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LECTURES ON PROTEINS
Introduction
Proteins are one of the four different types of macromolecules, in addition to carbohydrates, lipids, or fats, and nucleic acids, such as DNA and RNA. The word protein is obtained from the Greek word “ prōteios ”, which means holding first position. They are called proteins because they have great biological significance. Proteins are synthesized by plants and animals to play a role in their physiology. Proteins are required for the synthesis of RNA and DNA. They have a highly complex structure. They are polymers of chains of amino acids linked together by polypeptide chains. Proteins are species-specific; that is, the proteins of one species differ from those of another species. They are also organ-specific; for instance, within a single organism, muscle proteins differ from those of the brain and liver.There are 20 different amino acids. Out of these, nine amino acids cannot be synthesized by our body and these are therefore called as essential amino acids. All biological proteins can be used as food proteins. Proteins are found in each and every cell of the body. They are involved in most of the body’s functions and life processes. The sequence of amino acids in a protein is determined by the DNA.
Classification of Proteins
A-Based on composition B- Based on Structure i. Simple proteins/ Homoproteins ii. Conjugated proteins/ Heteroproteins iii. Derived proteins i. Fibrous proteins (rod shaped), eg. meat proteins ii. Globular proteins (spherical shaped), eg. enzymes I. Simple proteins (i) Albumins: Soluble in water, coagulable by heat and 1 precipitated at high salt concentrations. Examples – Serum albumin, egg albumin, lactalbumin (Milk), leucosin (wheat), legumelin (soyabeans). (ii) Globulins: Insoluble in water, soluble in dilute salt 1 solutions and precipitated by half 1 saturated salt solutions. Examples – Serum globulin, vitellin (egg yolk), tuberin (potato), myosinogen (muscle), legumin (peas).
(iii) Glutelins: Insoluble in water but soluble in dilute 1 acids and alkalis. Mostly found in plants. Examples – Glutenin (wheat), oryzenin (rice). (iv) Prolamines: Insoluble in water and absolute alcohol 1 but soluble in 70 to 80 per cent alcohol. Examples – Gliadin (wheat), zein (maize). (v) Protamines: Basic proteins of low molecular weight. 1 Soluble in water, dilute acids and alkalis, j Not coagulable by heat. Examples – Salmine (salmon sperm). (vi) Histones: Soluble in water and insoluble in very dilute ammonium hydroxide. Examples – Globin of hemoglobin and thymus histones. (vii) Scleroproteins: Insoluble in water, dilute acids and alkalis. Examples – Keratin (hair, horn, nail, hoof and feathers), collagen (bone, skin), elastin (ligament). II. Conjugated Proteins (i) Nucleoproteins: Composed of simple basic proteins (protamines or histones) with nucleic acids, found in nuclei. Soluble in water. Examples – Nucleoprotamines and nucleohistones. (ii) Lipoproteins: Combination of proteins with lipids, such as fatty acids, cholesterol and phospholipids etc. Examples – Lipoproteins of egg-yolk, milk and cell membranes, lipoproteins of blood. (iii) Glycoproteins:
They are denatured proteins formed by the action of heat. X-rays, ultraviolet rays etc. Cooked proteins, coagulated albumins. B. Secondary derivatives (i) Proteoses: Formed by the action of pepsin or trypsin. Precipitated by saturated solution of ammonium sulphate, incoagulable by heat. Examples – Albumose from albumin, globulose from globulin. (ii) Peptones: Further stage of cleavage than the proteoses. Soluble in water, incoagulable by heat and not precipitated by saturated ammonium sulphate solutions. (iii) Peptides: Compounds containing two or more amino acids. They may be di-, tri-, and porypeptides. Examples – Glycyl-alanine, leucyl-glutamic acid.
