Biochemistry of Carbohydrates PDF

Biochemistry of Carbohydrates Dr/ Nashwa Abdelghaffar Abdel Rahman Maghraby Medical Biochemistry Department ▪ Carbohydrates are substances containing carbon, hydrogen and oxygen ▪ Carbohydrates are aldehyde (CHO) or ketone (C=O) derivatives of polyhydric alcohols (have more than on...

Biochemistry of Carbohydrates Dr/ Nashwa Abdelghaffar Abdel Rahman Maghraby Medical Biochemistry Department ▪ Carbohydrates are substances containing carbon, hydrogen and oxygen ▪ Carbohydrates are aldehyde (CHO) or ketone (C=O) derivatives of polyhydric alcohols (have more than one OH group) or compounds which yield these derivatives on hydrolysis  Importance of carbohydrates 1. The chief source of energy. 2. Important structural components in animal and plant cells. 3. Important part of nucleic acids and free nucleotides and coenzymes. 4. Major antigens are carbohydrates in nature, e.g., blood group substance. 5. Has a biological role as a part of hormones and their receptors and enzymes. Monosaccharides 4. Hexoses: monosaccharides containing 6 carbon atoms. a. Aldohexoses: glucose, mannose, and galactose. b. Ketohexose: fructose. Importance (functions) of Hexoses: 1- Glucose: “grape sugar” - - It is is the most important sugar of carbohydrate: - Glucose is the main sugar in blood - Glucose is one of major sources of energy in the body. - In the liver and other tissues, glucose is converted to all carbohydrates in the body e.g. glycogen, galactose, - It is produced by hydrolysis of starch, dextrins, glycogen, sucrose, maltose and lactose - It appears in urine in diabetes mellitus.  2- Galactose - It can be converted into glucose in the liver. - It is synthesized in mammary gland to make the lactose of milk (milk sugar) - It enters in structure of glycoproteins, glycolipids and mucopolysaccharides in its amine form  3- Fructose: “fruit / semen sugar ” - It is called(or levoulose) because is levorotatory - It is the sweetest sugar known. - It is the main sugar in bee's honey and fruits. - - It is the main sugar of semen - It is obtained from inulin and sucrose hydrolysis - It can be converted into glucose in the liver. 4- Mannose - A constituent of many glycoproteins and sialic acid - Isolated from plant mannans; hence the name Ring (cyclic) structure of sugars  Glyceraldehyde ▪ Is the carbon atom to which 4 different groups or atoms are attached. ▪ Glyceraldehyde is called the reference sugar because it contains one asymmetric carbon atom. ▪ Any substance containing asymmetric carbon atom shows 2 Properties, ✓ Optical activity and ✓ Optical isomerism.  Optical activity ▪ It is the ability of substance to rotate plane polarized light either to the right or to the left. ✓ If the substance rotates plane polarized light to the right so it is called: dextrorotatory or d or (+). ✓ If substance rotates it to the left so it is called: levorotatory or I or (-). ▪ Glucose contains 4 asymmetric carbon atoms. It is dextrorotatory, so it is sometimes named dextrose. ▪ Fructose contains 3 asymmetric carbon atoms. It is levorotatory. so it is sometimes called: LevuIose  Optical isomerism ▪ It is the ability of substance to present in more than one form (isomer). ▪ Isomers are substances which have the same molecular formula but differ in ✓ Distribution of their. atoms into groups (Structural isomerism). ✓ Distribution of these groups and atoms in the space around carbon atoms (Stereo-isomerism).  