Chemistry of Proteins PDF

# Proteins ## Occurrence Proteins are present in every cell of humans, animals, plant tissues, tissue fluids and in micro organisms. They account for about 50% of the dry weight of a cell. The term protein is derived from the Greek word proteios meaning holding first place or rank in living matter...

# Proteins ## Occurrence Proteins are present in every cell of humans, animals, plant tissues, tissue fluids and in micro organisms. They account for about 50% of the dry weight of a cell. The term protein is derived from the Greek word proteios meaning holding first place or rank in living matter. ## Medical and Biological Importance Proteins perform wide range of essential functions in mammalians. 1. Proteins are involved in the transport of substances in the body. * Example: Haemoglobin transports oxygen. 2. Enzymes which catalyze chemical reactions in the body are proteins. 3. Proteins are involved in defence function. They act against bacterial or viral infection. * Example: Immunoglobulins. 4. Hormones are proteins. They control many biochemical events. * Example: Insulin. 5. Some proteins have role in contraction of muscles. * Example: Muscle proteins. 6. Proteins are involved in the gene expression. They control gene expression and translation. * Example: Histones. 7. Proteins serve as nutrients. Proteins are also involved in storage function. * Examples: Casein of milk, Ferritin that stores iron. 8. Proteins act as buffers. * Example: Plasma proteins. 9. Proteins function as anti-vitamins. * Example: Avidin of egg. 10. Proteins are infective agents. * Example: Prions which cause mad cow disease are proteins. 11. Some toxins are proteins. * Example: Enterotoxin of cholera microorganism. 12. Some proteins provide structural strength and elasticity to the organs and vascular system. * Example: Collagen and elastin of bone matrix and ligaments. 13. Some proteins are components of structures of tissues. * Example: a-keratin is present in hair and epidermis. In order to understand how these substances though they are all proteins play such diverse functions their structures, and composition must be explored. ## Chemical Nature of Proteins All proteins are polymers of aminoacids. The aminoacids in proteins are united through "Peptide" linkage. Sometimes proteins are also called as polypeptides because they contain many peptide bonds. ## Properties of Proteins 1. Proteins have high molecular weight, e.g., the lactalbumin of milk molecular weight is 17000 and pyruvate dehydrogenase molecular weight is $7 \times 10^6$. 2. Proteins are colloidal in nature. 3. Proteins have large particle size. 4. Different kinds of proteins are soluble in different solvents. 5. Proteins differ in their shape. 6. Some proteins yield amino acids only on hydrolysis where as others produce amino acids plus other types of molecules. 7. Charge properties: Charge of a protein depends on the surroundings like amino acids. So, by changing the pH of surroundings the charge of protein can be altered. This property is used for separation of proteins. * **Isoelectric point:** Proteins have characteristic isoelectric points. At the isoelectric point its net charge is zero because the number of positive charges are equal to number of negative charges. So proteins are insoluble or have minimum solubility at isoelectric point. This property is used for the isolation of casein from milk. The isoelectric point for casein is 4.6. If the pH of the surrounding is raised above the isoelectric point, the protein is negatively charged i.e., it exists as anion. Likewise, if the pH of the surrounding is lowered, the protein is positively charged i.e., it exist as cation. Further, proteins do not move in an electrical field at isoelectric point like amino acids. However, if the pH of the medium is raised or lowered protein moves towards anode or cathode respectively. This property is exploited for the separation of proteins. 8. Proteins act as buffers: Since proteins are amphoteric substances, they act as buffers. Hemoglobin (Hb) of erythrocytes and plasma proteins are important buffers. Hb accounts for 60% of buffering action with in erythrocytes and plasma proteins contributes to 20% of buffering action of blood. ## Classification of Proteins There is no single universally satisfactory system of protein classification so far. 1. One system classifies proteins according to their composition or structure. 2. One system classifies them according to solubility. 3. One system classifies them according to their shape. 4. Classification of proteins based on their function also found in literature. ### Classification of proteins based on their composition Proteins are divided into three major classes according to their structure. 