PROTEINS

Proteins are high molecular weight (5000 to 25,000,000) substances containing the elements C, H, O, and N. They are macromolecules which also contain other elements like S, P, and a few, such as hemoglobin, contain some other elements. Other than water, proteins are the chief constituents of all cells of the body. The word protein is derived from the Greek word proteios which means primary or of first importance. About half the dry weight of living materials is protein. They are the source to replace N as almost 15 g of N is lost everyday by an adult chiefly as urinary urea. Sources of proteins: Plants synthesize proteins from inorganic substances present in the air and in the soil. Animals cannot synthesize proteins from such sources. Animals obtain proteins from plants or from other animals who have obtained them from other plants. Animals excrete waste materials containing many N compounds. These N cpds along with decaying animal and plant matter are converted into soluble N cpds by soil bacteria. Plants, then use these soluble N cpds to manufacture more protein thus completing a cycle. Functions of Proteins: (Most impt type of cpd in the body.) 1. Building of new cells 2. Maintenance of existing cells 3. Replacement of old cells hormones 4. Valuable source of energy 5. Involved in the regulation of metabolic processes 6. Involved in the catalysis of biochemical reactions (enzymes) 7. Involved in the transport of oxygen (hemoglobin) 8. Involved in the bodys defense against infection (antibodies) 9. Involved in the transmission of hereditary characteristics (nucleoproteins) 10. Involved in the transmission of impulses (nerves) 11. Involved in muscular activity (contraction) 12. Components of skin, hair, and nails, and connecting and supporting tissues Composition of proteins: Proteins are polymers built up from smaller units called amino acids. Hydrolysis of proteins yields amino acids. Polymers are chain-like molecules produced by the linking together of a number of small units, chiefly -amino acids. O RCHCOH NH2

-amino acid where R can be many different groups

Amino acids: Most proteins produce approximately 20 -amino acids. Alpha amino acids have both an amino and a carboxylic acid group attached to the same -carbon, that is the carbon atom next to the acid group. The 2nd C from the acid group is called *+ carbon; then come the *+ gamma and *+ delta carbons. There are 20 known amino acids that can be produced by the hydrolysis of proteins. All these amino acids, except glycine, which has no chiral C, have the L-configuration. The body can synthesize some, but not all, of the amino acids it needs. Those that it cannot synthesize must be supplied from the diet. These are called the nutritionally essential amino acids. They are the ffg: 1. leucine 2. isoleucine Mnemonic 3. threonine Pvt. Mat Hill 4. tryptophan or Pvt. Tim Hall H-histidine 5 6 3 42 7 18 5. phenylalanine a-arginine 6. valine Adequate amounts of amino 7. methionine acids are required to maintain the 8. lysine the proper N balance in the body. Semi-essential amino acids Arginine and histidine are synthesized partially by the body but not at the rate to meet the requirement in growing children, pregnant and lactating women.

Non-essential amino acids These can be synthesized by the body and may not be required in the diet. These amino acids are derived from the carbon skeletons of lipids and carbohydrates during their metabolism or from the transamination of essential amino acids. Deficiency in one or more essential amino acids in the diet reduces protein synthesis leading to failure in growth of the child, negative nitrogen balance in adults and fall in plasma proteins and hemoglobin levels. Amino acids can be divided into two groups, polar and nonpolar, depending on the polarity of the R group attached to the -carbon. If the R-group is non-polar, then the amino acid will be less soluble in water than the one containing a polar group. An R-group that is polar, such as OH, SH, NH2, or COOH, produces an amino acid that is polar. Such amino acids are soluble in water.

Polar A.A. COOH H2NCH H Glycine (gly)

COOH COOH H2NCH H2NCH O CH2COOH CH2C aspartic acid (asp) NH2 asparagine (asn)

COOH H2NCH NH CH2CH2CH2NHC arginine (arg) NH2 COOH COOH COOH H2NCH H2NCH H2NCH CH2 CH2CH2COOH CH2OH SH glutamic acid (glu) serine (ser) Cysteine (cys) COOH COOH COOH H2NCH H2NCH O H 2 N C H HCOH CH2CH2C CH2 CH3 NH2 C=CH threonine (thr) glutamine (gln) HN N C

COOH H2NCH CH2 Tyrosine (tyr)

H histidine (his)

