Enzymes and Protein Drugs

Chapter 21

Enzymes and Protein Drugs

ENZYMES

Enzymes are organic catalysts produced in the body by living organisms. They perform many complex chemical reactions that make up life processes. Enzymes are lifeless and when isolated, they still exert their characteristic catalytic effect. Their chemical composition varies, and they do show several common properties. They are colloids, soluble in water and dilute alcohol but are precipitated by concentrated alcohol. Most enzymes act best at temperatures between 35 and 40°C; temperatures above 65°C, especially in the presence of moisture, destroy them, whereas their activity is negligible at 0°C. Certain heavy metals, formaldehyde, and free iodine retard the enzymes activity. Their activity is markedly affected by the pH of the medium in which they act or by the presence of other substances in this medium. They are highly selective in their action.

The enzymes are proteins having molecular weight from about 13,000 to 8,40,000. At present they are divided according to their action by a complex system established by the Commission on Enzymes of the International Union of Biochemistry. Six major classes are recognized; each has 4 to 13 subclasses, and each enzyme is assigned a systematic code number (B.C.) composed of 4 digits. The major classes.

Enzymes are found in combination with inorganic or organic substances that have an important part in the catalytic action. If these are nonprotein organic compounds, they are known as coenzymes. If they are inorganic ions, they are referred to as activators. Coenzymes are integral components of a large number of enzyme systems. Several vitamins (thiamine, riboflavin, nicotinic acid) have a coenzymatic function.

Enzymes are obtained from plant and animal cells, and many have been purified. They are used as therapeutic agents and as controlling factors in certain chemical reactions in industry. Pepsin, pancreatin, and papain are used therapeutically as digestants. Hyaluronidase facilitates thediffusion of injected fluids. Streptokinase and streptodornase dissolve clotted blood and purulent accumulations. Zymase and rennin are used in the fermentation and cheese industries; and penicillinase inactivates the various penicillins.

The names used to designate enzymes usually end in -ase or -in. The important enzymes are given hereunder.

Properties of Enzymes

Enzymes are sensitive to heat and are denatured by excess heat or cold, i.e. their active site becomes permanently warped, thus the enzyme is unable to form an enzyme substrate complex. This is what happens when you fry an egg, the egg white (augmentin, a type of protein, not an enzyme), is denatured.

Enzymes are created in cells but are capable of functioning outside of the cell. This allows the enzymes to be immobilized, without killing them.

Enzymes are sensitive to pH, the rate at which they can conduct reaction is dependent upon the pH of where the reaction is taking place, for example, pepsin in the stomach has an optimum pH of about 2, whereas salivary amylase has an optimum pH of about 7.

Enzymes are reusable and some enzymes are capable of catalysing many hundreds of thousands of reactions, for example, catalase working on hydrogen peroxide, try putting some liver into hydrogen peroxide.

Enzymes will only catalyse one reaction, for example, invertase will only produce glucose and fructose, when a glucose solution is passed over beads of enzyme.

Enzymes are capable of working in reverse, this act as a cut off point for the amount of product being produced. If there are excess reactants, the reaction will keep going and be reversed, so that there is no overload or build up of product.

DIASTASE

Synonym

Amylase

Biological Source

It is an amylolytic enzymes present in the saliva (salivary diastase or ptyalin and pancreatic diastase or amylopsin) found in the digestive tract of animals and also in malt extract. Diastase hydrolyses starch, glycogen and dextrin to form in all three instances glucose, maltose, and the limit-dextrin. Salivary amylase is known as ptyalin; although humans have this enzyme in their saliva, some mammals, such as horses, dogs, and cats, do not. Ptyalin begins polysaccharide digestion in the mouth; the process is completed in the small intestine by the pancreatic amylase, sometimes called amylopsin. The amylase of malt digests barley starch to the disaccharides that are attacked by yeast in the fermentation process.

Description

Uses

It is used as a digestant, used in the production of predigested starchy foods and also for the conversion of starch to fermentable sugars in fermentation.

PEPSIN

Biological Source

It is the enzyme prepared from the mucous membrane of the stomach of various animals like pig, sheep, or calf. The commonly used species of pig is Sus scrofa Linn, belonging to family Suidae.

The stomach consists of an outer muscular layer and an inner mucous layer. The inner surface is covered with a single layer of epithelial cells which also lines the piths present on them. The piths are about 0.2 mm in diameter, and each pith has two to three narrow tubular ducts opening at the base. The epithelial layer is made of either the parietal cell or the central cell. The central cells are mainly covered with almost cubical shape and secrete pepsinogen and rennin zymogen, whereas the parietal cells are round or oval shaped cells, and they secrete the hydrochloric acid to activate the zymogen to produce rennin and pepsin. Pepsin is the first in a series of enzymes that digest proteins. Pepsin binds with protein chains and breaks it up into small pieces. Pepsin cleaves proteins preferentially at carboxylic groups of aromatic amino acids such as phenylalanine and tyrosine but does not cleave at bonds containing amino acids like valine or alanine. Pepsin mainly cleaves C-terminal to F, L, and E, and it does not cleave at V-, A-, or G-terminals. Structurally, the active site is located in a deep cleft within the molecule. Optimal activity of pepsin is at pH of 1.8 –3.5, depending on the isoform. They are reversibly inactivated at about pH 5 and irreversibly inactivated at pH 7–8.

