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

Chapter I. BIOMOLECULES AND CELLULAR STRUCTURES

CHAPTER 2. PROTEINS AND PEPTIDES

2.2. STRUCTURE AND AMINO ACID COMPOSITION OF PROTEINS AND PEPTIDES

Molecular Weight of Proteins. Proteins are macromolecular compounds; their molecular weight (m.w.) ranges from several thousand to several million a.m.u. (Daltons). Individual proteins consist of several hundred amino acid residues.

Proteins may consist of one or more separate polypeptide chains joined by covalent (disulfide) and non-covalent bonds. Proteins containing a single polypeptide chain have a molecular weight ranging from 5-6 to 50 kD; proteins with a higher m.w. generally consist of several polypeptide chains that form protomers (subunits) — multichain (oligomeric) proteins (Table 2.1).

Class="center">Table 2.1. Molecular weight of proteins

Peptides (oligopeptides, Polypeptides) differ from actual proteins in their molecular weight (less than 5-6 kD) and corresponding physicochemical properties.

Shape of Protein molecules

The polypeptide chains underlying the Covalent Structure of protein molecules are capable of forming ordered Conformations stabilized by Hydrogen Bonds and other weak physicochemical interactions. These highly ordered conformations establish specific Levels of Protein structural Organization (see below), which are reflected in the various architectural forms of protein molecules.

Based on their molecular shape, proteins are divided into globular (spherical) and fibrillar (elongated).

The formation of globular and Fibrillar Proteins will be discussed in greater detail when examining the mechanisms governing higher levels of protein structural organization.

Amino Acid Composition of proteins and peptides

Hydrolysis of natural PROTEINS AND PEPTIDES releases about 20 different α-L-Amino Acids, THE POSITION OF each in the polypeptide chain being encoded by a nucleotide triplet in the genomic DNA.

The amino acids that make up natural proteins and peptides (Proteinogenic Amino Acids) share a common chemical structure, represented by the standard structural (a) and projection (b) formulas shown below (Fig. 2.1).

Fig. 2.1. Structural (a) and projection (b) formulas of proteinogenic amino acids. The projection formula of an L-amino acid is shown.

Structural Features of proteinogenic amino acids:

1) the amino group, hydrogen ion, and side chain (R-group) are attached to the carbon atom located at the α-position relative to the carboxyl group, meaning Natural Amino Acids are α-amino acids; Certain amino acids (Lysine, Arginine) possess an additional amino group located at the terminal (ω-) position of the R radical;

2) in terms of their absolute configuration, proteinogenic Amino acids are L-series stereoisomers (L-amino acids). D-amino acids are not found in natural proteins; they occur in bacterial and plant sources and are constituents of certain Antibiotics (gramicidin, actinomycin D). Optical isomers of Amino acids differ in taste (L-isomers are bitter or tasteless, D-isomers are sweet), which indicates the stereospecificity of taste receptors.

Classification of proteinogenic amino acids

Natural α-Amino acids can be divided into classes depending on the Chemical Structure of the side R radical:

The modern rational classification, based on the polarity and charge of the R radical, comprises four classes of amino acids (Table 2.2):

I — amino acids with nonpolar (hydrophobic) R-groups;

II — amino acids with polar (hydrophilic) uncharged R-groups;

III — amino acids with negatively charged R-groups (acidic amino acids);

IV — amino acids with positively charged R-groups (basic amino acids).

In addition to the twenty amino acids listed in Table 2.2, derivatives of these Amino acids have been found in certain proteins, notably 4-hydroxyproline, 5-hydroxylysine, N-methyllysine, 3-methylhistidine, phosphoserine, phosphothreonine, and diiodotyrosine. Chemical modification (hydroxylation, phosphorylation, iodination) of the respective amino acids occurs after their incorporation into polypeptide chains (Post-translational protein modification).

Table 2.2. Amino acids making up proteins

Properties of proteinogenic amino acids

1. Acid-base properties of amino acids.

Amino acids are amphoteric electrolytes that can dissociate to form ionic species—anions or cations. In an aqueous environment, amino acids exist as an equilibrium mixture comprising anionic, cationic, and zwitterionic (dipolar ion) forms, as shown in Fig. 2.2.

Fig. 2.2. Anionic (a), cationic (b), and zwitterionic (c) forms of amino acids In aqueous solutions.

The aforementioned reactions for the formation of amino acid anions, cations, and zwitterions correspond strictly to the acid-base dissociation scheme of monoaminomonocarboxylic amino acids, which contain a single α-amino and a single α-carboxyl group. In this simplest case, equilibrium between positively and negatively charged molecules can theoretically be achieved even in neutral solutions, i.e., at pH=7.

At the same time, certain amino acids possess R side chains containing additional functional groups capable of dissociation:

- acidic groups of Asp, Glu;

- basic groups of Lys, Arg, His.

Thus, the net charge of amino acid molecules (and, accordingly, of the proteins and peptides they comprise) is determined by the ratio between the number of free acidic and basic groups, their degree of dissociation (pK_a), and the ambient pH.

