Textbook - BIOLOGICAL CHEMISTRY - Gubsky Yu.I. - 2000

Chapter I. BIOMOLECULES AND CELLULAR STRUCTURES

CHAPTER 2. PROTEINS AND PEPTIDES

2.5. METHODS FOR ISOLATION AND ANALYSIS OF PROTEINS AND PEPTIDES

2.5.2. Methods for determining the amino acid composition and primary structure of proteins and peptides

Class="center">Study of the Amino Acid Composition of Proteins

Investigation of The amino acid composition (identification of individual amino acid residues and determination of their quantitative ratios) is the first step in establishing the Introduction/19.html">Primary Structure of a protein or peptide.

The stages of studying the amino acid composition of proteins (Peptides) are as follows:

a) Hydrolysis of the polypeptide chain of the protein or peptide under investigation.

Hydrolysis of the polypeptide chain is carried out by acid Cleavage

of peptide bonds. The standard hydrolysis Procedure involves boiling the protein in Hydrochloric acid (5.7 mol/L) at 105-110 °C for 24 hours;

b) Separation of the Amino Acids from the hydrolysate.

This is performed via Ion-exchange Chromatography of the hydrolysate components on sulfopolystyrene resins bearing cationic groups — SO₃H^-Na⁺ on their surface. When a mixture of amino acids is passed through a chromatographic Column packed with sulfopolystyrene, the Amino acids are sorbed at specific levels of the column. Subsequent elution of the column with Buffer solutions at different pH values results in the fractional washing out (elution) of specific amino acids;

c) identification and Quantitative determination of individual amino acids.

This is carried out using reactions with ninhydrin or fluorescamine, which react with amino acids to form corresponding colored or fluorescent complexes (see Section 2.2).

The method for quantitative Analysis of the amino acid composition of PROTEINS AND PEPTIDES was first proposed in 1958 by W. Stein and S. Moore and is currently implemented using automated amino acid analyzers.

Elucidation of the Primary Structure of Proteins and peptides

Determining The sequence of amino acid residues in protein and peptide molecules (elucidation of the primary structure) consists of the following stages:

1. Analysis of N- and C-terminal amino acid residues

Since the polypeptide chain forming the primary structure of any protein has a free α-NH₂ group at one end and a free α-carboxyl group at the other, the identification of N- and C-terminal amino acid residues makes it possible to estimate the number of polypeptide chains comprising the given protein molecule.

Identification of N-terminal amino acids is carried out using chemical Reagents that covalently bind to the terminal α-amino group of the peptide chain. Following subsequent acid hydrolysis of the "labeled" peptide, the chemically modified N-terminal amino acid residue is identified chromatographically.

The following reagents are used to modify N-terminal amino acids in proteins and peptides:

- 2,4-dinitrofluorobenzene (DNFB) — a reagent that interacts with the α-amino group of the terminal amino acid to form a DNP-derivative of the protein or peptide. Subsequent acid hydrolysis of the polypeptide arylation product yields a mixture of the DNP-derivative of the N-terminal amino acid, which is identified chromatographically, and free amino acids that make up the studied protein (Fig. 2.15):

Fig. 2.15. Scheme of formation of DNP derivatives of N-terminal amino acids.

- dansyl chloride (1-dimethylaminonaphthalene-5-sulfonyl chloride, DNS) is a reagent that reacts with the N-terminal amino acid to form DNS derivatives of proteins and peptides. Subsequent acid hydrolysis of the DNS-Polypeptides enables chromatographic identification of DNS-amino acids.

Identification of C-terminal amino acids is carried out via enzymatic Hydrolysis of Proteins and peptides using Carboxypeptidases—Proteolytic Enzymes that specifically cleave the C-terminal amino acid from the polypeptide chain.

2. Fragmentation of the Polypeptide Chain and Separation of Peptide Fragments

Cleavage of the studied protein or peptide into shorter fragments is typically achieved by Enzymatic hydrolysis using proteolytic

enzymes such as Trypsin, Chymotrypsin, or other proteases (Pepsin, Elastase, Papain, etc.). The resulting peptide fragments consist of 10–20 amino acid residues and can be separated using one of the chromatographic Methods (in particular, High-Performance Liquid Chromatography — HPLC) or Electrophoresis.

3. Determination of Amino Acid Sequences in Peptides

The principal modern method for deciphering Amino acid sequences is stepwise degradation of polypeptide chains using phenylisothiocyanate (PITC).

Proposed by the Swedish biochemist P. Edman between 1950 and 1956, it comprises the following stages:

- interaction of PITC with the α-amino group of the N-terminal amino acid in the target peptide, yielding a phenylthiocarbamoyl (PTC)-peptide;

- acidic cleavage of the phenylthiohydantoin (PTH)-derivative of the N-terminal amino acid from the PTC-peptide, followed by its identification (Fig. 2.16):

Fig. 2.16. Scheme of peptide analysis by the Edman method.

A unique feature of the Edman method is the sequential shortening of the studied peptide from the N-terminus by a single monomer without breaking the peptide bonds between the remaining amino acid residues. Consequently, this sequence of operations can be repeated to analyze the entire peptide through successive cleavage of its N-terminal amino acid residues. Automation of the method, implemented in a specialized device known as a "sequenator" (derived from the English word "sequence"), enables highly efficient Analysis of Protein hydrolysis products and Natural peptides.