Preparation

The mucous membrane is separated from the stomach either by the process of stripping or it is scrapped off, and it is placed in acidified water for autolysis at 37°C for 2 hours. The liquid obtained after autolysis consist of both pepsin and peptone. It is then filtered and sodium or ammonium salts are added to the liquid till it is half saturated. At this point only the pepsin separates out, and the peptone remains in the solution. The precipitates are collected and subjected to dialysis for the separation of salts. Remaining amount of pepsin if any in the aqueous solution is precipitated by the addition of alcohol into it. The pepsin is collected and dried at low temperature.

Description

Pepsin occurs in pale yellow colour, they are odourless or with very faint odour, translucent grains and slightly bitterin taste. It is soluble in dilute acids, water, and physiological salt (NaCl) solution. It is best active at a temperature of 40°C with pH 2–4. Pepsin is unstable above pH 6. The enzyme gets denatured at a temperature of 70°C and in the presence of alcohol and sodium chloride. Pepsin can be stored for 1–2 years at 2–8°C.

Uses

It is used in the deficiency of gastric secretion. Pepsin is also used in the laboratory analysis of various proteins; in the preparation of cheese, and other protein-containing foods.

PANCREATIN

Pancreatin is a digestive enzyme extracted from the pancreas of certain animals like hog, Sus scrofa (Suidae), or ox, Bos taurus (Bovidae) that is used to supplement loss of or low digestive enzymes, often used in people with cystic fibrosis. It is also known as pancreatinum and pancreatic enzymes.

Pancreatin is made up of the pancreatic enzymes trypsin, amylase, and lipase. Pancreatin is very similar to another enzyme known as pancrelipase. The primary difference between these two enzymes is that pancrelipase contains more active lipase enzyme than pancreatin. The trypsin found in pancreatin works to hydrolyse proteins to oligopeptides, amylsas hydrolyses starches to oligosaccharides and the disaccharide maltose, and lipase hydrolyses triglycerides to fatty acids and glycerols.

Pancreatin is an effective enzyme supplement for replacing missing pancreatic enzymes used in a number of essential body processes.

Pancreatin enzymes have two important functions in the body: digestion of foods and routine cancer eradication. Pancreatin is a mix of many different enzymes and those involved in the digestion of proteins are also used to help eliminate cancers that occur. Cancer is often a disease of protein metabolism because the pancreatin enzyme cancer defence mechanism can be overwhelmed by consuming protein rich foods at inappropriate times or in excessive amounts. The body needs a time span each day approaching 12 hours or more without protein consumption for its pancreatin cancer defence mechanism to work optimally. Pancreatin enzymes can be made ineffective by contact with acids or alcohols. A diet comprised mostly of refined foods and meats may result in an acidic body chemistry that depletes these enzymes. Cancer, once established, ensures its survival by continuously generating acid as it inefficiently metabolizes food. Consuming alcoholic beverages can also interfere with the defence mechanism. Many popular cosmetics that contain acids or alcohols are a special concern for skin cancer. Mercury leakage from amalgam tooth fillings is also debilitating too many enzyme functions. It has also been claimed to help with food allergies, celiac disease, autoimmune disease, cancer, and weight loss.

TRYPSIN

Biological Source

Trypsin is a proteolytic enzyme produced by Ox pancreas, Bos taurus, belonging to family Bovidae.

It is one of the three principal digestive proteinases which along with other proteinases like pepsin and chymotrypsin break the dietary protein molecules to their amino acids and peptide component. Trypsin cleaves proteins at the carboxyl side like ‘C-terminals’ of the basic amino acids lysine and arginine. Trypsin is an endopeptidase which cleavage occurs within the polypeptide chain and not the terminal amino acids located at the ends of polypeptides. The aspartate residue located in trypsin is responsible for attracting and stabilizing positively charged lysine and/or arginine.

Production

Trypsin is produced by pancreas in the form of trypsinogen. Trypsin is then transported to the small intestine, where the proteins are cleaved into polypeptides and amino acids. As trypsin is an autocatalytic enzyme, it by itself catalyses the conversion of trypsinogen to trypsin. Another enzyme (enterokinase) is also required in small amount to catalyse the initial reaction of trypsinogen to trypsin.

Process of digestion by trypsin gets started in stomach and is continued to the small intestine where the environment is slightly alkaline. Trypsin has maximum enzymatic activity at pH 8.