Biological Functions of Proteins
Proteins perform the following functions in the body of an organism: a- Communication via hormones (e.g.,insulin). Hormones are long-distance chemical signals released by endocrine cells (like the cells of your pituitary gland). They control specific physiological processes, such as growth, development, metabolism, and reproduction. While some hormones are steroid-based (see the article on lipids), others are proteins. These protein-based hormones are commonly called peptide hormones. b- structural (e.g., collagen in skin or keratin in hair) c- biochemical catalysts (e.g.,enzymes), d- transportation (e.g., hemoglobin to transport oxygen in the blood) e- defense (e.g., antibodies, immunoglobulins), f- storage (e.g., globulins in seeds). Very rarely do our needs from the protein match those of the plant/animal we are going to eat. Our needs are:
firstly nutritional – of the 20 amino acids common in nature, we can synthesize 1 0 of them and must gain the rest ( 10 ) from dietary sources and secondly functional – we rely on proteins to change the texture of our food for example by forming gels, or by stabilizing foams and emulsions. The only area where the proteins’ evolved function overlaps with our needs is when we use proteins as enzymes in bioprocessing (e.g., making corn syrup from the action of amylase enzymes on starch). We are also interested in how proteins interact with other food components (e.g., flavor binding) or have toxic or anti-nutritional properties (e.g., botulinum toxin is a protein).
Amino Acids
Now before we move on to the structure of proteins, it is very necessary to first know what amino acids are, because they are the main building blocks of proteins. There are 20 amino acids out of which 1 0 can be synthesized by the body and the rest are obtained from outside souces and these 09 amino acids are called as the essential amino acids. Humans can produce 10 of the 20 amino acids. The others must be supplied in the food. Failure to obtain enough of even 1 of the 10 essential amino acids, those that we cannot make, results in degradation of the body's proteins—muscle and so forth—to obtain the one amino acid that is needed. Unlike fat and starch, the human body does not store excess amino acids for later use—the amino acids must be in the food every day. The 10 amino acids that we can produce are alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine. Tyrosine is produced from phenylalanine, so if the diet is deficient in phenylalanine, tyrosine will be required as well. The essential amino acids are arginine (required for the young, but not for adults), histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. These amino acids are required in the diet. Plants, of course, must be able to make all the amino acids. Humans, on the other hand, do not have all the the enzymes required for the biosynthesis of all of the amino acids. The figure below shows the anatomy of an amino acid:
determining the structure and function of the polypeptide, and the protein it's part of. The peptide bond is broken through hydrolysis into their original amino acid. Every amino acid also has another atom or group of atoms bonded to the central atom, known as the R group, which determines the identity of the amino acid. For instance, if the R group is a hydrogen atom, then the amino acid is glycine, while if it’s a methyl (CH3) group, the amino acid is alanine. The twenty common amino acids are shown in the chart below, with their R groups highlighted in blue. The properties of the side chain determine an amino acid’s chemical behavior (that is, whether it is considered acidic, basic, polar, or nonpolar). The four groups of R-groups in amino acids are: 1 - Non-Polar or Hydrophobic R-groups
- Polar but uncharged R-groups
- Polar because of negatively charged R- groups
- Polar because of positively charged R- groups 1 - Non-Polar or Hydrophobic R-groups: These R-groups have limited solubility in water as they are hydrophobic or ‘water hating’. This group contains both aliphatic R-groups as in glycine, alanine, valine leucine, isoleucine and methionine. Aromatic or ring structures are found in phenylalanine, tryptophan and proline. Glycine Alanine (Ala)
Valine Leucine Iso-Leucine Methionine Phenylanaline Tryptophan Proline
- Polar because of negatively charged R- groups: This group includes 2-dicaboxylic acids known as glutamic acid and aspartic acid. At neutral pH their 2 carboxylic groups give a net - 1 negative charge to these compounds. Glutamic Acid Aspartic Acid
- Polar because of positively charged R- groups: This group contains lysine, arginine and histidine. Histidine contains imidazole group. They can dissociate in the pH range of 6-12. They contain a net +1 charge. Lysine Arginine Histidine
The simplest level of protein structure, primary structure , is simply the sequence of amino acids in a polypeptide chain. For example, the hormone insulin has two polypeptide chains, A and B, shown in diagram below. (The insulin molecule shown here is cow insulin, although its structure is similar to that of human insulin.) Each chain has its own set of amino acids, assembled in a particular order. For instance, the sequence of the A chain starts with glycine at the N-terminus and ends with asparagine at the C-terminus, and is different from the sequence of the B chain. B- Secondary Structure of proteins The next level of protein structure, secondary structure , refers to local folded structures that form within a polypeptide due to interactions between atoms of the backbone. (The backbone just refers to the polypeptide chain apart from the R groups – so all we mean here is that secondary structure does not involve R group atoms.) The most common types of secondary structures are the α helix and the β pleated sheet. Both structures are held in shape by hydrogen bonds, which form between the carbonyl O of one amino acid and the amino H of another.