Structural isomerism - They are isomers that have different structure due to different ways of arrangement of atoms and groups forming the molecule. - Types of structural isomerism are 4 : ✓ Ring isomerism ✓ Functional group isomerism ✓ Positional isomerism ✓ Chain isomerism  Ring isomerism Functional group isomerism (Aldose-Ketose isomerism) EX: - glucopyranose & glucofuranose, EX: - Fructose & glucose - fructopyranose & fructofuranose - erythrose & erythrulose -ribose&ribulose,  Stereo-isomerism - They are molecules having the same structure but differ in position of their different groups and atoms in the space, i.e., in spatial configuration. - The number of stereoisomers = 2n, where n is the number of asymmetric carbon atoms. - There are four types of stereo-isomerism as follows: ✓ D and L isomerism (enantiomers): ✓ Anomers ✓ Epimers ✓ Geometric isomerism D and L isomerism (enantiomers): ▪ They differ in distribution of H and OH groups around the sub-terminal asymmetric carbon atoms. ✓D form has the OH group to the right of the sub-terminal carbon atom (i.e., adjacent to the terminal primary alcohol group-reference carbon atom). ✓L form has the OH on the left ▪ Metabolizable sugars in human body are D-forms only. ▪ Examples are ✓ D- glyceraldehyde and L- glyceraldehyde ✓D-glucose and L-glucose. or ✓D-mannose and L-mannose. Anomers: - They are stereoisomers which differ in distribution of H and OH group around the asymmetric anomeric carbon atom after cyclization of the molecule ✓ C1 in aldoses or ✓ C2 in ketoses - Examples - and -glucose are anomers. Epimers: ▪ Epimers are isomers having more than asymmetric carbon, all are same except only one is different. ✓ Glucose & Mannose are epimers at carbons 2. ✓ Glucose & Galactose are epimers at carbons 4. 1. Sugar acids 2. Sugar alcohols 3. Deoxy sugars 4. Amino sugars 5. Amino sugar acids  Sugar acids - Are produced by oxidation of carbonyl carbon, last carbon or both - L-Ascorbic acid (vitamin C) is an important sugar acid  CHO COOH H C OH H C OH HO C H bromine water, O2 HO C H H C OH H C OH H C OH H C OH CH 2OH CH 2OH D-Glucose D-Gluconic acid CHO CHO H C OH H C OH HO C H H2O2 HO C H H C OH Dil. Nitric acid H C OH H C OH H C OH CH 2OH COOH D-Glucose D-Glucuronic acid CHO COOH H C OH H C OH HO C H O2 HO C H H C OH Conc. Nitric acid H C OH H C OH H C OH CH 2OH COOH D-Glucose D-Glucaric acid  Sugar alcohols Are produced by Reduction of carbonyl carbon Mannitol is injected intravenously to reduce intracranial hypertension in cases of meningitis, cerebral hemorrhage or thrombosis and Is used in kidney function testing  CHO CH2OH CHO CH2OH H2 H C OH H C OH H C OH H C OH HO C H H2 HO C H Na amalgum, H2SO4 CH2OH CH2OH H C OH H C OH Na amalgum, H2SO4 Glyceraldehyde Glycerol H C OH H C OH CH2OH CH2OH Glucose Sorbitol CHO CH2OH CH2OH CH2OH CH2OH H C OH H C OH H C OH C=O HO C H H C OH H2 H C OH HO C H HO C H HO C H H2 H C OH Na amalgum, H2SO4 H C OH H C OH OR H C OH H C OH Na amalgum, H SO 2 4 CH 2OH CH2OH H C OH H C OH H C OH Ribose Ribitol CH2OH CH2OH CH2OH Fructose Sorbitol Mannitol CHO CH 2OH CHO CH 2OH HO C H HO C H H C OH H C OH HO C H H2 HO C H HO C H H2 HO C H H C OH H C OH HO C H Na amalgum, H2SO4 Na amalgum, H2SO4 HO C H H C OH H C OH H C OH H C OH CH 2OH CH 2OH CH 2OH CH 2OH Mannose Mannitol Galactose Dulcitol  3- Deoxysugars ▪ Are produced by replacement of hydroxyl groups by hydrogen atom i.e. one oxygen is missed. ▪ This may occur at either of three places: 1. At C2 gives Deoxy sugar proper, e.g., deoxyribose that enters in structure of DNA., 2. At C6 gives methyl pentoses (Methylose), ✓ L-galactose gives L-fucose enter in the structure of glycoproteins, e.g., blood group substance 3. At C3, as in Sialic acid,  4- Amino sugars ▪ In these sugars, the hydroxyl group attached to C2 is replaced by ✓ an amino or ✓an acetyl-amino group. ▪ Amino sugars enter in glycoproteins. ▪ Examples: ✓ Glucosamine: It enters in heparin and hyaluronic acid ✓ Galactosamine: It enters in chondroitin sulphate. ✓ Mannosamine: It enters in neuraminic and sialic acids..  