Chemical Composition

It has a similar structure as that of other pancreatic proteinase like chymotrypsin and also has the similar mechanisms of action. They differ only in their specificity. Trypsin is active against peptide bonds in protein molecules that have carboxyl groups donated by amino acids like the arginine and lysine, whereas chymotrypsin are active against the carboxyl group denoted by tyrosine, phenylalanine, tryptophan, methionine, and leucine. Trypsin is considered the exceptional of all other proteolytic enzyme due to its attack on restricted number of chemical bonds. Trypsin is widely employed as a reagent for the orderly and unambiguous cleavage of proteins in which amino-acid sequence is to be determined.

Uses

In a tissue culture lab, trypsin is used to resuspend cells adherent to the petri dish wall during the process of harvesting cells. It is also used to harvest corn and oats. Trypsin is vital in a cow’s diet, without it they would not be able to digest the grass they eat.

HYALURONIDASE

Synonym

Spreading factor, hyalase.

Biological Source

Hyaluronidase is an enzyme product prepared from mammalian testes which shows the capability of hydrolysing hyaluronic acid like mucopolysaccharides. Skin is considered as the largest store of hyaluronidase in the body.

Preparation

Hyaluronidase enzyme is found in type-II Pneumococci, in group A and C hemolytic streptococci, S. aureus, and Clostridium welchii. Hyaluronidase manufacturers define their product in terms of turbidity reducing (TR) units or in viscosity units. Prepared solution for injection usually contains 150 TR units or 500 viscosity units dissolved in 1 ml. of isotonic NaCl solution.

Characteristics

Hyaluronidase for injection consists of not more than 0.25 μg of tyrosine for each USP hyaluronidase unit. Due to its action on hyaluronic acid, it promotes diffusion and hastens absorption of subcutaneous infusions. It depolymerises and catalyses hyaluronic acid and similar hexosamine-containing polysaccharides.

Chemical Constituents

Hyaluronidases are a group of enzymes such as 4-lycanohydrolase, hyaluronate 3-glycanohydrolase, and hyaluronate lyase. They are mucopeptides composed of alternating N-acetylglucosamine and glucuronic acid residues. Hyaluronidases catalyse the breakdown of hyaluronic acid.

Uses

Hyaluronidase for injection is used in the conditions of hypodermoclysis. It is used as a spreading and diffusing agent. It promotes diffusion, absorption, and reabsorption

UROKINASE

Synonym

Uroquinase.

Biological Source

Urokinase is serine protease enzyme isolated from human urine and from human kidney cells by tissue culture or by recombinant DNA technology.

Preparation

Urokinase is a fibrinolytic enzyme produced by recombinant DNA using genetically manipulated E. coli cells. It is produced firstly as prourokinase q.v. and then converted to active form by plasmin or kallikrein. Urokinase used medicinally is also purified directly from human urine. It binds to a range of adsorbents such as silica gel or kaolin which can be use to initially concentrate and purify the product. It can be further purified by precipitation with sodium chloride or ethanol or by chromatography. Human urokinase needs sterile filtration, a septic filling and freeze drying.

Characteristics

Urokinase enzyme occurs in two different forms as single and double polypeptide chain forms. It has a half-life of 10–16 minutes after intravenous administration. These enzymes act on an endogenous fibrinolytic system.

Chemical Constituents

Urokinase enzymes are serine proteases that occur as a single low molecular weight (33 kDa) and double, high molecular weight (54 kDa) polypeptide chain forms. They differ in molecular weight considerably. A single chain is produced by recombinant DNA technique and is known as SCUPA.

Uses

Urokinase is used in the treatment of pulmonary embolism, coronary artery thrombosis and for restoring the potency of intravenous catheters. It is generally administered intravenously in a dose of 4,400 units/kg body weight per hour for twelve hours.

STREPTOKINASE

Synonym

Estreptokinase, plasmin kinase.

Biological Source

Estreptokinase, plasmin kinase is a purified bacterial protein produced from the strains of group C β-hemolytic S. griseus.

Preparation

Streptokinase is a bacterial derived enzyme of serine protease group. The ancestral protease activity lies within the first 230 amino-acid residues at the N-terminal part of the protein that evolves from serine protease due to the replacement of histamine at 57th amino acid by glycine. The amino terminal residue polypeptide chain shows sequence homology to serine protease. Duplication and fusion of gene generate an ancestral streptokinase gene. Streptokinase is produced by fermentation using streptococcal culture and is isolated from the culture filtrate. It is produced in the form of a lyophilized powder in sterile vials containing 2,50,000 to 7,50,000 IUs.

Characteristics

Streptokinase is a bacterial protein with half-life of 23 minutes. Its anisolylated plasminogen activator complex (APSAC) has a higher half-life of six hours.

Chemical Constituents

Streptokinase is the purified bacterial protein with about 484 amino-acid residues.