OH Non-polar A.A. COOH COOH COOH H2NCH H2NCH H2NCH CH3 CH CH2 alanine (ala) CH3 CH2CH3 CH isoleucine (ile) CH3 CH3 leucine (leu) COOH COOH H2NCH C H CH2CH2SCH3 HN CH2 proline(pro) methionine (met) H2C COOH CH2 COOH H2NCH H2NCH CH COOH CH2 H3C CH3 H2NCH Valine (val) CH2

Phenylalanine (phe)

N Tryptophan (trp)

These units are joined together through the peptide bonds (CONH). The peptide linkage is formed between two amino acids by the release of one water molecule. The amino group of the first amino acid and the carbonyl group of the next amino acids are involved in the formation of peptide bonds. Amphoteric Nature Amino acids contain the COOH group, which is acidic, and the NH2 group, which is basic. In solution, the carboxyl group can donate a H+ to the amino group, forming a dipolar ion, called a zwitterion.

RCHCOOH NH2 Amino acid

RCHCOONH3+ zwitterion form of an amino acid

Amino acids are amphoteric compounds; that is, they can react with either acids or bases. When an amino acid is placed in a basic solution, it forms a negatively charged ion that will be attracted toward a positively charged electrode. In acid solution, the amino acid forms a positively charged ion that will be attracted toward a negatively charged electrode. OHRCHCOOH RCHCOO RCHCOO+ + + NH3 H NH3 NH2 positively charged ion zwitterion negatively charged ion (in acid solution) (in basic solution) Since amino acids are amphoteric, proteins, which are made up of amino acids, are also amphoteric. This amphoteric nature of proteins accounts for their ability to act as buffers in the blood; they can react with either acids or bases to prevent an excess of either. At a certain pH, amino acids will not migrate toward either the positive or the negative electrode. At this pH, amino acids will be neutral; there will be an equal number of + and ions. This point is called the isoelectric point. Proteins, which are composed of amino acids, also have an isoelectric point, which is different for each protein. At its isoelectric point, a protein has a minimum solubility, a minimum viscosity, and also a minimum osmotic pressure. At a pH above the isoelectric point, a protein has more negative than positive charges. At a pH below isoelectric point, a protein has more positive than negative charges. Peptide Bonds When two amino acids are joined together by the peptide bond the result is a dipeptide.

O CH3CHCOH + HNHCH2COOH NH2 alanine glycine

amine part of glycine rxts with acid part of alanine

O peptide bond CH3CHCNHCH2COOH + H2O NH2 alanylglycine (ala-gly)

or acid part of glycine react with the amine part of the alanine

O peptide bond NH2CH2CNHCHCOOH + H2O CH3 glycylalanine (gly-ala)

When three amino acids combine, the product is called a dipeptide. When many amino acids join together, the product is called a polypeptide.

Each polypeptide can have any number of any one or different types of amino acids which can be present in any sequence. The individual amino acid of a peptide is called the amino acid residue. Each polypeptide has one free carboxylic acid ( COOH) at one end which is called the C-terminal and a free amino group at the other end called N-terminal. For just two amino acids, glycine and alanine, two different combinations have already been indicatedglycylalanine and alanylglycine, in which the first member of each group acts as the one furnishing the OH from the acid group. For three different amino acidssuch as glycine, alanine and valinethere are 6 possible combinations ( or tripeptide linkages). 1. glycylalanylvaline (gly-ala-val) 4. alanylvalylglycine (ala-val-gly) 2. glycylvalylalanine (gly-val-ala) 5. valylglycylalanine (val-gly-ala) 3. alanylglycylvaline (ala-gly-val) 6. valylalanylglycine (val-ala-gly) Example: A dipeptide composed of glycine, alanine and serine.