β - PLEATED SHEETS
C- Tertiary structure of proteins The overall three-dimensional structure of a polypeptide is called its tertiary structure. The tertiary structure is primarily due to interactions between the R groups of the amino acids that make up the protein. R group interactions that contribute to tertiary structure include hydrogen bonding, ionic bonding, dipole-dipole interactions, and London dispersion forces – basically, the whole gamut of non- covalent bonds. For example, R groups with like charges repel one another, while those with opposite charges can form an ionic bond. Similarly, polar R groups can form hydrogen bonds and other dipole-dipole interactions. Also important to tertiary structure are hydrophobic interactions , in which amino acids with nonpolar, hydrophobic R groups cluster together on the inside of the protein, leaving hydrophilic amino acids on the outside to interact with surrounding water molecules. Finally, there’s one special type of covalent bond that can contribute to tertiary structure: the disulfide bond. Disulfide bonds , covalent linkages between the sulfur-containing side chains of cysteines, are much stronger than the other types of bonds that contribute to tertiary structure. They act like molecular "safety pins," keeping parts of the polypeptide firmly attached to one another. D- Quaternary structure of proteins Many proteins are made up of a single polypeptide chain and have only three levels of structure (the ones we’ve just discussed). However, some proteins are made up of multiple polypeptide chains, also known as subunits. When these subunits come together, they give the protein its quaternary structure.
When a protein loses its higher-order structure or native state (which is the most stable state), but not its primary sequence, it is said to be denatured. The protein chain unfolds due to denaturation. Denatured proteins are usually non-functional. The biological activity of proteins is lost after denaturation. For some proteins, denaturation can be reversed. Since the primary structure of the polypeptide is still intact (the amino acids haven’t split up), it may be able to re-fold into its functional form if it's returned to its normal environment. Other times, however, denaturation is permanent. One example of irreversible protein denaturation is when an egg is fried. The albumin protein in the liquid egg white becomes opaque and solid as it is denatured by the heat of the stove, and will not return to its original, raw-egg state even when cooled down. This is an example where denaturation is desirable, Partial denaturation improves the digestibility of proteins. Denaturation is also desirable in the production of yoghurt or curd. External agents that can cause denaturation are:
- Physical agents (temperature, moisture content, pressure and shear force)
- Chemical agents (pH, organic solutes, organic solvents, detergents and chaotropic salts) 1 Physical agents a) Temperature: When a protein is heated above a critical temperature, it undergoes transition from native state to denatured state. This denaturation is irreversible but at low temperature, the denaturation may occur but it is reversible. Thermal denaturation of globular proteins is mostly reversible but it becomes irreversible if the protein is exposed to high temperature for a long time. b) Moisture content: it also affects the denaturation of proteins. Dry protein powders are not denatured easily. Addition of water increases the chances of denaturation of proteins. c) Pressure: Globular proteins are more flexible as compared to fibrous proteins. The volume of proteins decreases during denaturation by pressure. Fibrous proteins have lesser air spaces, therefore they are more stable to denaturation by pressure. Denaturation by pressure is highly reversible. Once pressure is removed, proteins will again go back to thir natural state but complete regeneration will take several hours. d) Shear Force: High mechanical shear generated by shaking, whipping, kneading, etc., can cause denaturation of proteins. Flexible proteins get denatured easily by shear force. During whipping, air bubbles get absorbed into the surface of proteins. Combination of high temperature and high shear force causes irreversible denaturation of proteins.
- Chemical Agents a) pH: Proteins get denatured at low PH (acidic conditions) as well as at high pH (alkaline conditions). At extreme pH values, electrostatic repulsion occurs between the protein chain. This causes swelling and unfolding of the protein chain. Denaturation by pH is mostly reversible but sometimes it becomes irreversible. b) Organic Solutes: These organic solutes bind with protein chain and cause denaturation. Examples are urea, guanidine hydrochloride etc. which can cause denaturation of proteins. Urea causes denaturation at a concentration of 4-6 molarity and guanidine hydrochloride causes denaturation at a concentration of 3-4 molarity. c) Organic Solvents: Organic solvents affect the polar and non-polar R groups of the protein chain. They can cause denaturation at high concentration. Most of the non-polar groups of the proteins are soluble in organic solvents. d) Detergents: Detergents such as sodium dodecyl sulfates are powerful denaturation agents. They can bind with the protein chain and cause denaturation. Denaturation by detergents is irreversible because of strong binding between the protein chain and the detergent.