Uses

Streptokinase is the first available agent for dissolving blood clots. It binds to plasminogen in a 1:1 ratio and changes molecular conformation. Thus, the complex formed becomes an active enzyme and promotes the activity of fibrinolytic enzyme plasmin. Plasmin breaks fibrin clots. Anistreptase or the anisolylated plasminogen streptokinase activator complex (APSAC) can also be used in a similar way for degrading blood clots. Streptokinase and anistreptase are both used in the treatment of pulmonary embolism, venous, and arterial thrombosis and coronary artery thrombosis. It is also sometimes administered along with heparin to counter act a paradoxical increase in local thrombin.

BROMELIN

Synonyms

Bromelin, bromelain.

Biological Source

Bromelin is a mixture of proteolytic enzymes isolated from the juice of Ananas comosus (pineapple), belonging to family Bromeliaceous.

Geographical Source

Pineapple is a native of tropical America. It is grown in almost all parts of the world including India, China, Thailand, United States, Brazil, Philippines, Mexico, Hawaii, and Taiwan.

Cultivation, Collection, and Preparation

Bromelin is found in pineapple fruit juice and stem. Pineapple is perennial, and it does not have a natural period of dormancy. It is propagated through suckers, slips, and crowns. In India it is planted in August, the plant generally flowers in February–March, and the fruit ripens during July–October.

The fruits must be left on the plant to ripen for the full flavors to develop. Dark green unripe fruits gradually change to yellow and finally to deep orange. The fruits are cut off. The enzyme bromelain does not disappear as the fruit ripens. The enzyme from fruit and stem are known as fruit bromalin and stem bromalin, respectively. It is isolated from pineapple juice by precipitation with acetone and also with ammonium sulphide.

Characteristics

The optimum pH of bromelain is 5.0–8.0. In solution pH below 3.0 and above 9.5 inactivates the enzyme. The optimum temperature is between 50 and 60°C, still it is effective between 20 and 65°C too. The moisture content should not exceed 6%. It is obtained in light browncoloured powder.

Chemical Constituents

Bromelain is not a single substance, but rather a collection of enzymes and other compounds. It is a mixture of sulphur-containing protein-digesting enzymes, called proteolytic enzymes or proteases. It also contains several other substances in smaller quantities, including peroxidase, acid phosphatase, protease inhibitors, and calcium.

Uses

Bromelain is an effective fibrinolytic agent; bromelain inhibits platelet aggregation and seems to have both direct as well as indirect actions involving other enzyme systems in exerting its antiinflammatory effect. Antibiotic potentiation is one of the primary uses of bromelain in several foreign countries; it can modify the permeability of organs and tissues to different drugs. The potentiation of antibiotics and other medicines by bromelain may be due to enhanced absorption, as well as increased permeability of the diseased tissue which enhances the access of the antibiotic to the site of the infection. It is also thought that the use of bromelain may provide a similar access to specific and nonspecific components of the immune system, therefore, enhancing the body’s utilization of its own healing resources. Bromelain has been used successfully as a digestive enzyme following pancreatectomy, in cases of exocrine pancreas insufficiency and in other intestinal disorders. Research has indicated that bromelain prevents or minimizes the severity of angina pectoris and transcient ischemic attacks (TIA); it is useful in the prevention and treatment of thrombosis and thrombophlebitis. If administered for prolonged time periods, bromelain also exerts an antihypertensive effect in experimental animals. It may even be useful in the treatment of AIDS to stop the spread of HIV. It has no major side effects, except for possible allergic reactions.

SERRATIOPEPTIDASE

Synonym

Serrapeptase, serrati peptidase.

Biological Source

Serrati peptidase is a proteolytic enzyme isolated from nonpathogenic enterobacteria Serratia E 15. It is also produced by the larval form of the silk moth.

Preparation

Serratiopeptidase is produced by fermentation technology by using nonpathogenic enterobacteria species such as Serratia E 15. The larvae of silk moth produce this enzyme in their intestine to break down cocoon walls. It can thus be obtained from the silk moth larvae.

Characteristics

Serratiopeptidase is very much vulnerable to degradation in the acidic pH. When consumed in unprotected tablet or capsule, it is destroyed by acid in stomach. However enteric coated tablets facilitate its absorption through intestine. One unit of the enzyme hydrolyses casein to produce colour equivalent to 1.0 μmol of tyrosine per minute at pH 7.5 and 35°C.

Chemical Constituents

Serratiopeptidase is a proteolytic enzyme of protease type XXVI. The preparation contains 7.1 units/mg solid.

Uses

Serratiopeptidase is the most widely prescribed antiinflammatory enzyme in developed countries and also in India. It eliminates inflammatory oedema and swelling, accelerate liquefaction of pus and sputum, and enhance the action of antibodies. It is also used as a fast wound healing agent. It is proving to be a superior alternative to the nonsteroidal antiinflammatory drugs traditionally used to treat rheumatoid arthritis and osteoarthritis. It has wide ranging applications in trauma surgery, plastic surgery, respiratory medicine, obstetric and gynaecology

PAPAIN

Synonyms

Papayotin, vegetable pepsin, tromasin, arbuz.

Biological Source

Papain is the dried and purified latex of the green fruits and leaves of Carica papaya L., belonging to family Caricaceae.