5- Amino sugar acids ▪ Formed by addition of amino sugars and some acids. ▪ Examples: ✓ Neuraminic acid = Mannosamine + pyruvic acid. 9C ✓ Sialic acid or N-acetylneuraminic acid (NANA) enters in glycolipid.  Disaccharides 1. Reducing Disaccharides ✓ Maltose ✓ Isomaltose ✓ Cellobiose ✓ Lactose 2. Non-Reducing Disaccharides ✓Sucrose ✓Trehlose  Disaccharides ▪ The most Important disaccharide are: 1. Maltose = α-glucose + α -glucose (α 1 - 4 glycosidic bond ). 2. Isomaltose = α - glucose + α -glucose (α 1 - 6 glycosidic bond). 3. Trehalose = α - glucose + α -glucose (α 1 - 1 glycosidic bond). 4. Cellobiose = β - glucose + β -glucose (β 1 - 4 glycosidic bond). 5. Lactose = β- glucose + β - galactose (β 1 - 4 glycosidic bond). 6. Sucrose = α -glucose + β -fructose (α 1 - β 2 glycosidic bond). ▪ Properties: all disaccharide except (trehalose & Sucrose) showing the following characters : ✓ It is a reducing agent (can reduce Benedict’s reagent). ✓ It can be present in α and β forms. ✓ It can form characteristic osazone crystals.  A. Reducing Disaccharides Maltose (malt sugar) It consists of 2 -glucose units linked by -1,4-glucosidic linkage It is produced during digestion of starch. It is hydrolyzed by acids and in human intestine by maltase enzyme. CH 2OH CH 2OH O O H H OH..... H H H 1 4 H H OH H OH O H..... OH OH H OH H OH -Glucose Glucose − and −Maltose  Lactose (milk sugar) ▪ It is formed of β -galactose and β -glucose linked by 1,4-glucosidic linkage ✓ β -galactose participates by C1 ✓ β -glucose participates by C4 ▪ It is excreted in urine of pregnant and lactating females ▪ It is the most suitable sugar for baby feeding as a sweetener for milk because: 1. It is the least sweet sugar so that the baby can nurse a large amount of mother’s milk without getting his appetite lost. 2. Because it has a B-glycosidic linkage it is non-fermentable sugar, so it does not form gases and not cause colic to the infant. 3. It has a laxative effect and prevents constipation and non-irritant to the stomach and does not cause vomiting. 4. Unabsorbed sugar is used as a food for large intestinal bacteria that form a number of vitamins that benefits the baby. 5. It is easily digested and helps absorption of the minerals of milk.  Cellobiose - It is formed of 2 -glucose units linked by -1,4-glucosidic linkage. - It is a reducing disaccharide. - It is the building unit of cellulose. CH 2OH CH 2OH O O H OH H H H 1 O 4 H OH H OH H H OH H OH H OH -Glucose -Glucose Cellobiose  B. Non-reducing Disaccharides 1. Sucrose (invert sugar) It is table sugar and sugar of cane and molasses It is formed of -glucose linked to -fructose by --1,2-linkage. It is a fermentable sugar. The 2 anomeric carbons (C1 of glucose and C2 of fructose) are involved in the linkage, therefore it is: CH 2OH O H ✓ Non-reducing sugar. -Glucose H H 1 OH H ✓ Non osazone forming. OH H OH ✓ Not mutarotating. CH 2OH O O ✓ Not having  or -forms. -Fructose OH 2 H H CH 2OH OH H  It is a dextrorotatory sugar but when it is hydrolyzed by sucrase enzyme or by acid hydrolysis (HCl) the mixture of sugars produced is levorotatory. This is because the levorotatory power of fructose (-92.5) cancels the dextrorotatory power of glucose (+52.5) since they are at equal proportions in the product. This is why this sugar is called invert sugar. - Sucrase enzyme is therefore, also called invertase enzyme.  