CH2 H2N
gly

H N C O CH CH3

O C N H

CH2OH CH COH O ser C-terminal CH3 HC CH3 CH N COH H O val

ala

N-terminal H N C O gly
STRUCTURE OF PROTEINS

CH2 H2N

O C CH CH3 ala

1. Primary structurerefers to the number and sequence of the A.A in the protein. (Peptide bonds) Example is the 1o structure of insulin. A slight change in the amino acid sequence, such as the replacement of a single amino acid with another,can change the entire protein. Example of this is the protein hemoglobin that is composed of 146 amino acids in proper sequence to form the chain. Amino acid #6 is glutamic acid. If it is changed to Valine, a new type of protein, Hemoglobin S, is produced and causes the genetic disease sickle cell anemia. 2. Secondary structurerefers to the regular recurring arrangement of the amino acid chain. (a) -helix, occurs when the amino acids form a coil, or spiral. The coil consists of loops of amino acids held together by H-bonds (between the H of the NH2 of one AA and the O of the C=O of the acid part of another AA. Each turn of the helix contains an average of 3.6 amino acids. Such a structure is both flexible and elastic. Example of this helical structure is hair and wool. *When AA are coiled, they can form either a right- or left-handed spiral. Since AA in proteins are all of the L-configuration, the coils always are right-handed. (b) -pleated sheet (also called the pleated sheet) consists of parallel strands of polypeptides held together by hydrogen bonds. Such a structure is flexible but not elastic. Example of this is one found in silk. It is strong, but resistant to stretching. This type of structure is less common than the helix. 3. Tertiary structurerefers to the specific folding and bending of the coils into specific layers or fibers. The tertiary structure gives proteins their specific biologic activity. They are stabilized by several types of bonds: (a) salt bridges formed between +ly and ly charged groups within the protein moleculeexamples are the carboxyl

and amino side chains found in glutamic acid, lysine, arginine and aspartic acid (b) H-bonds can form between different segments of the coil. (c) Disulfide bonds can form between cysteine groups in different parts of the coil. (d) Hydrophobic bonds can be formed. Generally, non-polar amino acids are folded on the inside of the protein. (e) Hydrophilic or polar group interactions between polar amino acids generally formed on the outside 4. Quaternary structuresome proteins have this structure, which occurs when 2 or more protein units, each with its own 10, 20, and 30 structure, combine to form a more complex unit. Example is hemoglobin. It consists of 2 identical chains and 2 identical chains. Each chain enfolds a heme, which is an iron-containing group.

NITROGEN IN PROTEIN
The average %N present in protein is 16%; that is, about 1/6 of protein is N. Because protein is the major food that contains N, the chemist can determine the amount of protein present in a food substance by determining the amount of N present. The amount of protein in the food can therefore be calculated by multiplying the weight of N by 6 and converting this to a percentage of the total. For example, suppose that a 100-g sample of food yielded 4 g of N on chemical analysis. Since the amount of total nitrogen in protein is one sixth of the total amount of protein present, the amount of protein present is 6 x 4 g or 24 g. Then the percentage of protein present in the original 100-g sample is 24%. Melamine, C3H6N6a white, crystalline compound that is slightly soluble in water, melts @ 3540C and is a cyclic trimer of cyanamide; used to make melamine resins and in tanning of leather. Also known as cyanurotriamide, cyanurotriamine and cyanuramide; 1,3,5-Triazine-2,4,6-triamine.

NH2 N H2 N
CLASSIFICATION OF PROTEINS
1. Simple proteinson hydrolysis, simple proteins yield only amino or derivatives of amino acids. 2. Conjugated proteinson hydrolysis, conjugated proteins yield amino acids + some other type of cpd. Conjugated protein consists of a simple protein combined with a non-protein compound. 3. Derived proteinsare produced by the action of chemical, enzymatic and physical forces on the other two classes of protein. Derived proteins include proteoses, peptones, polypeptides, tripeptides, and dipeptides. They also can be hydrolyzed to amino acids.

N N NH2

CLASSIFFICATION ACCDG. TO SOLUBILITY

Proteins are classified accdg to their soly in various solvents and whether they are coagulated by heat.

SIMPLE PROTEINS
Type of Protein Albumins Globulins Solubility Soluble in water and salt solutions Slightly soluble in H2O; Soluble in salt solns Insol in all neutral solvents and in dilute acid and alkali Soluble in all salt solns; insol in very dilute NH4OH Coagulated by heat yes yes Examples Egg albumin; serumalbumin; lactalbumin Serum globulin; Lactoglobulin; Vegetable globulin Keratin in hair, nails, feathers; collagen Nucleohistone in thymus gland; globin in hemoglobin

Albuminoids Histones

no no

CONJUGATED Type Nucleoproteins

PROTEINS Prosthetic group (Non- Protein portion of the combination) Nucleic acids

