrated in the pH = 6.4 buffer. Predict the order of elution for the three proteins shown in the accompanying table. At pH = 6.4, which protein(s) do you predict will remain bound to the column?

b. List two different ways you could change the buffer to elute the bound protein(s) and achieve proper separation of the proteins.
c. You are surprised to observe that the patient's thrombin flows through the CM-cellulose column at pH = 6.4 and does not bind. Confident in your technique, you suspect the patient's thrombin is different from wild-type thrombin. Using a different buffer system, you manage to purify some of the patient's thrombin, and you submit the purified sample for amino acid sequencing. The sequence analysis shows that the patient's thrombin contains a mutation in the enzyme active site. A lysine residue in the wild type has been mutated to an asparagine in the patient's thrombin. Does this mutation explain the anomalous CM- cellulose binding behavior you observed?
d. How many nucleotide changes are required at the level of the gene for a lysine to asparagine mutation? Do you think Lys-to- Asn (or Asn-to-Lys) is likely to be a frequently observed mutation in proteins?
e. Based on their side-chain structures, compare and contrast the potential of Lys and Asn to form noncovalent interactions. In other words, can each form H bonds and/or salt bridges and/or van der Waals contacts?