2. Trehalose It is formed of 2 -glucose units linked by -1,1-glucosidic linkage. Present in ✓ a highly toxic lipid extracted from Mycobacterium tuberculosis. ✓ It is the major sugar of insect hemolymph ✓ fungi and yeast. CH2OH O H OH H H H H H OH H 1 1 OH H HOH2C O OH OH O H OH H -Glucose -Glucose Trehalose All the following are sugar alcohols, EXCEPT: a- Galactitol b- Mannitol c- Xylulose d- Sorbitol Ribitol is: a. Deoxysugar b- Sugar alcohol c- Amino sugar d- Sugar acid Sorbitol is produced by reduction of: a- Glucose or fructose b- Glucose or galactose c- Glucose or mannose d- Galactose or fructose 1. A triose sugar is (A) Glycerose (B) Ribose (C) Erythrose (D) Fructose 2. A pentose sugar is (A) Dihydroxyacetone (B) Ribulose (C) Erythrose (D) Glucose 3. The number of isomers of glucose is (A) 2 (B) 4 (C) 8 (D) 16 4. Two sugars which differ from one another only in configuration around a single carbon atom are termed (A) Epimers (B) Anomers (C) Optical isomers (D) Stereoisomers 1. Isomers differing as a result of variations in configuration of the —OH and —H on carbon atoms 2, 3 and 4 of glucose are known as (A) Epimers (B) Anomers (C) Optical isomers (D) Steroisome 2. The most important epimer of glucose is (A) Galactose (B) Fructose hexose (C) Arabinose (D) Xylose 3. α-D-glucose and β -D-glucose are (A) Stereoisomers (B) Epimers (C) Anomers (D) Keto-aldo pairs 4. Compounds having the same structural formula but differing in spatial configuration are known as (A) Stereoisomers (B) Anomers (C) Optical isomers (D) Epimers 5. The sugar found in DNA is (A) Xylose (B) Ribose (C) Deoxyribose (D) Ribulose The sugar found in milk is: a- Galactose b- Glucose. c- Fructose. d- Lactose. The glycosidic linkage seen in sucrose is: a- Alpha linkage b- Beta 1-4 linkage c- Alpha 1-6 linkage d- Alpha 1-2 linkage Sucrose consists of: a- α Glucose + β glucose b- α Glucose + β fructose c- α Glucose + β galactose d- α Glucose + β mannose Which is a non-reducing sugar? a- Maltose b- Sucrose c- Lactose d- Isomaltose The glycosidic linkage seen in lactose is: a- Alpha 1-4 linkage b- Beta 1-4 linkage c- Alpha 1-6 linkage d- Alpha 1-2 linkage The glycosidic linkage seen in lactose is: a- Alpha 1-4 linkage b- Beta 1-4 linkage c- Alpha 1-6 linkage d- Alpha 1-2 linkage The glycosidic linkage seen in maltose is: a- Alpha 1-4 linkage b- Beta 1-4 linkage c- Alpha 1-6 linkage d- Alpha 1-2 linkage Hydrolysis of maltose will give rise to: a. Glucose only b- Glucose and fructose c- Glucose and galactose d- Glucose and mannose 24. Lactose by hydrolysis gives: a- 2 glucose molecules b- Glucose and mannose. c- Fructose and galactose. d- Glucose and galactose.  Biochemistry of Carbohydrates Dr/ Nashwa Abdelghaffar Abdel Rahman Maghraby Medical Biochemistry Department Polysaccharides  Polysaccharides They contain more than 10 monosaccharide units per molecule and give monosaccharides on acid hydrolysis. They are classified into: Homopolysaccharides & Heteropolysaccharides  Homopolysaccharides ▪ They yield only one type of monosaccharides on hydrolysis and they are named according to the type of that monosaccharide ▪ For example - Pentosans + H2O  Pentoses - Hexosans + H2O  Hexoses  Hexosans I. Glucosans: They produce only glucose on hydrolysis. - They include A. Starch B. Dextrins C. Dextran D. Glycogen E. Cellulose II. Fructosans: They produce only fructose on hydrolysis. Ex inulin III. Galactosans:They produce only galactose on hydrolysis. Ex agar agar IV. N-acetyl-glucosan: They produce only N-acetyl-glucosamine on hydrolysis. Ex chitin of insects.  