Pharmacognosy with the Basics of Plant Biochemistry - Kovalyov, V. M. 2004

Special Part
Peptides and Proteins

Peptides, Polypeptides, and peptones are substances whose molecules consist of α-amino acid residues linked by peptide bonds —C(O)—NH—.

Proteins are high-molecular-weight natural Organic compounds, also composed of Amino Acids, that form The basis of the Structure and function of living organisms.

Structure and Classification of Peptides and Proteins

A peptide bond is formed by the dehydration reaction between the carboxyl group (—COOH) of one Amino Acid and the amino group (—NH₂) of another amino acid. Depending on the number of amino acid residues comprising a peptide, they are classified as dipeptides, tripeptides, and so on. Polypeptides containing 2 to 10 amino acid residues are called oligopeptides, while those with more than 10 are referred to as polypeptides.

Conventionally, peptides are considered to contain up to 100 amino acid residues, while proteins contain more than 100. This corresponds to peptide molecular masses of up to 10 thousand; proteins have molecular masses ranging from 10 thousand to 1 million and even higher.

High-molecular-weight peptides and proteins exhibit four LEVELS OF STRUCTURAL Organization. The Nature of The amino acid residues and their sequence constitute the Primary Structure. This, in turn, dictates The formation of more highly organized structures. The Secondary structure refers to the configuration of the polypeptide chain, most commonly forming an α-Helix or a β-sheet. The secondary structure is stabilized by Hydrogen Bonds between peptide groups that are closely located within the amino acid residue chain. The tertiary structure is the three-dimensional arrangement of the secondary structure. This structure is maintained not only by hydrogen bonds but also by Other types of interactions, such as ionic, hydrophobic, and Disulfide Bonds. The first three levels of structural organization are characteristic of all protein molecules. The quaternary structure refers to macromolecules composed of several non-covalently linked polypeptide chains (subunits). This fourth level describes the association and spatial arrangement of these subunits.

Peptides are found in all types of organisms. In their pure form, oligopeptides are typically crystalline substances and decompose when heated to 200-300 °С. They are highly soluble in Water, dilute acids, and bases, but practically insoluble in organic Solvents, with the exception of those composed of hydrophobic amino acid residues. Oligopeptides are closer to amino acids in their properties, while polypeptides are closer to proteins.

In living organisms, peptides can exist in a free state, such as Glutathione and carnosine. Many of them possess specific biological activity. Peptides include Hormones, Antibiotics, Vitamins, toxins, inhibitors, enzyme activators, and their derivatives. In laboratory settings, peptides are obtained by incomplete Hydrolysis of Proteins, while physiologically active ones are synthesized from amino acids.

Most proteins have the following elemental composition: 50.6-54.5 % carbon, 6.5-7.3 % hydrogen, 21.5-23.5 % oxygen, 15-17.6 % nitrogen, 0.3-2.5 % sulfur; many proteins also contain phosphorus, iron, and zinc. Virtually all proteins are composed of L-amino acids, with the exception of Glycine. Unlike proteins, peptides have a more diverse Amino Acid Composition and often include D-amino acid residues, as well as containing cyclic fragments and branched chains in their structure. Protein molecules do not pass through semipermeable membranes and have a low diffusion capacity. Proteins are amphoteric electrolytes, possessing free carboxyl (acidic) and amide (basic) groups. The Solubility of proteins varies greatly. Protein solutions in water are hydrophilic colloids, characterized by significant viscosity and low osmotic pressure. Many proteins are capable of crystallization.

The detailed Chemical Structure of proteins is not yet fully understood, so they are classified by chemical composition into simple and complex proteins. Simple proteins (albumins, globulins, Histones, glutelins, prolamins, protamines, protenoids) consist solely of amino acids. Complex proteins (proteids), In addition to the protein part, contain a non-protein component, known as a prosthetic group. Complex proteins include the following types: Glycoproteins, which contain CARBOHYDRATES; Lipoproteins, which contain Lipids; Chromoproteins, which contain pigments; Phosphoproteins, which contain phosphoric acid; Nucleoproteins, which contain Nucleic Acids; and Metalloproteins, which contain metals.

Based on their spatial form, proteins are divided into globular and fibrous. Globular proteins are more characteristic of plants. They possess an α-helical structure and typically have a spherical shape. An example of a globular protein is albumin (egg white protein). Almost all Enzymes belong to the globular protein Class. Fibrous proteins are prevalent in animal organisms. They are characterized by a β-sheet structure and a fibrous composition. This group includes β-keratin (which forms the basis of Hair and horny tissue) and Collagen (Connective Tissue). Globular proteins are highly soluble in water and salt solutions, forming colloids; fibrous proteins are insoluble in water.