The plant is cultivated in Sri Lanka, Tanzania, Hawai, and Florida. The plant is 5–6 m in height bearing fruits of about 30 cm length and a weight up to 5 kg. The epicarp adheres to the orange-coloured, fleshy sarcocarp, which surrounds the central cavity. This cavity contains a mass of nearly black seeds.

Preparation

It is distributed throughout the plant, but mostly concentrated in the latex of the fruit.

The latex is obtained by making two to four longitudinal incisions, about 1/8 inch deep, on the surface on four sides of nearly mature but green fruits while still on the tree. The incisions are made early in the morning, at intervals of three to seven days. The latex flows freely for a few seconds but soon coagulates. The exudate is collected in nonmetallic containers. The latex is dried as soon as possible after collection. Rapid drying or exposure to sun or higher temperature above 38°C produce dark colour product with weak in proteolytic activity. The use of artificial heat yields the better grade of crude papain. The final product should be creamy white and friable. It is sealed in air-tight containers to prevent loss of activity. If 10% common salt or 1% solution of formaldehyde is added before drying, the product retains its activity for many months.

Fully grown fruits give more latex of high enzyme potency than smaller or immature fruits. The yield of Papain varies from 20 to 250 g per tree. The yield of commercial Papain from latex is about 20%.

Characteristics

Papain occurs as white or greyish-white, slightly hygroscopic powder. It is incompletely soluble in water and glycerol. It may digest about 35 times its weight of lean meat. Best grades render digestion of 200–300 times their weight of coagulated egg albumin in alkaline media. A temperature range of 60–90°C is fav
ourable for the digestive process with 65° the optimum point. Best pH is 5.0, but it functions also in neutral or alkaline media. It is activated by reduction (HCN and H2 S) and inactivated by oxidation (H2 O2 , iodoacetate).

Chemical Constituents

Papain contains several enzymes such as proteolytic enzymes peptidase I capable of converting proteins into dipeptides and polypeptides, rennin-like enzyme, clotting enzyme similar to pectase and an enzyme having a feeble activity on fats.

The enzymes, papain, papaya proteinase, and chymopapain, have been isolated in crystalline form from the latex. Papain is atypical protein digesting enzyme with isoelectric point. It contains 15.5% nitrogen and 1.2% sulphur. Crystalline papain is most stable in the pH range 5–7 and is rapidly destroyed at 30°C below pH 2.5 and above pH 12. Papain is a protein of 212 amino acids and having a molecular weight of about 23,000 daltons. It is resistant to heat, inactivated by metal ions, oxidants and reagents which react with thiols, and is an endopeptidase activated by thiols and reducing moieties, for example, cysteine, thiosulphate, and glutathione.

The leaves possess dehydrocarpaines I and II, fatty acids, carpaine, pseudocarpaine, and carotenoids.

The fruits yield lauric, myristoleic, palmitoleic and arachidic acids, malonated benzyl-p-o-glucosides, 2-phenyl ethyl glucoside, and 4-hydroxy-phenyl-2-ethyl glucoside.

Uses

Papain is used to prevent adhesions; in infected wounds; internally as protein digestant, as anathematic (nematode), to relieve the symptoms of episiotomy (incision of vulva), in meat industry for tenderizing beef, for treatment of dyspepsia, intestinal and gastric disorders, and diphtheria, for dissolving diphtheria membrane; in surgery to reduce incidence of blood clots where thromboplasma is undesirable and for local treatment of buccal, pharyngeal, and laryngeal disorders.

It is used in digestive mixtures, liver tonics, for reducing enlarged tonsils, in prevention of postoperative adhesions, curbuncles, and eschar burns. It is an allergic agent causing severe paroxysmal cough, vasomotor rhinitis and dyspnea. It is a powerful poison when injected intravenously. In industry it is used in the manufacture of proteolytic preparations of meat, lever, and casein, with dilute alcohol and lactic acid as meat tenderizer, as a substitute for rennet in cheese manufacture, in brewing industry for making chillproof bear, for degumming natural milk, in preparation of toothpastes and cosmetics, in tanning industry for bathing skin and hides, and as an ingredient in cleansing solutions for soft contact lenses.

Test

Papain is reacted with a gelatin solution at 80°C in the presence of an activating cysteine chloral hydrate solution for an hour. The solution is cooled to 4°C for long time. The treated solution must not regel in comparison to a blank solution under identical conditions.

Adulteration

Commercial papain is often adulterated with arrowroot starch, dried milk of cactus, gutta percha, rice flour, and pepsin.

PROTEINS

A protein is a complex, high molecular weight organic compound that consists of amino acids joined by peptide bonds. The word protein is derived from greek ‘protos’ meaning ‘of primary importance’. Proteins are essential to the structure and function of all living cells. Many proteins are enzymes or subunits of enzymes. Other proteins play structural or mechanical roles, such as those that form the struts and joints of the cytoskeleton, serving as biological scaffolds for the mechanical integrity and tissue signalling functions.

