Question

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.

1. 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?

   Table 5.3 says

Cytochrome C pI: 10.6

Lactoglobin pI: 5.2

Thrombin (wind type) pI 7.1

1. List two different ways you could change the buffer to elute the bound protein(s) and achieve proper separation of the proteins.

2. 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?

3. 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?

4. 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?

Answer 1

a) As information provided above for Ion exchange chromatography, a column is used of CM Cellulose which is a cation exchanger it posses a negative charge on it. For which pH should be between 6-10 and here the pH is 6.4. And to elute out proteins increase in pH is recommended in cation exchange. Here pI for these three protein is given. pI is a pH where a net charge is zero on a biomolecule.  To elute out increasing a pH is necessary. If pH above the pI then the molecule will have a negative charge so Lactoglobulin will not bind the column it will directly pass out. Thrombin and Cytochrome C will be bonded to the column. After increasing, pH Thrombin will elute out and Cytochrome C will be as a bound state if we stop increasing the pH.  

b) Elusion of protein is done by -

1) Changing the pH

2) Increasing the ionic strength of the buffer by salt gradient is the best way. (Na+/Cl-)

The proteins having the lowest net charge at the selected pH will be the first eluted from the column as ionic strength increases, the proteins with the highest charge at a certain pH will be eluted last.

C) The mutation in Thrombin will affect the binding with CM cellulose as it bears a negative charge on it the lysine is positively charged amino acid. When the amino acid composition will be changed in a protein there will be an effect on the charge of protein which will affect the binding. Lysine provides the positive charge which helps in binding with the negatively charged column but due to mutation charge will lose on protein and it will not bind.

D) At the gene level, any one out of two nucleotide substitution is required for a change in Lys-Asn as the last nucleotide in a codon with A, G with U or C. As AAA and AAG for lysine and AAU and AAC are for Aspargine.

Here the mutation is due to nucleotide substitution, and one nucleotide substitution is common in many cases but only when the repair system fails to detect it. As mutations are not frequent as DNA damage repair system works. If it fails to repair the damage then only this is the possibility.

E) Lysine is a charged amino acid and the charged molecule forms the ionic interaction in which H bonds/salt bridge can be formed by lysine at NH3 position. And the similar thing with the Arginine as it also has NH3 which will participate in electrostatic interaction and hydrogen bonding.

Van der walls interaction occurs when two molecules are taken near and it occurs in every case if we bring them closer.

Answer 2

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