Examples Chromosomes

Glycoproteins Phosphoproteins Chromoproteins

Carbohydrates Phosphate Chromophore group (color-producing grp) Lipids Metals

Lipoproteins Metalloproteins

Mucin in saliva Casein in milk Hemoglobin, hemo-cyanin; flavoproteins cytochrome Fibrin in blood Ceruloplasmin (with Cu) Siderophilin (with Fe) in blood plasma

Nucleoproteins are proteins combined with nucleic acids. Glycoproteins are proteins containing CHO in varying amounts. Glucose is not found in glycoproteins, except for collagen. Glycoproteins are present in most organisms, including animals, plants, bacteria, viruses and fungi. Human cell membranes are about 5% carbohydrate, which is present as glycoproteins and glycolipids. Glycophorin is a glycoprotein found in membranes of human erythrocytes. Heparin, which inhibits clotting of blood, is also a glycoprotein. Functions of glycoproteins: 1. in cell membranes 2. structural proteins (collagen) 3. lubricants (mucin and mucous membranes) 4. transportation molecules for vitamins, lipids, minerals and trace elements 5. immunoglobulins as interferon 6. hormones such as thyrotropin (TSH) 7. enzymes such as hydrolases and nucleases 8. hormone receptor sites 9. for specification of human blood types

LIPOPROTEINS
Lipoproteins are proteins containing lipids; they are present in cell membranes. Lipids such as cholesterol and triglycerides are not soluble in water and so must be complexed to a water-solube carrier protein, which is called lipoprotein. Plasma lipoproteins consist of a neutral lipid core of triglyceride and cholesterol, protein and phospholipid. The relative proportions of nonpolar lipid, protein, and polar lipid determine the density, size and charge of the resulting lipoproteins. The density of lipoproteins has been used to classify them: 1. Chylomicronsproduced in the intestinal mucosa and are used to transport dietary lipids into the blood plasma via the thoracic lymph duct. They are responsible for the creamed-tomato-soup appearance of blood following a meal containing fats. 2. Very low density lipoproteins (VLDL) transport glycerides synthesized by the liver to other parts of the body. 3. Intermediate low density lipoproteins (IDL) from the breakdown of VLDL 4. Low density lipoproteins (LDL)end products of breakdown of VLDL; they provide cholesterol for cellular needs. LDL is thought to promote coronary heart disease by first penetrating the coronary artery wall and then depositing cholesterol to formatherosclerotic plaque. 5. High density lipoproteins (HDL)involved in the catabolism of other lipoproteins. They incorporate the cholesterol and phospholipid released by a lipoprotein. HDLs may also remove excess cholesterol from peripheral tissues. 6. Lipoprotein(a), similar to LDL; The striking similarity of lipoprotein(a) to human plasminogen has stimulated intense studies as to a possible link between atherosclerosis and thrombosis. Primarily, results have indicated that lipoprotein(a) is an independent risk factor (similar to total cholesterol) for coronary heart disease. Elevated LDL levels have been associated with an increased risk of developing coronary artery disease, whereas elevated HDL levels appear to reduce the risk. Women have higher HDL levels than men (55 vs. 45 mg/100 mL), and this may account for womens lower rate of heart disease. Aerobic exercise increases HDL levels (marathon runners average 65 mg/100 mL).

CLASSIFICATION ACCORDING TO FUNCTION Type of Protein Example Use

1. Structural 2. Contractile 3. Storage 4. Transport 5. Hormones 6. Enzymes 7. Protective 8. Toxins Collagen Keratin Myosin, actin Ferritin Hemoglobin Serum albumin Insulin Pepsin Gamma globulin Fibrinogen Venoms In connective tissues In hair and nails In muscle contraction In storage of iron needed to make hemoglobin In carrying oxygen In carrying fatty acids In metabolism of CHO In digestion of protein In antibody formation In blood clotting Poisons

FUNCTIONS OF PROTEINS
Among all biomolecules, proteins have the most variety of functions. Proteins provide: 1. Structure, protection and support; Examples are collagen (connective tissues) fibroin (silk protein) elastin (blood vessels) 2. Defense and protection against chemical and mechanical injury. Keratin in the skin provide protection. Fibrinogen and thrombin induce blood clotting. Antibodies or immune-globulin protects man from infection. 3. Regulation through the body hormones. Examples are insulin, glucagon, growth and sex hormones. 4. Catalysis through enzymes. Enzymes are proteins that direct and regulate the speed of biochemical reactions. Examples are amylase, oxidase, phosphatase, etc. 5. Transport as carrier of ions or molecules across cell membranes or between cells. Other transport proteins are hemoglobin, carrying O2 to the tissues and lipoproteins, LDL and HDL, that transport lipids from the liver and intestines to other organs.

