A heptapeptide upon HCL hydrolysis produced equimolar amounts of ASP, CYS, GLU,

Question

A heptapeptide upon HCL hydrolysis produced equimolar amounts of ASP, CYS, GLU, LYS, PHE, TYR, VAL and ammonia. Exposure of the intact heptapeptide to FDNB followed by hydrolysis produced DNP-VAL. Treatment with trypsin produced a tripeptide , T1 and a tetrapeptide, T2. T2 had an absorbance peak near 260 nm and produced ammonia on hydrolysis. T1 had an absorbance peak near 275 nm ( which was greater than that of T1) and tested positive for sulfur. Exposure of the heptapeptide to chymotrypsin produced two tripeptides ( CT1 and CT2) which both had UV absorbances and ASP. CT1 contained sulfur and had the greater absorbance of the two chymotrypsin produced tripeptides. CT2 produced ammonia upon hydrolysis. When exposed to electrophoresis at pH6, T2, and CT1 were cations, T1 was an anion, and CT2 has essentially no charge. Deduce the sequence of the heptapeptides from the data.

Explanation / Answer

If a heptapeptide was composed of three glycines, two alanines, a leucine and a valine, many possible primary structures could be written. On the other hand, if partial hydrolysis gave two known tripeptide and two known dipeptide fragments, as shown on the right, simple analysis of the overlapping units identifies the original primary structure. Of course, this kind of structure determination is very inefficient and unreliable. First, we need to know the structures of all the overlapping fragments. Second, larger peptides would give complex mixtures which would have to be separated and painstakingly examined to find suitable pieces for overlapping. It should be noted, however, that modern mass spectrometry uses this overlap technique effectively. The difference is that bond cleavage is not achieved by hydrolysis, and computers assume the time consuming task of comparing a multitude of fragments. 3. N-Terminal Group Analysis Over the years that chemists have been studying these important natural products, many techniques have been used to investigate their primary structure or amino acid sequence. Indeed, commercial instruments that automatically sequence peptides and proteins are now available. A few of the most important and commonly used techniques will be described here. Identification of the N-terminal and C-terminal aminoacid units of a peptide chain provides helpful information. N-terminal analysis is accomplished by theEdman Degradation, which is outlined in the following diagram. A free amine function, usually in equilibrium with zwitterion species, is necessary for the initial bonding to the phenyl isothiocyanate reagent. The products of the Edman degradation are a thiohydantoin heterocycle incorporating the N-terminal amino acid together with a shortened peptide chain. Amine functions on a side-chain, as in lysine, may react with the isothiocyanate reagent, but do not give thiohydantoin products. A major advantage of the Edman procedure is that the remaining peptide chain is not further degraded by the reaction. This means that the N-terminal analysis may be repeated several times, thus providing the sequence of the first three to five amino acids in the chain. A disadvantage of the procedure is that is peptides larger than 30 to 40 units do not give reliable results. 4. C-Terminal Group Analysis Chemical Analysis Complementary C-terminal analysis of peptide chains may be accomplished chemically or enzymatically. The chemical analysis is slightly more complex than the Edman procedure. First, side-chain carboxyl groups and hydroxyl groups must be protected as amides or esters. Next, the C-terminal carboxyl group is activated as an anhydride and reacted with thiocyanate. The resulting acyl thiocyanate immediately cyclizes to a hydantoin ring, and this can be cleaved from the peptide chain in several ways, not described here. Depending on the nature of this final cleavage, the procedure can be modified to give a C-terminal acyl thiocyanate peptide product which automatically rearranges to a thiohydantoin incorporating the penultimate C-terminal unit. Thus, repetitive analyses may be conducted in much the same way they are with the Edman procedure. Enzymatic Analysis Enzymatic C-terminal amino acid cleavage by one of several carboxypeptidase enzymes is a fast and convenient method of analysis. Because the shortened peptide product is also subject to enzymatic cleavage, care must be taken to control the conditions of reaction so that the products of successive cleavages are properly monitored. The following example illustrates this feature. A peptide having a C-terminal sequence: ~Gly-Ser-Leu is subjected to carboxypeptidase cleavage, and the free aminoacids cleaved in this reaction are analyzed at increasing time intervals. The leucine is cleaved first, the serine second, and the glycine third, as demonstrated by the sequential analysis. Of course, fourth and fifth units will also be released as time passes, but these products are not shown.

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