They are obtained from both plant and animal sources. In plants they are stored in the form of aleurone grains. In animals they are present in structural material in the form of collagen (connective tissue), keratin (hair, wool,hairs, feathers, and horns), elastin (epithelial connective tissue), casein (milk), and plasma proteins. Casein, gelatin, heparin, and hemoglobin are pharmaceutically important proteins of animal origin.

Proteins are generally large molecules, having molecular masses of up to 3,000,000 (the muscle protein titin has a single amino-acid chain 27,000 subunits long). However, protein masses are generally measured in kiloDaltons (kDa). Such long chains of amino acids are almost universally referred to as proteins, but shorter strings of amino acids are referred to as ‘polypeptides’, ‘peptides’, or rarely, ‘oligopeptides. The dividing line is undefined, though ‘polypeptide’ usually refers to an amino-acid chain lacking tertiary structure which may be more likely to act as a hormone (like insulin), rather than as an enzyme (which depends on its defined tertiary structure for functionality).

There are about 20 different amino acids, eight of which must be present in the diet. The eight essential amino acids required by humans are: leucine, isoleucine, valine, threonine, methionine, phenylalanine, tryptophan, and lysine. For children, histidine is also considered to be an essential amino acid. Unlike animal proteins, plant proteins may not contain all the essential amino acids in the necessary proportions, and so the proteins derived from plants are grouped as incomplete and from animals are grouped as complete. However, a varied vegetarian diet means a mixture of proteins are consumed, the amino acids in one protein compensating for the deficiencies of another.

The structure of protein could be differentiated into four types:

Primary structure: the amino-acid sequence
Secondary structure: highly patterned substructures– alpha helix and beta sheet–or segments of chain that assume no stable shape. Secondary structures are locally defined, meaning that there can be many different secondary motifs present in one single protein molecule.
Tertiary structure: the overall shape of a single protein molecule; the spatial relationship of the secondary structural motifs to one another
Quaternary structure: the shape or structure that results from the union of more than one protein molecule, usually called protein subunits in this context, which function as part of the larger assembly or protein complex.

Proteins are sensitive to their environment. They may only be active in their native state, over a small pH range, and under solution conditions with a minimum quantity of electrolytes. A protein in its native state is described as folded and that is not in its native state is said to be denatured. Denatured proteins generally have no well-defined secondary structure. Many proteins denature and will not remain in solution in distilled water also they are denatured due to heat, changes in pH, treatment of organic solvents or by ultra violet radiation.

Proteins are essential for growth and repair. They play a crucial role in virtually all biological processes in the body. All enzymes are proteins and are vital for the body’s metabolism. Muscle contraction, immune protection and the transmission of nerve impulses are all dependent on proteins. Proteins in skin and bone provide structural support. Many hormones are proteins. Protein can also provide a source of energy. Generally the body uses carbohydrate and fat for energy but when there is excess dietary protein or inadequate dietary fat and carbohydrate, protein is used. Excess protein may also be converted to fat and stored. The important proteins are given hereunder.

MALT EXTRACT

Synonym

Diastase, malt extract.

Biological Source

Malt extract is the extract obtained from the dried barley grains of one or more varieties of Hordeum vulgare Linne, family Poaceae.

Geographical Source

Barley is widely cultivated throughout the world. The major producers are United States, Russia, Canada, India, and Turkey. It is also cultivated in highlands of China and Tibet.

Cultivation, Collection, and Preparation

Barley is one of the oldest cultivated cereals. It is an annual erect stout herb resembling wheat. The crop becomes ready for harvest in about four months after sowing. The grains are threshed out by beating with sticks or trampling by oxen. Dried barley grains are artificially germinated by keeping their heaps wet with water in a warm room. When the caulicle of the grains starts protruding out, the germinated grams are dried. Dry germinated barley or dry malt is subjected to extraction. The malt is infused with water at 60°C. An infusion is concentrated below 60°C under reduced pressure and then dried. Less purified malt extract contains sugars, and amylolytic enzymes. Its further purification affords diastase

Characteristics

Malt extract contains enzymes, which are most active in neutral solution. The acidic conditions destroy the activity. It converts starch into disaccharide maltose. The enzyme is destroyed by heat. Many heat sterilized malt extracts do not contain diastase. It is completely soluble in cold water, more readily in warm water. The aqueous solution shows flocculant precipitate on standing. Limit for arsenic should not exceed one part per million.

Chemical Constituents

Malt extract contains dextrin, maltose, traces of glucose and about 8% of amylolytic enzyme diastase.

Uses

Malt extract and purified diastase, both are used as amylolytic enzymes and as an aid in digesting starch. They are used as bulk producing laxatives.

GELATIN

Synonyms

Gelfoam; puragel; gelatinum.

Biological Source

Gelatin is a protein derivative obtained by evaporating an aqueous extract made from bones, skins, and tendons of various domestic animals. Some important sources are Ox, Bos taurus, and Sheep, Ovis aries belonging to family Bovidae.