CLASSIFICATION ACCORDING TO SHAPE

1. Globular proteinsconsists of polypeptides folded into the shape of a ball. They have a length-to-width ratio of less than 10. They are soluble in water or form colloidal dispersions and have an active function. Examples are hemoglobin, albumin, and the globulins. 2. Fibrous proteinsconsist of parallel polypeptide chains that are coiled and stretch out. They have a length-to-width ratio greater than 10. Fibrous proteins are insoluble in water. Examples include collagen,fibrin, and myosin.

PROPERTIES OF PROTEINS
1. Colloidal Nature Proteins form colloidal dispersions in water. Being colloidal, proteins will pass through a filter paper but not through a membrane. This inability of proteins to pass through a membrane is of great importance in the body. Proteins present in the bloodstream cannot pass through the capillaries and should remain in the bloodstream. Since proteins cannot pass through membranes, there should be no protein material present in the urine. The presence of protein in the urine indicates damage to the membrane in the kidneyspossibly nephritis. NEPHRITIS OR NEPHROSIS IS A DISEASE OF THE KIDNEYS IN WHICH THE FILTRATION PROCESS PERMITS PROTEIN MOLECULES FROM THE BLOOD TO BE DISCHARGED IN THE URINE. THIS DEPLETION OF PROTEINS IN THE BLOOD INTERFERES WITH THE PASSING OF FLUID FROM NORMAL TISSUE AND CAUSES SWELLING THROUGHOUT THE BODY, PARTICULARLY AROUND THE EYES, HANDS AND FEET. WHEN THERE ARE NO OTHER COMPLICATIONS, THE CONDITION IS NOT LIFE THREATENING AND CAN EASILY BE TREATED. 2. Denaturation of protein refers to the unfolding and rearrangement of the secondary and tertiary structures of a protein without breaking the peptide bonds. A protein that is denatured loses its biologic activity. When the conditions for denaturation are mild, the protein can be restored back to its original conformation by carefully reversing the conditions that caused the denaturation. This is called reversible denaturation. If the conditions that caused the denaturation are drastic, the process is irreversible.

Denaturation agents
Physical agents (a) Heat: Water is necessary for denaturation and coagulation by heat. The steps in heat coagulation are: 1stdenaturation; 2ndflocculation; 3rdcoagulation. (b) Light: Clouding of the lens of the eye in old-age cataract is probably due to denaturating of the globulins present in the lens. Glass workers are particularly subject to cataract presumably as a result of infrared rays emanating from the molten glass. Change of fibrinogen to fibrin in blood clotting may be partly due to light. (c) Surface action (d) High pressure (e) Mechanical agitation Chemical agents (a) Organic solvents (alcohol, acetone, ether) (b) Acids and alkalies (c) Salts of heavy metals (d) Enzymes (e) Detergents (f) Urea and guanidine

Alteration of Properties due to Denaturation I. Physical (a) (b) (c) (d) Increase in the viscosity of the soln Increase in the rate of diffusion Cannot be crystallized Decreased in solubility

II. ChemicalSome groups in the denatured protein molecules become exposed and readily detectable. Among these groups are the sulfhydryl (SH), disulfide (SS) and the phenolic group. They are shielded in the native state. Denaturation decreases the solubility of proteins. III. Biological (a) Increased digestibility by proteolytic enzymes has been found in certain denatured proteins. (b) Enzymatic or hormonal activity is usually destroyed. (c) Modification of the specific immunological properties.