I. Glucosans A.Starch ▪ It is the stored form of carbohydrate of plants. ▪ never exists in animals. ▪ It is present in ✓ Cereals such as wheat and rice ✓ Tubers such as potatoes and sweet potatoes. ▪ It is in the form of starch granules ▪ Each granule is composed of Amylose ( core: 20%) - Straight chain compound present in the form of a helix formed of a large number of -glucose units linked by -1,4-glucosidic bond CH 2OH CH 2OH O O H H H H H H 4 1 4 1 OH H OH O O O H OH H OH n Amylose  Amylopectin( shell: 80%) ▪ It is branched chains formed of a large number of -glucose units linked by -1,4- glucosidic linkage along the branch and by -1,6-glucosidic linkage at the branching point that occur periodically every 24-30 glucose units. CH 2OH CH 2OH O O H H H H H H 4 1 4 1 OH H OH O O O H OH H OH CH CH 2OH CH 2OH CH 2OH 62 O O O O H H H H H H H H H H H H 1 4 1 4 1 4 4 1 OH H OH OH H OH O O O O O H OH H OH H OH H OH n Amylopectin  B.Dextrins ▪ Products of hydrolysis of starch ex. corn and rice syrup. ▪ They include amylodextrin, erythrodextrin, achrodextrin that can be distinguished by the color with iodine (I2). ▪ They have sweet taste and are used as demulcents. ▪ They are easily digested than starch HCl or Amylase Starch Maltose + Amylodextrin, violet with iodine HCl or Amylase Maltose + Erythrodextrin, red with iodine HCl or Amylase Maltose + Achrodextrin, colorless with iodine HCl or Amylase Maltase 2 Glucose Maltose  C. Dextran ▪ It t is synthesized by certain bacteria having sucrose in its media. ▪ A compound formed of -glucose units linked by -1,4, -1,3- and -1,6-linkage present in the form of a network ▪ It has a great biochemical importance, ✓ It is used as plasma substitute to restore blood pressure in cases of shock. ✓ Iron used for treatment of iron deficiency anemia is used as dextran ferrous sulfate intramuscular injection. ✓ An anticoagulant Sodium dextran sulfate is an anticoagulant. (compare with dextrins and dextrose)  D. Glycogen: (also called animal starch) ▪ Glycogen Is present mainly in liver and muscles. ▪ Liver glycogen: It maintains normal blood glucose concentration especially during the early stage of fast (between meals). After 12- 18 hours fasting, liver glycogen is depleted. ▪ Muscle glycogen: It acts as a source of energy within the muscle itself especially during muscle contractions ▪ Glycogen is bomopolysaccharide formed of branched a D glucose units (α 1,4 and α 1,6). ▪ Its structure is similar to amylopectin a branched tree with α-1,4-glucosidic along the branch and α1,6-glucosidic linkage at the branching point. ▪ Each branch Is made of 12-14 glucose units. The glycogen tree is shorter and more branched than amylopectin.  E. Cellulose Structural polysaccharide ▪ It forms the skeleton of plant cells and does not enter in animals cell structures. ▪ It enters in structure of Linen, cotton and paper that are nearly pure cellulose. ▪ It is the major food for herbivorous animal where it is fermented into volatile fatty acids by cellulase of microorganisms in their rumen. ▪ It is a straight chain molecule formed of a large number of -glucose units linked by - 1,4-glucosidic linkage ▪ It gives cellobiose on hydrolysis with HCl. ▪ It is indigestible but is very essential in food for: - Prevention of constipation by increasing the bulk of stools. - Anticancer Its fermentation by large intestinal bacteria give ✓ volatile fatty acids that is anticancer for colon cells and ✓some water soluble vitamins. ✓ It adsorbs toxins present in foods and prevents its absorption into the body.  