Preparation

The process of manufacture of gelatin vary from factory to factory. However, the general outline of the process is given below.

Raw material

Bones, skins, and tendons of Bovideans is collected and subjected to liming operation.

Liming Process

The raw material is first subjected to the treatment known as ‘liming’. In this process, the skins and tendons are steeped for fifteen to twenty and sometimes for 40 days in a dilute milk of lime. During this, fleshy matter gets dissolved, chondroproteins of connective tissues gets removed and fatty matter is saponified. The animal skin is further thoroughly washed in running water.

Defattying

In case of bones, the material is properly ground and defatted in close iron cylinders by treatment with organic solvents such as benzene. The mineral and inorganic part of the bone is removed by treatment with hydrochloric acid.

Extraction

The treated material from bones, skins and tendons is boiled with water in open pans with perforated false bottom. This process can also be carried out under reduced pressure. The clear liquid runs of again and again and is evaporated until it reaches to above 45 per cent gelatin content.

Setting

The concentrated gelatin extract is transferred to shallow metal trays or trays with glass bottom. It is allowed to set as a semisolid jelly.

Drying

The jelly is transferred to trays with a perforated wire netting bottom and passed through series of drying compartments of 30–60°C increasing each time with 10°C. About a month is taken for complete drying.

Bleaching

In case of darker colour, finished product is subjected to bleaching by sulphur dioxide. Bleaching affords a light coloured gelatin.

Characteristics

Gelatin occurs as a colourless or slightly yellow, transparent, brittle, practically odourless, tasteless sheet, flakes or course granular powder. In water it swells and absorbs 5–10 times its weight of water to form a gel in solutions below 35–40°C. It is insoluble in cold water and organic solvents, soluble in hot water, glycerol, acetic acid; and is amphoteric. In dry condition it is stable in air, but when moist or in solution, it is attacked by bacteria. The gelatinizing property of Gelatin is reduced by boiling for long time. The quality of gelatin is determined on the basis of its jelly strength (Bloom strength) with the help of a Bloom gelometer. Jelly strength is used in the preparation of suppositories and pessaries.

Commercially two types of gelatin, A and B, are available. Type A has an isoelectric point between pH 7 and 9. It is incompatible with anionic compounds such as Acacia, Agar and Tragacanth. Type B has an isoelectric point between 4.7 and 5, and it is used with anionic mixtures. Gelatin is coloured with a certified colour for manufacturing capsules or for coating of tablets. It may contain various additives.

Chemical Constituents

Gelatin consists of the protein glutin which on hydroysis gives a mixture of amino acids. The approximate amino-acid contents are: glycine (25.5%), alanine (8.7%), valine (2.5%), leucine (3.2%), isoleucine (1.4%), cystine and cysteine (0.1%), methionine (1.0%), tyrosine (0.5%), aspartic acid (6.6%), glutamic acid (11.4%), arginine (8.1%), lysine (4.1%), and histidine (0.8%). Nutritionally, gelatin is an incomplete protein lacking tryptophan. The gelatinizing compound is known as chondrin and the adhesive nature of gelatin is due to the presence of glutin.

Chemical Tests

Biuret reaction: To alkaline solution of a protein (2 ml), a dilute solution of copper sulphate is added. A red or violet colour is formed with peptides containing at least two peptide linkages. A dipeptide does not give this test.

Xanthoproteic reaction: Proteins usually form a yellow colour when warmed with concentrated nitric acid. This colour becomes orange when the solution is made alkaline.

Millon’s reaction: Millon’s reagent (mercuric nitrate in nitric acid containing a trace of nitrous acid) usually yields a white precipitate on addition to a protein solution which turns red on heating.

Ninhydrin test: To an aqueous solution of a protein an alcoholic solution of ninhydrin is added and then heated. Red to violet colour is formed.

On heating gelatin (1 g) with soda lime, smell of ammonia is produced.

A solution of gelatin (0.5 g) in water (10 ml) is precipitated to white buff coloured precipitate on addition of few drops of tannic acid (10%). 7. With picric acid gelatin forms yellow precipitate.

Uses

Gelatin is used to prepare pastilles, pastes, suppositories, capsules, pill-coatings, gelatin sponge; as suspending agent, tablet binder, coating agent, as stabilizer, thickener and texturizer in food; for manufacturing rubber substitutes, adhesives, cements, lithographic and printing inks, plastic compounds, artificial silk, photographic plates and films, light filters for mercury lamps, clarifying agent, in hectographic matters, sizing paper and textiles, for inhibiting crystallization in bacteriology, for preparing cultures and as a nutrient.

It forms glycerinated gelatin with glycerin which is used as vehicle and for manufacture of suppositories. Combined with zinc, it forms zinc gelatin which is employed as a topical protectant. As a nutrient, Gelatin is used as commercial food products and bacteriologic culture media.

