A) To properly analyze thrombin present in the blood sample, you need to first r

Question

A) To properly analyze thrombin present in the blood sample, you need to first remove two protein impurities that disrupt the activity assay (cytochrome c and lacaglobin). You have CM-cellulose and phosphate buffer (pH=6.4) already on hand. You decide to lead the sample onto a column using the CM-cellulose equilibrated with the phosphate buffer. Based on the table below, predict the order of elution for the three proteins. At pH=6.4, which protein(s) would you predict to remain bound to the column?

Protein: pI

cytochrome c: 10.6

Lacaglobin: 5.2

Thrombin (wild-type): 7.1

B) List two different ways you could change the buffer to elute the bound protein(s), thereby successfully separating the three proteins.

C) When you run the patient's blood sample and analyze the results, you find that the patients thrombin does not bind to the CM-cellulose. Since you trust your reagents and your technical abilities, you instead conclude that the patient must have a mutation in their thrombin gene that gives it different properties relative to the wild-type. After implementing a different buffer system, you are able to purify some of the patient's thrombin and submit it for sequencing. The results reveal that the patients' thrombin contains a mutation in the enzyme active site wherein a lysine residue has been replaced by an asparagine. Does this mutation explain the anomalous CM-cellulose binding behavior you observed?

Explanation / Answer

A) ans= CM - cellulose ellutes oppositely charged molecules first.So, at pH = 6.4(acidic) , firstly the cytochrome >wild type Thrombin> Patient' s protein will ellute out.

B) ans= We can change the buffer, which is of basic pH , so that Lacaglobin( pH 5.2) get elluted first.,which may get bound to the gel due to the acidic pH of itself as well as of the previously used buffer then ,

We can change the buffer which is mildly acidic , so that the basic molecules,( cytochrome & Thrombin) also get elluted out.

c) Ans= Obviously, yes! Lysine is a positively charged molecule, which is found in wild type of Thrombin, &

Argenine is a negatively charged molecule, which has been found in the patient's Thrombin sample.

Thus,this mutant Thrombin couldn't bind to CM-cellulose at acidic pH because,Argenine has already made the mutant Thrombin acidic in nature.

Related Questions

Navigate

• Browse (All)
• Browse A
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.