Protein Chemistry - Part 1 - General Protein Chemistry - Ashmarin I. P. 1968

Chemical reactions of proteins, determination of terminal and functional groups in proteins
Determination of N- and C-terminal amino acids

As is well known, The amino acid residues located at the ends of polypeptide chains are classified into N-terminal residues, i.e., those possessing a free NH₂ group, and C-terminal residues, i.e., those with a free carboxyl group. Methods for determining both types of terminal Amino Acids are divided into chemical and enzymatic approaches.

We have already discussed the principal chemical methods for determining N-terminal groups (Sanger's dinitrofluorobenzene method, Edman's phenylisothiocyanate method, and the pipsyl chloride method). Here, we need only note that Sanger's and Edman's methods are the most widely used for these purposes.

For the enzymatic determination of N-terminal groups, aminopeptidases are occasionally employed—Enzymes that sequentially cleave amino acids from the N-terminus of a polypeptide chain. The best-studied and most commonly used enzyme is leucine aminopeptidase, isolated from porcine Kidneys. This enzyme has been utilized to study the N-terminal sequences of Insulin and Ribonuclease. Experiments with synthetic Peptides and amino acid amides have demonstrated that N-terminal amino acids adjacent to Lysine, Arginine, and aromatic amino acids are cleaved very slowly. This relatively narrow Specificity of the enzyme complicates its application, while the specificities of other aminopeptidases remain insufficiently studied.

Regarding The Study of C-terminal amino acids, Hydrazinolysis is virtually the only sufficiently reliable chemical technique available. Unfortunately, as noted above, the hydrazides of Certain amino acids readily convert into free amino acids, which can significantly distort analysis results and limits the applicability of this accessible and straightforward method. Consequently, the enzymatic method is more frequently used to determine C-terminal amino acids.

Carboxypeptidase is employed for the enzymatic Determination of C-Terminal Groups. Carboxypeptidase cleaves only those peptide bonds in Proteins that are adjacent to a free α-carboxyl group. Consequently, when the enzyme acts on a protein, the C-terminal amino acid is the first to be cleaved from the polypeptide chain, followed by the Cleavage of the adjacent one, and so on. Thus, carboxypeptidase brings about the stepwise degradation of the protein starting from the C-terminus.

While the method is straightforward to perform, it requires rigorous purification of the enzyme; even the slightest contamination by proteinases (such as Trypsin or Chymotrypsin) severely distorts the results. Therefore, a specific inhibitor of these enzymes, diisopropyl fluorophosphate—which has no effect on carboxypeptidase—is usually added to the incubation medium. The protein is incubated with carboxypeptidase for various periods, after which the enzyme is inactivated, the proteins are precipitated, and the released amino acids are quantitatively determined in the protein-free filtrate. A plot of amino acid concentration versus time in the protein-free filtrate is constructed for each amino acid. Naturally, the highest rate of accumulation should be observed for the terminal amino acid, a somewhat lower rate for the subsequent one, and so on. In this manner, it is possible to establish The sequence of several amino acid residues (7–10) starting from the C-terminus of the polypeptide chain.