CASEIN

Biological Source

Casein is a proteolytic enzyme obtained from the stomachs of calves. It is extracted from the proteins of the milk; in the milk, casein is structured in voluminous globules. These globules are mainly responsible for the white colour of the milk. According to various species, the casein amount within the total proteins of the milk varies.

The casein content of milk represents about 80% of milk proteins. The principal casein fractions are alpha (s1)and alpha (s2)-caseins, β-casein and κ-casein. The distinguishing property of all casein is their low solubility at pH 4.6. The common compositional factor is that caseins are conjugated proteins, most with phosphate group(s) esterified to serine residues. These phosphate groups are important to the structure of the casein micelle. Calcium binding by the individual caseins is proportional to the phosphate content.

Within the group of caseins, there are several distinguishing features based on their charge distribution and sensitivity to calcium precipitation:

Alpha (s1)-casein: (molecular weight 23,000; 199 residues, 17 proline residues)

Two hydrophobia regions, containing all the proline residues, separated by a polar region, which contains all but one of eight phosphate groups. It can be precipitated at very low levels of calcium.

Alpha (s2)-casein: (molecular weight 25,000; 207 residues, 10 prolines).

Concentrated negative charges near N-terminus and positive charges near C-terminus. It can also be precipitated at very low levels of calcium.

β-casein: (molecular weight 24,000; 209 residues, 35 prolines).

Highly charged N-terminal region and a hydrophobia C-terminal region. Very amphiphilic protein acts like a detergent molecule. Self-association is temperature-dependent; will form a large polymer at 20°C but not at 4°C. Less sensitive to calcium precipitation.

κ-casein: (molecular weight 19,000; 169 residues, 20 prolines).

Very resistant to calcium precipitation, stabilizing other caseins. Rennet cleavage at the Phe l05 – Met l06 bond eliminates the stabilizing ability, leaving a hydrophobia portion, para- κ-casein and a hydrophilic portion called κ-casein glycomacropeptide (GMP), or more accurately, caseinomacropeptide (CMP).

Characteristics

The isoelectric point of casein is 4.6. The purified protein is water insoluble. While it is also insoluble in neutral salt solutions, it is readily dispersible in dilute alkalis and in salt solutions such as sodium oxalate and sodium acetate. Casein does not coagulate on heating. It is precipitated by acids and by a proteolytic enzyme (rennet).

Chemical Constituents

Milk consists of 80% of milk proteins (casein). The major constituents of casein are alpha (s1) and alpha (s2)-caseins, β-casein and kappa-casein. These caseins are conjugated proteins with phosphate group(s) which are esterified into serine residues they have a low solubility at pH 4.6.

Uses

It is used in the manufacture of binders, adhesives, protective coatings, plastics (such as for knife handles and knitting needles), fabrics, food additives, and many other products. It is commonly used by bodybuilders as a slow-digesting source of amino acids. There is growing evidence that casein may be addictive for some individuals, particularly those on the autism spectrum or having schizophrenia.

COLLAGEN

Synonym

Ossein.

Biological Source

It is the protein which consists of major portion of white fibres in connective tissues of the animal body specifically, from the tendons, skin, bones, and teeth.

Characteristics

The molecule of collagen is similar to three strand rope, each strand consisting of polypeptide chain with molecular weight of 10,000. These three strands are left-handed helices and are wrapped together in a right-handed superhelix. The strands are held together by hydrogen bonds, which give the molecule its strength. Collagen fibres range from 10 to 100 μm in diameter and visible by microscope as banded structure in the extra cellular matrix of connective tissues.

Chemical Constituents

Glycine and proline are the important amino acids in the central core of the triple helical molecule of collagen. It can be differentiated from other accompanying fibrous proteins like elastin and reticulin. Elastin is highly crossed linked hydrophobic protein. Collagen is characterized by the presence of glycine, proline, hydroxyproline, and hydroxylysine and low tyrosine and sulphur contents, whereas elastin contains nonpolar amino acids like valine, isoleucine, and leucine. Various types of collagen exist depending upon the amino-acid sequence. Collagen is converted to gelatin by boiling with water.

Uses

It is used in the preparation of sutures, as a gel in food casings and in photographic emulsions.

FICIN

Biological Source

Ficin is found in the latex of the plants of the Genus Ficus. Commercial ficin is purified from the latex of the fig tree, Ficus glabatra or Ficus carica. Refined ficin micro granulate is a protease, which can be used when a degradation of proteolytical stuff is required.

Characteristics

The optimum pH of ficin depends on the substrate and its concentration. Generally, the optimum pH is between 5 and 8, although ficin keeps its activity over the range of pH 4–9 at 60°C. Though the optimum temperature of ficin is 45–55°C, it is effective in temperatures between 15 and 60°C. It is obtained as a white to yellow microgranular powder. The moisture content should not exceed 6%.

Uses

It is generally used in alcohol and beer industries, hydrolization of proteins, meat processing, baking industry, pet food, health food, contact lens cleaning. Cancer treatment, antiarthritis, digestive aid, etc.