- Chaotropic Salts: A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules. This has an effect on the stability of the native state of other molecules in the solution, mainly macromolecules such as proteins, by weakening the hydrophobic effect. At low concentration they make the protein more stable. At high concentration they may or may not cause denaturation. F¯^ < SO 4 ¯^2 < Cl¯^ ……They make the protein more stable. Br¯^ < I¯^ < ClO 4 ¯^ < SCN¯^ < Cl 3 C¯^ < COO¯^ ………They cause denaturation.
Functional Properties of Proteins
They are those properties that affect the behavior of proteins during processing. Function Food Protein 1 Solubility Beverages Whey proteins 2 Viscosity Soups, gravy Gelatin
- Water bonding Cakes, bread Egg proteins, cereal proteins 4 Gelation (gel formation) Cheese, bakery products Egg proteins, milk proteins
Protein load: The amount of protein absorbed at the oil and water interphase is known as the protein load. After addition of protein, emulsion will be formed, oil and water phase will mix together. Emulsion Stability: Stable emulsions will not separate into oil and water phases. Emulsion stability is checked under extreme conditions such as a high centrifugal force. For testing emulsion stability, the emulsion is centrifuged at 1300 x g (or Relative Centrifugal Force ( RCF ) is the term used to describe the amount of accelerative force applied to a sample in a centrifuge. RCF is measured in multiples of the standard acceleration due to gravity at the Earth's surface (x g).) for 5 min. After that the emulsion is checked whether oil layer has separated or not. Emulsion Stability= Thickness of layer of oil/Total amount of emulsion taken x 100
- Foaming: The amount of foam produced by whipping of protein solution is called foamability. The time for which the foam remains stable is called foam stability. Foamability decreases with the addition of salt, sugar, oil, etc. At isoelectric point of protein, lesser foam will be produced but it will be very stable. Bubbling of air at high speed causes large air bubbles. At moderate speeds causes medium and small sized bubbles.
Nutritional Properties of Proteins
As we already know that essential amino acids are those amino acids which are not produced by our body but they are obtained from outside through diet. Ideal amount of amino acids which should be there in 1 g of protein: Amino Acid Wt, mg Leucine 93 Isoleucine 46 Lysine 66 Threonine 43 Tryptophan 17 Valine 55 Histidine 26 Phenylalanine + Tyrosine 72 Methionine + Tyrosine 42
Cereal grains are rich in Methionine but deficient in Lysine whereas legumes are rich in Lysine but deficient in Methionine. Therefore, cereals and legumes are consumed together to balance the amounts of Methionine and Lysine. The quality of a protein depends up on the amount of essential amino acids present in it as well as its digestibility. Animal proteins such as milk protein, egg protein, meat protein are better in duality than plant proteins. Dual proteins are formed say, by blending soy protein with milk proteins (whey and casein) and offer several advantages over individual proteins. Protein Digestibility (%) Protein Efficiency Ratio (PER) Biological Value (BV) Egg 97 3.9 94 Milk 95 3.1 84 Meat/ Fish 94 3 - 3.5 74 - 76 Rice (Polished) 88 - - Rice 75 2 73 Whole Wheat 86 1.5 65 Wheat Flour 96 - - Peas 88 2.65 - Corn cereal 70 - - Beans 78 - - Digestibility (%) = Food Nitrogen absorbed in body/ Total Nitrogen consumed x 100 Protein Efficiency Ratio (PER) = weight gain/ grams of protein consumed Net Protein Retention (NPR) = weight gain in protein receiving group- weight loss in protein free group Net Protein Utilization (NPU) = BV x D Net Protein Value (NPV) = NPU x Amount of protein in food. Protein digestibility corrected amino acid score (PDCAAS) In 1989, FAO and WHO jointly stated that the quality of a protein can be determined by calculating the percentage of content of the 1st^ limiting essential amino acid present in the test protein to the same amino acid content in a reference protein [1, 2, 10]. The reference values were based upon the requirement of essential amino acids by children of preschool age. The percentage is subsequently corrected for true fecal digestibility of test
