3 PART BIOCHEM QUESTION. PLEASE HELP AND EXPLIN. THANK YOU! 26. (6 points) You a

Question

3 PART BIOCHEM QUESTION. PLEASE HELP AND EXPLIN. THANK YOU!

26. (6 points) You are trying to determine the sequence of a protein that you know is pure. Give the most likely explanation for each of the following experimental observations. a) Dansyl chloride identifies Ala and Leu as amino terminal residues, in roughly equal amounts. b) Your protein has an apparent MW of 80,000, as determined by SDS-PAGE. After treatment of the protein with -mercaptoethanol, the same technique reveals two proteins of MW 35,000 and 45,000. c) Gel filtration chromatography experiments indicate the native protein has an apparent MW of 160,000.

Explanation / Answer

Ans. #a. Presence of two amino terminal residues (Ala, Leu) indicates that there are atleast two different polypeptide chains in the protein.

Since the number of both the N-terminal residues is equal to each other, both the polypeptide chains are equal in number.

#b. 2- mercaptoethanol (a commonly used reducing agent used in SDS-PAGE) breaks disulfide bridges (-S-S-) in protein resulting two cysteine residues with free thiol groups.

Presence of two bands at 35000 and 45000 Da after treatment of 80000kDa protein indicates the presence of two polypeptides in the protein. That is, 80000 Da protein consists of two polypeptide chain, one of 35000 Da and another of 45000 Da – both covalently linked through one or more disulfide bonds.

Let’s label the constituent polypeptide chains as follow-

            80000 kDa protein = Chain A (35000 Da) + Chain B (45000 Da) = (AB)

#c. Given, Gel filtration chromatography gives MW of 160000 Da.

Thus, total mass of the protein in its natural conformation is 160000 Da.

However, the MW of protein in SDS-PAGE = 80000 Da.

SDS denatures proteins by disrupting non-covalent interactions (H-bonds, hydrophobic interactions, ionic interactions, etc.) binds to all polypeptides chains and provide them uniform negative charge i.e. all polypeptide chains bear almost equal negative charge per unit length. That is, all linearized polypeptide chains have the same charge density.

# The comparison of gel-filtration chromatography and SDS-PAGE indicates that the overall protein consists of two copies of the (AB) heterodimer. Moreover, the two (AB) dimers are held together with non-covalent interactions only.

Conclusion: Overall protein composition-

            (AB)—(AB) =(AB)2

Polypeptide chains A and B are linked by disulfide bonds.

            (AB) is associated with the other (AB) heterodimer with non-covalent bonds.

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