[cvc121]

I am having some trouble with a tetrapeptide digest (w/ chymotrypsin) problem and would greatly appreciate any guidance.

The question is as follows:

The tetrapeptide His-Trp-Arg-Val (relevant pKa values are 2.1, 6.0, 9.5, and 12.5) was digested with chymotrypsin and adjusted to pH = 0.5 to fully protonate all digestion products. The digestion products were then applied to a strong cation exchange column (sulfonic acid). The products were eluted from the column using a pH gradient from 0.5 to 13. Write the primary amino acid sequence of the products in the order they would elute from the column. Indicate the pH value at which each product elutes from the column.

Here is my reasoning and attempt at the solution:

Chymotrypsin cleaves after aromatic residues (Trp, Tyr, Phe). In this case, it would cleave after the tryptophan amino acid and result in two dipeptides as digestion products: His-Trp and Arg-Val.

I have determined the isoelectric point (pI) of His-Trp to be 7.75 and Arg-Val to be 11.0.

Since the digestion products will elute from the column when the pH reaches the pI, His-Trp (pI = 7.75) will come out first followed by Arg-Val (pI = 11.0). Therefore, would the primary amino acid sequence of the products in the order they would elute from the column be His-Trp-Arg-Val? His-Trp would elute from the column at a pH of 7.75 and Arg-Val at pH = 11.0.

Am I on the right track? Is my reasoning sound? All help is greatly appreciated. Thanks.

[Babcock_Hall]

There are two dipeptides, but the way you phrased you answer, it almost sounds as if you have four amino acids as products.  I think that your reasoning is OK in a general way, but I would say that peptides elute when the pH is in the vicinity of the pI of the peptide, without being overly specific.

[cvc121]

Thank you for the response. So should the primary amino acid sequence not be written as His-Trp-Arg-Val since this seems as though there are four amino acids as products? I know that I have two dipeptides, but shouldn't the primary amino sequence be represented in the way that I have shown.

[Babcock_Hall]

You have written the primary sequence of the original peptide as His-Trp-Arg-Val, which is fine.  The question asked for the "digestion products."  I would notate them as you did:  His-Trp and Arg-Val.

Suppose for a minute that one subjected this to complete hydrolysis under conditions that did not destroy Trp.  Then one would have four amino acids.  I often separate amino acids and peptides with commas, but I don't know how universal this is.  I would write products of complete hydrolysis as Arg, His, Trp, and Val.  Let me know if this answers your question or if clarification is needed.