1. **Simple proteins:** Simple proteins are made up of amino acids only. On hydrolysis, they yield only amino acids. * Examples: Human plasma albumin, Trypsin, Chymotrypsin, pepsin, insulin, soyabean trypsin inhibitor and ribonuclease. 2. **Conjugated proteins:** They are proteins containing non-protein part attached to the protein part. The non-protein part is linked to protein through covalent bond, non-covalent bond and hydrophobic interaction. The non-protein part is loosely called as prosthetic group. On hydrolysis, these proteins yield non-protein compounds and amino acids. * Conjugated protein → Protein + Prosthetic group The conjugated proteins are further classified into subclasses based on prosthetic groups. | Subclass | Prosthetic group | Examples | Type of linkage | |--------------------|-------------------|--------------------------------------------------------------------------------|----------------------| | Lipoproteins | Lipids | Various classes of Lipoproteins. Lipovitellin of egg | Hydrophobhic | | Glycoproteins | Carbohydrates | Immunoglobulins of blood, Egg albumin | Covalent | | Phosphoproteins | Phosphorus | Casein of milk, vitellin of egg yolk | Covalent | | Nucleoproteins | Nucleic acids | Chromatin, Ribosomes | Non-covalent | | Hemoproteins/ | Heme | Hemoglobin, Myoglobin, Cytochromes | Non-covalent | | Chromoproteins | | | | | Flavoproteins | Flavin nucleotides | FMN, FAD, Dehydrogenase | Covalent | | Metalloproteins | Iron | Ferritin, Cytochromes | Non-covalent | | Visual pigments | Retinal | Rhodopsin | Covalent | 3. **Derived proteins:** As the name implies this class of proteins are formed from simple and conjugated proteins. There are two classes of derived proteins. * (i) Primary derived proteins: They are formed from natural proteins by the action of heat or alcohol etc. The peptide bonds are not hydrolysed. They are synonymous with denatured proteins. * Example: Coagulated proteins like cooked-egg albumin. * (ii) Secondary derived proteins: They are formed from partial hydrolysis of proteins. * Examples: Proteoses, peptone, gelatin, and peptides. ### Proteins classification according to their solubility 1. **Albumins:** Soluble in water and salt solutions. * Examples: Albumin of plasma, egg albumin and lactalbumin of milk. 2. **Globulins:** Sparingly soluble in water but soluble in salt solutions. * Examples: Globulins of plasma, ovoglobulins of egg, lactoglobulin of milk. 3. **Glutelins:** Soluble in dilute acids and alkalies. * Examples: Glutenin of wheat, oryzenin of rice, zein of maize. 4. **Protamins:** Soluble in ammonia and water. * Examples: Salmine from salmon fish, sturine of sturgeon. 5. **Histones:** Soluble in water and dilute acids. * Example: Histones present in chromatin. 6. **Prolamines:** Soluble in dilute alcohol and insoluble in water and alcohol. * Examples: Gliadin of wheat, zein of corn. 7. **Sclero proteins:** Insoluble in water and dilute acids and alkalies. * Examples: Collagen, elastin and keratin. ### Classification of proteins based on shape Proteins are divided into two classes based on their shape. 1. **Globular proteins:** Polypeptide chain(s) of these proteins are folded into compact globular (Spherical) shape. * Examples: Haemoglobin, myoglobin, albumin, lysozyme, chymotrypsin. 2. **Fibrous proteins:** Poly peptide chains are extended along one axis. * Examples: a-keratin, ẞ-keratin, collagen and elastin. ## Protein Structure Since proteins are built from amino acids by linking them in linear fashion, it may be viewed as proteins having long chain like structures. However, such arrangement is unstable and polypeptide or protein folds to specific shape known as conformation, which is more stable. Various stages involved in the formation of final conformation from linear chain are divided into four levels or orders of protein structure. They are ### 1. Primary Structure The linear sequence of amino acid residues in a polypeptide chain is called as primary structure. Generally disulfide bonds if any are also included in the primary structure. Bonds responsible for the maintenance of primary structure are mainly peptide bonds and disulfide bonds. Both of them are covalent bonds. - A diagram showing the primary structure of insulin is presented. ### 2. Secondary Structure Folding of polypeptide chain along its long axis is called as secondary structure of protein. Folding of polypeptide chain can be ordered, disordered or random. Secondary structure is often referred as conformation. So, proteins has ordered secondary structure or conformation and random or disordered secondary structure or conformation. #### Ordered