A. Separate cleavage reactions of polypeptide by CNBr and chymotrypsin yield fra

Question

A. Separate cleavage reactions of polypeptide by CNBrand chymotrypsin yield fragments with the following amino acid sequences. What is the sequence of the intact polypeptide?

CNBr treatment

1. Arg-Ala-Tyr-Gly-Asn

2. Leu-Phe-Met

3.Asp-Met

Chymotrypsin

1. Met-Arg-Ala-Tyr

2. Asp-Met-Leu-Phe

3. Gly-Asn

B. The pKa of the carboxylic acid is around 3, and the pKa of the protonated amino group is around 10. Which of the structures below best represents an amino acid at neutral pH (i.e.,pH = 7)?

Select one:

a. +H3NCHRCOOH

b. +H3NCHRCOO-

c. H2NCHRCOOH

d. H2NCHRCOO-

Explanation / Answer

ANSWER A.
Separate cleavage reactions of polypeptide by CNBr and Chymotrypsin yield fragments with the given amino acid sequences. Using the concept of chromosome walking wherein we cross-check overlapping stretches across the two enzymes, we get the following pattern:

CNBr Treatment: Asp-Met + Leu-Phe-Met + Arg-Ala-Tyr-Gly-Asn
Chymotrypsin: Asp-Met-Leu-Phe + Met-Arg-Ala-Tyr + Gly-Asn
(with overlapping regions separately underlined)

ANSWER B.
The average of those two pKa's will yield an isoelectric point that comes out to be (10+3)/2 = 6.5
Since pH 7 is very close and similar to pH 6, for all practical purposes, we can assume that isoelectric point will be achieved and both ions will be activated to yield zwitterion configuration. Hence answer is B. H3+NCHRCOO-

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