Comparison between Starch, Glycogen and Cellulose Starch Glycogen Cellulose 1. Nature: Stored form of carbohydrate in Stored form of carbohydrates in Structural form of carbohydrate in plants. animals. plant cells but prevents constipation in human. 2. Source: Cereals, e.g., wheat, rice, and Muscles and liver Linen and cotton are nearly pure tubers, e.g., potatoes. cellulose. 3. Solubility: Amylose is water soluble and Water soluble forming colloidal Water insoluble. amylopectin is insoluble. solution. 4. Nature of the chains: Amylose is helical straight chain (- - Highly branched chain. Each Straight chain (large number of - glucose units linked by -1,4-glucosidic branch is composed of 12-14 glucose units linked by -1,4- bonds). glucose units. glucosidic bonds). Amylopectin is branched chain. Each - It similar to amylopectin but its chain is composed of 24-30 glucose units trees are shorter and have more linked together by α-1,4 glycosidic bond branches than amylopectin tree. and a α1 ,6 glycosidic bond at branching points. 5. Reaction with iodine: Amylose gives blue color and Gives red color. No color. amylopectin gives red color. 6. Digestibility: Is hydrolyzed by HCl or amylase into Digestible by amylase into dextrins Non-digestible but HCl hydrolysis dextrins and maltose. and maltose. gives cellobiose.  II. Fructosans ▪ They are formed of fructose as a building unit such as Inulin ▪ Inulin: is formed of fructose only and present in onions and dahlia tubers. ▪ It is not metabolizible in human body, therefore, it is used in evaluation of kidney function as a part of inulin clearance test.  III. Galactosans ▪ They are formed of galactose as the building units such as agar-agar. ▪ Agar-Agar is a galactosan present in seaweed. ▪ Biochemical importance: 1) It is used for growth of bacteria and mammalian cells in culture. 2) It imbibes water and increases intestinal contents to prevent and treat constipation. 3) Some electrophoresis gels is formed of it.  IV. N-acetyl-glucosan: - It is a homopolysaccharide formed of -N-acetyl-glucosamine units such as chitin of insects. Chitin: - It is present in the exoskeleton of insects and crustaceans. 20  Heteropolysaccharides ▪ Heteropolysaccharldes contain different sugar units and ▪ They include glycosaminoglycans or (mucopolysaccharides) 21  Glycosaminoglycans, GAGs (mucopolysaccharide) Criteria 1. They are formed of repeating disaccharide units (Acidic sugar- Amino sugar)n  The acidic sugar The acidic sugar ✓ D-glucuronic acid or ✓ its epimer, L-induronic acid.  Amino sugar Amino sugar is ✓D-glucosamine or ✓ D-Galactosamine in which the amino group is usually acetylated. The amino sugar may also be sulfated at carbon 4 or 6. 2. GAGs often contain sulfate group.. 3. They are very negatively charged: because of The uronic acid and sulfate residues. 3. They are unbranched and contain no N-acetyl neuraminic acid NANA. 4. Most of GAGs are present extracellularly except heparin. 5. Most of them form the structural components of connective tissue such as bone, elastin and collagen. 6. They act as Lubricants and cushion for other tissues because they have the property of holding large quantities of water. 7. When a solution of glycosaminoglycans is compressed, the water is “squeezed out” and the glycosaminoglycans are forced to occupy a smaller volume, when the compression is released, the glycosaminoglycans return to their original hydrated volume because of the repulsion of their negative charges. This property is the cause of resilience of synovial fluid and the vitreous humor of the eye. Classification Mucopolysaccharides (GAGS) can be classified into two types A) Sulfur-free mucopolysaccharides Their sugar units are not sulfates, e.g., 1- hyaluronic acid B) Sulfur-containing mucopolysaccharides 2- Chondroitin 4 - and 6 sulfate. 