5. Protein Techniques

You perform multiple tests to derive the amino acid sequence of a purified peptide (see results below). Which of the following peptides listed best represents the sequence of the unknown peptide?

Draw out each of the peptide fragments that would be generated if the peptide is treated with trypsin.

Draw out the resulting peptide fragments that would be generated if the peptide is treated with chymotrypsin.

What would the resulting peptide fragments be if the following peptide were treated with excess Pepsin? 

                                H—E—L—P—M—E—P—L—E—A—S—E

Which is the expected result of chymotrypsin cleavage of the following peptide? 

                            Lys—Gly—Phe—Thr—Tyr—Pro—Asn—Trp—Ser—Tyr—Phe

A) The octapeptide AVGWRVKS was digested with the enzyme trypsin. Would ion exchange or size-exclusion chromatography be most appropriate for separating the fragments? Explain. 

a. Ion-Exchange chromatography.

b. Size-exclusion chromatography.

B) Suppose the same peptide was digested with chymotrypsin. Which would be the optimal separation technique? Explain.

a. Ion-Exchange chromatography.

b. Size-exclusion chromatography.

A nonapeptide was determined to have the following amino acid composition: (Lys)₂, (Gly)₂, (Phe)₂, His, Thr, Met.  The native peptide was incubated with 1-fluoro-2,4-dinitrobenzene (FDNB) and then hydrolyzed; 2,4-dinitrophenylhistidine was identified by HPLC.  When the native peptide was exposed to cyanogen bromide (CNBr), an octapeptide and free glycine were recovered.  Incubation of the native peptide with trypsin gave a pentapeptide, a tripeptide, and free Lys.  2,4-Dinitrophenyl-histidine was recovered from the pentapeptide, and 2,4-dinitrophenylphenylalanine was recovered from the tripeptide.  Digestion with the enzyme pepsin produced a dipeptide, a tripeptide, and a tetrapeptide.  The tetrapeptide was composed of (Lys)₂, Phe, and Gly.  The native sequence was determined to be:

*Recall: Pepsin cleaves N-terminal peptide bond of F, Y, W & L residues.*

The following reagents are often used in protein chemistry. Match the reagent with the purpose for which it is best suited. Some answers may be used more than once or not at all and more than one reagent may be suitable for a given purpose.