3. Chemical Agents or Conditions That Cause Protein Denaturation (a) Alcohol coagulates or precipitates all types of protein except prolamines. Alcohol is used as a disinfectant because of its ability to coagulate the protein present in bacteria. Alcohol denatures protein by forming hydrogen bonds that compete with the naturally occurring H-bonds in the protein. Such a pxs is not reversible. As an antiseptic, alcohol, specifically ethanol works best at a concentration of 70 % in water. Pure alcohol coagulates protein on contact. If pure alcohol is poured over a germ, it would penetrate the cell wall of that organism in all directions, coagulating the protein just inside the cell wall. This ring of coagulated protein would then prevent the alcohol from penetrating farther into the cell, and no more coagulation would take place. At this time the cell would become dormant, but not dead. Under proper conditions, the organism could again begin to function. However, if only 70% is poured over a germ, the diluted alcohol also coagulates the protein but at a slower rate, so that it penetrates all the way through the cell before coagulation can block it. Then all the cells are coagulated, and the organism dies. (b) Salts of Heavy Metals Heavy metal salts, such as mercuric chloride or silver nitrate (lunar caustic), precipitate protein. These denature protein irreversibly by disrupting the salt bridges and the disulfide bonds present in the protein. They are very poisonous if taken internally because they coagulate and destroy proteins present in the body. Antidotes for metallic poisons: egg white, milk and other liquid protein. The heavy metal salts react with the egg white and precipitate out. The egg white colloid has a charge opposite to that of the heavy metal ion and so attracts it. The ppt thus formed must be removed from the stomach by an emetic or the stomach will digest the egg white and return the poisonous material to the system. An emetic is a medicine used to induce vomiting. Or it can be removed by a stomach tube to prevent the digestion of protein and the liberation, re-solution and absorption of the poisonous metal. AgNO3 is used in cauteries. It precipitates the proteins of as silver salts. To cauterize is to destroy dead or abnormal tissue by applying a caustic, intenseheat or cold. Dilute silver nitrate solution is used as a disinfectant in the eyes of newborn infants. (c) Heat. Gentle heating causes reversible denaturation of protein, whereas vigorous heating denatures protein irreversibly by disrupting several types of bonds. Egg white, a substance containing a high percentage of protein, coagulates on heating. Heat coagulates and destroys protein present in bacteria. Hence, sterilization of instruments and clothing for use in operating rooms requires the use of high temperatures. The presence of protein in the urine can be determined by heating a sample of urine, which will cause the coagulation of any protein material that is present. denature protein irreversibly by disrupting salt bridges and H-bonds. Tannic acid has been used extensively in the treatment of burns. When this substance is applied to a burn area, it causes the protein to precipitate as a tough covering, thus reducing the amount of water loss from the area. It also reduces exposure to air. Newer drugs have taken the place of tannic acid for burns, but an old-fashioned remedy still in use for emergencies involves the use of wet tea bags which contain tannic acid. Tannic acid is also used to relieve diarrhea. Commercially, it is used to precipitate collagen in hides, thus yielding leather. Picric acid is used in the treatment of burns because it produces an astringent effect on the tissue, diminishes secretion of the mucous membranes and prevents absorption of toxins.

(e) Radiation. Ultraviolet or x-rays can cause protein to coagulate. The radiation denatures irreversibly by disrupting the H-bonds and the hydrophobic present in the protein. In the human body the skin absorbs and stops the UV rays from the sun so they do not reach the inner cells. Proteins in cancer cells, which are rapidly dividing cells, are more susceptible to radiation than those present in normal cells, so x-irradiation is used to destroy cancerous tissues. (f) pH Changes in pH can disrupt H-bonds and salt bridges causing irreversible denaturation. Proteins are coagulated by such strong acids as concd HCl, H2SO4, and HNO3. Casein is precipitated from milk as a curd when it comes in contact with the HCl of the stomach. Hellers ring test is used to detect the presence of albumin urine. A layer of concd HNO3 is placed carefully under a sample of urine in a test tube. If albumin is present, it will precipitate out as a white ring at the interface of the two liquids. If acid or base remains in contact with protein for a long period of time, the peptide bonds will break. (g) Oxidizing and reducing agents. OA such as bleach and nitric acid and RA such as sulfites and oxalates denature protein irreversibly by disrupting disulfide bonds. (h) Salting Out. Most proteins are insoluble in saturated salt solutions and precipitate out unchanged. To separate protein from a mixture of other substances, the mixture is placed in a saturated salt solution such as NaCl, Na2SO4, or (NH4)2SO4. The protein precipitates out and is removed by filtration. The protein can then be purified from the remaining salt by the process of dialysis.