Conformation of Polypeptides The polypeptide chain of some proteins may exist in highly ordered conformation. The conformation is maintained by hydrogen bonds formed between peptide residues. - A diagram showing the secondary structure of a protein is presented. - A detailed explanation of intra chain hydrogen bonds is presented. ### 3. Tertiary Structure Three-dimensional folding of polypeptide chain is called as tertiary structure. It consists of regions of a-helices, ẞ-pleated sheet, ẞ-turns, motifs and random coil conformations. Inter-relationships between these structures are also a part of tertiary structure. Tertiary structure of a protein is mainly stabilized by non-covalent bonds. - A schematic diagram showing tertiary structure and different types of secondary structures of a protein molecule is presented. - A diagram showing the non-covalent bonds present in tertiary structure is presented: - Hydrophobic interactions - Electrostatic bonds - Internal hydrogen bonds - Vander waal's interactions ### 4. Quaternary Structure Proteins containing two or more polypeptide chains possess quaternary structure. These proteins are called as oligomers. The individual polypeptide chains are called as protomer, monomers or subunits. The protomers are united by forces other than covalent bonds. Occasionally, they may be joined by disulfide bonds. - A diagram illustrating the quaternary structure of a tetramer is presented. - A detailed explanation of the forces that stabilize these aggregates are presented: - Hydrogen bonding - Electrostatic interactions - Hydrophobic interactions - Vander waals interactions - Disulfide bridges * Examples: - Haemoglobin consist of 4 polypeptide chains. - Hexokinase contains 2 subunits. - Pryuvate dehydrogenase contains 72 subunits. ## Determination of Protein Structure The primary structure of protein directs specific folding (secondary structure) and its tertiary structure. If there is a change in one of the amino acids of protein, then conformation of polypeptide chain alters, which results change in biological function. Further, the sequence of amino acids in proteins that gives them their striking specific biological actions. Therefore knowledge of primary structure of a protein is required for the understanding of relationship of a protein's structure to its function at molecular level. ### Determination of Primary Structure of Protein 1. **Sanger's reagent** Sanger used FDNB (1-Fluoro-2, 4-Dinitrobenzene) to determine the amino acid sequence of a polypeptide chain from N-terminus. Sanger's reagent can be used to determine only one amino acid at a time because FDNB reacts with other amino acids. FDNB arylates free amino acid group and produces intense yellow 2, 4-dinitrophenyl residues of amino acids. These derivatives are separated by chromatography and identified. - A diagram depicting the Sanger's reaction is presented. 2. **Edman's reagent** Edman used phenylisothiocyanate (Edman's reagent) for the determination of amino acid sequence of a protein from the N-terminus. Edmans reagent not only identifies N-terminus but also when used repeatedly provides complete sequence of the polypeptide chain. In Edman's reaction, the polypeptide chain is shortened by only one residue and rest of the polypeptide remains intact. The reaction is repeated and second residue is determined. By continued repetition, complete sequence of protein is determined starting from N-terminus. - A diagram illustrating Edman's reaction is presented. ### Protein Folding Let us examine how polypeptide chain attains native conformation as soon as it comes out of protein synthesizing machinery. Though exact mechanisms involved in protein folding are not known due to extensive investigations carried out some information on protein folding mechanisms is available. ## Stages of Protein Folding Protein folding occurs by stages: 1. **Domains formation** a-helical, ẞ-pleated sheet, ẞ-bend containing domains are formed in the initial step of folding of polypeptide chain. This self assembling process mostly depends on primary structure. It involves extensive interaction among amino acids residues side chains of polypeptide chain. It is governed by thermodynamic principles like free energy etc. 2. **Molten globule** In the next step domains from molten globule state in which secondary structure predominates and tertiary structure is highly disordered. 3. **Native conformation** Finally native conformation develops from molten globule state after several minor conformational changes and rearrangements. 