In acidic solutions, the cationic form of amino acids predominates (molecules are positively charged), whereas in alkaline solutions, the anionic form prevails (amino acids are negatively charged). These PHYSICOCHEMICAL PROPERTIES OF amino acids determine their susceptibility to Electrophoresis, the Separation in a high-voltage direct-current electric field. When positive and negative charges are in equilibrium, The amino acid molecule exists in an isoelectric state. The characteristic pH value at which an amino acid carries a net zero charge is called the pH of the isoelectric point (pI).

2. Polarity of amino acid molecules.

Depending on the polarity of their R side chains (Table 2.2), amino acids interact to a greater or lesser extent with Water dipoles, thereby exhibiting hydrophilic or hydrophobic properties.

The polarity of amino acid functional groups, together with their acid-base properties, determines the structural features, as well as most of the physicochemical and, consequently, biological Properties of the proteins synthesized from these amino acids.

3. Optical Properties of amino acids.

The α-carbon atom of all proteinogenic amino acids, with the exception of glycine, is bonded to four different functional groups (making it an asymmetric atom) and serves as a chiral center of the molecule. Consequently, proteinogenic amino acids are optically active compounds capable of rotating the plane of polarized light.

4. Ability to form acid-amide bonds.

A characteristic chemical feature of amino acids is the ability of their α-amino and α-carboxyl groups to form an acid-amide (peptide) bond through the elimination of water molecules, thereby undergoing polycondensation:

The polyamides formed in these reactions are called peptides (specifically, dipeptides, tripeptides ... oligopeptides ... polypeptides, respectively).

STRUCTURE OF THE Peptide Group

The four atoms comprising the peptide group (-СО-NH-) lie in the same geometric plane, meaning they are coplanar. The carbonyl oxygen and the NH group hydrogen are in a trans-configuration (Fig. 2.3).

Fig. 2.3. Coplanar arrangement of the atoms of the peptide group in Peptide and Protein molecules.

The bond length between the carbonyl carbon and the amide nitrogen is 0.132 nm, which is shorter than a standard C-N single bond (0.147 nm) and has approximately 50% double-bond character. This partial double-bond nature results from the conjugation of the nitrogen lone pair of p-electrons with the π-electrons of the C=O double bond (p,π-conjugation) and the formation of a Resonance structure:

Based on The structure of the peptide bond and the peptide group, free rotation within the peptide chain is only possible around the -CHR groups located between adjacent coplanar peptide groups (Fig. 2.3). These structural constraints, along with the ability of certain functional groups in peptide chains to engage in strong and weak interactions, determine the Specific features of ordered protein conformation formation, as discussed below.

5. Chemical Reactions used for Amino acid analysis.

Due to The Diversity of their functional groups, α-amino acid molecules can participate in chemical reactions applied in analytical and clinical biochemistry for the identification and quantification of individual amino acids. These reactions (known as "color reactions") are used to determine both free amino acids found in biological samples (such as Blood Plasma and urine) and those within the amino acid composition Analysis of proteins and peptides.

Ninhydrin Reaction

When heated with α-amino acids, ninhydrin (triketohydrindene hydrate) induces their decarboxylation, yielding NH₃, СО₂, and an aldehyde—the product of The oxidative decarboxylation of the amino acid. Subsequently, the released ammonia reacts with reduced ninhydrin to form a blue-purple complex with a maximum absorption peak at λ_max = 570 nm. The ninhydrin reaction can detect as little as 1 nmol of an amino acid:

Fluorescamine Reaction

Another highly sensitive reagent for α-amino acids is fluorescamine, which forms fluorescent complexes with amino acids. The fluorescamine reaction is more sensitive than the ninhydrin reaction, allowing for the detection of amino acids in quantities of 10–50 pmol.

Spectrophotometric or spectrofluorometric measurement of amino acid complexes with ninhydrin or fluorescamine enables the Quantitative determination of amino acids not only as free metabolites but also within protein hydrolysates following chromatographic separation, which is utilized in the Analysis of the Introduction/19.html">Primary Structure of Proteins and peptides.

In addition to those mentioned above, clinical biochemistry employs the following amino acid "color reactions":

- Xanthoproteic reaction — characteristic of the benzene ring in cyclic amino acids (phenylalanine, tyrosine, tryptophan), which becomes nitrated upon Treatment with concentrated nitric acid, forming yellow nitro compounds;

- Millon's reaction — a specific test for tyrosine (an amino acid containing a phenolic hydroxyl group). Heating phenols and their derivatives with Millon's reagent (a mixture of mercury(I) and mercury(II) nitrates) yields brick-red mercury derivatives;

- Sakaguchi reaction — a test used to identify the guanidino group of arginine. The interaction of guanidine with α-naphthol and sodium hypochlorite under alkaline conditions produces red-colored compounds;

- Ehrlich's reaction — used to detect the indole ring of tryptophan, which reacts with p-dimethylaminobenzaldehyde in an acidic medium to yield purple-colored compounds;

- Fohring's reaction (Foll's test) — a characteristic reaction for Sulfur-Containing Amino Acids. Boiling a protein solution or relevant amino acids with an alkali in the presence of sodium plumbite produces a brownish-black precipitate of lead sulfide.