BIOLOGICAL Functions OF Proteins in Plants and Animals

Proteins are a crucial factor in the active manifestations of life, representing a biological form of matter in motion. The Diversity of their structure and the precision of their unique organization are combined with plasticity in proteins. All of this creates significant functional capabilities. Based on their biological functions, proteins are classified as follows:

enzymes — highly specific catalysts for biochemical reactions; structural proteins — the basis of bone and connective tissue, wool, etc. (e.g., collagen);

regulatory proteins — those that control the Biosynthesis of Proteins and nucleic acids, as well as hormones;

receptor proteins — located on the outer surface of Plasma Membranes and receive information about the state of the external environment;

transport proteins, or carrier proteins — participate in The Active Transport of ions, lipids, sugars, and amino acids across Introduction/36.html">Biological Membranes; this also includes Hemoglobin and Myoglobin, which transport oxygen;

bioenergetic proteins, or Proteins of the bioenergetic system — convert and utilize energy from Food Products and solar radiation (e.g., rhodopsin, Cytochromes);

nutritional and storage proteins — play an important role in the development and functioning of the Organism;

protective proteins — serve as defense systems in higher organisms; these include IMMUNOGLOBULINS (responsible for Immunity), Complement proteins (responsible for the lysis of foreign Cells and the activation of immunological functions), Blood clotting system proteins (Thrombin, fibrin), and antiviral interferon.

Methods for Protein Isolation and Study

The first step in protein isolation is obtaining the relevant Organelles (Ribosomes, Cell/35.html">Mitochondria, nuclei, cytoplasmic membrane, etc.) using differential centrifugation. Proteins are then brought into a soluble state by extraction with buffered salt solutions or detergents, sometimes with non-polar solvents. To prevent Protein Denaturation, the work is usually carried out at approximately 4 °С. Protease inhibitors are used to prevent proteolysis. Some proteins are stabilized with polyols, such as glycerol. Subsequently, fractional precipitation is employed using inorganic salts (usually ammonium sulfate), ethanol, acetone, or by altering the pH, Ionic strength, or Temperature of the solution. Purification is performed according to schemes specifically developed for individual proteins. The most common methods for separating protein mixtures are Gel chromatography, Ion-exchange chromatography, adsorption chromatography, Affinity Chromatography, and High-Performance Liquid Chromatography (HPLC). The criterion for protein purity is their homogeneity during Electrophoresis, chromatography, and ultracentrifugation. Highly sensitive immunochemical methods are used to detect impurities of other proteins (up to 10³ µg/mL of antigen impurity). Impurities of co-occurring enzymes are determined using specific substrates.

Structure Determination. Knowledge of a protein's primary structure is fundamental for determining its secondary and tertiary structures, elucidating the arrangement of functional groups in the protein's Active Site, and constructing a model of its function. For proteins, the amino acid composition of polypeptide chains, N- and C-terminal amino acid residues, and the Amino Acid Sequence are determined. Amino acid composition analysis involves complete hydrolysis of the protein or peptide and Quantitative determination of all amino acids in the hydrolysate. This determination is performed using an amino acid analyzer, where the amino acid mixture is separated on ion-exchange columns, and the content is assessed spectrophotometrically by reaction with ninhydrin or fluorometrically. An important step in determining the primary structure is the Cleavage of the macromolecule into peptide fragments using Proteolytic Enzymes or chemical Reagents with substrate Specificity. For example, Trypsin hydrolyzes only bonds involving the carboxyl group of Lysine or Arginine. In some cases, Partial Acid Hydrolysis is used for Protein Cleavage. The resulting peptides are then separated based on their physicochemical properties and molecular length. For fractionating short peptides (up to 15-20 amino acid residues), ion-exchange chromatography on cation exchangers is used. Further Separation and purification

are carried out using chromatography and electrophoresis on paper or in thin layers of Cellulose or silica gel. The main difficulty in fractionating large peptides (more than 20 amino acid residues) is their tendency to form high-molecular-weight aggregates. To prevent this, urea, guanidinium chloride, or detergents are added to Buffer solutions. Separation is often performed using gel or ion-exchange chromatography, and less frequently, HPLC. Covalent chromatography, based on the formation of a covalent bond between the peptide and the support, is used for peptides with chemically active groups.

For direct Analysis of the primary structure, a Sequencer is commonly used — an instrument that performs sequential automatic cleavage and analysis of N-terminal amino acid residues. Mass spectrometry is sometimes employed for determining the amino acid sequence of peptides. In some cases, a rapid and effective method for analyzing The nucleotide sequence of DNA is used, as the Primary structure of any protein is encoded in the corresponding segment of the DNA molecule.

Various modern Analytical Methods are used to determine the Spatial Structure of Proteins, including X-ray crystallography, neutron diffraction, UV, IR, NMR, and EPR spectroscopy. These studies involve either native proteins or those modified with various reagents carrying a free radical (a "spin label"). Theoretically, the spatial architecture of a protein can be predicted in general terms based on its primary structure. Such calculations are performed using computer software based on regularities derived from the statistical Analysis of proteins with known spatial structures. In A number of cases, these computational methods yield satisfactory results that help interpret data obtained by other techniques.