4. **Oligomer formation** In the case of multimeric or oligomeric proteins after attaining specific conformation protomers or sub-units may assemble into native like structure initially. After some realignments it ultimately gives rise to final conformation of oligomer. ## Additional Protein Folding Factors Though self association of polypeptide chain into ordered conformation is largely determined by amino acid sequence (primary structure) recent research has shown that in some cases folding of protein requires additional factors. Some of them are enzymes and some are protein factors. ### Protein Folding Enzymes Two protein folding enzymes are known: 1. **Disulfide isomerase** In the newly formed protein molecules -SH groups of cysteine residues may form several intra or inter disulfide linkages. However, only few disulfide linkages may be essential for proper protein folding. The disulfide isomerase favours formation of such disulfide linkages by breaking unwanted linkages formed. 2. **Cis-trans prolyl isomerase** It aids folding process by catalyzing inter conversion of cis-trans peptide bonds of proline residues of folding protein. ### Protein Factors #### Chaperons (Chaperonins) These proteins aid protein folding process by preventing formation of aggregates. Usually aggregate formation slows down protein folding process. Chaperons accelerate protein folding by blocking protein folding pathways of unproductive nature. They bind to hydrophobic parts of protein molecules and prevent formation of aggregates. They are also involved in protein refolding that occurs when proteins cross membrane structures. ## Denaturation of Proteins Denaturation is loss of native conformation. On denaturation, physical chemical and biological properties of a protein are altered. - A diagram outlining the process of protein denaturation is presented. - An explanation of some of the changes in properties is presented: - Decreased solubility - Unfolding of polypeptide chain - Loss of helical structure - Decreased or loss of biological activity - More susceptible to action of enzymes - Increased chemical reactivity - Dissociation of subunits in case of oligomeric proteins - An explanation outlining the causes of denaturation is presented: - High temperature - Extreme alkaline or acidic pH - Use of urea and guanidine at high concentration - UV radiation - Sonication - Vigorous shaking - Detergent like sodium dodecylsulfate also denatures protein - Treatment with organic solvents like ethanol, acetone etc. - Treatment with strong acids like trichloro acetic acid, picric acid and tungstic acid - Exposure to heavy metals like Pb2+, Ag2+ and Cu2+ - A brief overview of biomedical importance of protein denaturation is presented: - These properties are exploited for the separation of serum proteins from the other compounds of clinical importance. - Denaturation knowledge is required when activities of enzymes in biological fluids like blood are measured for diagnosis. - Purification of protein from mixture of proteins also needs denaturation properties. - Lead poisoning cases are treated with egg white to decrease toxicity of lead in the body. Many cases of the process of denaturation is irreversible. - Examples of Denaturation - When egg white is exposed to high temperature coagulum is formed because heat denatures egg albumin. The solubility of denatured protein is decreased. - Formation of coagulum when albumin is exposed to high temperature. - Heat treatment of trypsin results in loss of biological activity. - Monellin is a dimeric protein has sweet taste. On denaturation the sweet taste is lost. - Renaturation - Though denaturation is irreversible in majority of the cases, in few cases, renaturation is observed. - Example: Ribonuclease denatures on exposure to heat but come back to its native conformation when temperature is lowered. ## Plasma Proteins Plasma is non-cellular portion of blood. The total plasma protein level ranges from 6-7 gm/dl. Plasma contains many structurally and functionally different proteins. Plasma proteins are divided into two categories. 1. **Albumin:** Not precipitated by half-saturated ammonium sulfate. 2. **Globulin:** Precipitated by half-saturated ammonium sulfate. - The albumin constitutes over half of the total protein. Albumin level ranges from 3.5-5.5 gm/dl. Globulin ranges from 2-3 gm/dl. After the age of 40, albumin gradually declines with an increase in globulins. Albumin is found to be simple protein and a single entity. But globulin has been found to contain many components. Subglobulins are detected as bands on electrophoresis. They are α₁, α, β and y-globulins. Electrophoretic pattern is shown in (Fig. 3.12a). The different plasma protein bands are semi-quantitated using densitometer (Fig. 3.126). - A diagram depicting the electrophoretic pattern of plasma proteins is presented.