3. Keratan sulfate. 4. Dramatan sulfate. 5. Heparin 6. Heparan sulfate  References ✓ Harper’s Illustrated Biochemistry, 30 th edition, 2014 ✓ Lipincott’s illustrated reviews: Biochemistry, sixth edition, 2013 ✓ Kaplan Biochemistry and medical genetics, 2021 All the following are glucosans EXCEPT: a- Starch b- inulin c- Cellulose d- Glycogen Amylopectin is characterized by all of the following, EXCEPT: a- It is a branched polymer. b- It is structure is very near to glycogen. c- Contains a IA and a glucosidic bond. d- Hydrolysis by amylase gives maltose and fructose. Cellulose is characterized by all of the following, EXCEPT: a- it is a glucosans b- Linear polymer c- Consists of β-glucose units d- Easily digested as it contains β glucosidic bonds Cellulose is not digested as: a- It contains α-glucosidic bond. b- It contains β -galactosidic bond. c- It contains β -glucosidic bond. d- It contains α-fructosidic bond. Heparin is: a- A disaccharide b- An oligosaccharide c- An amino sugar d- A non-sulfated mucopolysaccharide e- A sulfated mucopolysaccharide Hyaluronic acid ¡s: a- Glycoprotein b- Sulfated glucuronic acid c- Repeated disaccharide formed of glucuronic acid and N-acetyl glucosamine d- High molecular weight positively charged Homopolysaccharide Chemistry of protein Lecture I By Dr. Nashwa Abdel-Ghaffar Addel-Rahman Lecturer at Medical Biochemistry  Objectives ❑ Amino acids ❑ Peptides ❑ Protein ❑ Methods of precipitation of proteins ❑ Separation techniques for proteins and amino acids Amino acids ▪ Amino acids are the structural units of proteins and are obtained from them by hydrolysis. ▪ Each amino acid (except proline and hydroxyproline) has the following 4 groups or atoms: attached to alpha (a)carbon: 1. Amino group: (NH2 ). 2. Carboxyl group: (COOH). 3. Hydrogen atom (H). 4. Side chain or radical group (R). The general formula of amino acid ▪ Although about 300 amino acids exist in nature, only 20 of them can polymerize in protein structure. ▪ All these amino acids are 1. alpha-amino acids. This means that the amino group is attached to the α- carbon atom (next to the carboxyl group). 2. L-Amino acids i.e. α-amino group is on the left side configuration. N.B. All amino acids present in mammals are L-amino acids. D-amino acids are found in the cell walls of bacteria  glycine 3. optically active (except glycine) , i.e., can rotate plane polarized light. This is because the 4 groups attached to α - carbon are different. 4. amphoteric, i.e., In α-amino acids, both-COOH and –NH2 groups are attached to the same (α ) carbon atom ✓ react as acid (by COOH) and ✓ as alkali (by NH2). N.B proline is not an amino acid. It is an imino acid as It contains imino group (-NH). Classification of Amino Acids  Chemical classification of the amino acid a) Neutral amino acids: these are amino acids, which contain one COOH and one -NH2 groups e.g. glycine, alanine. b) Acidic amino acids: contain more than one -COOH group. e.g. Aspartate and glutamate. c) Basic amino acids: contain more than one -NH2 group. e.g. ornithine, lysine, Arginine, citrulline and histidine.  I- Neutral amino acids ▪ Aliphatic amino acids: includes glycine& alanine. ▪ Branched chain amino acids: includes valine, leucine & isoleucine. ▪ Hydroxy amino acids: contain –OH group in their side chain e.g., serine, threonine, tyrosine, hydroxyproline and hydroxy-lysine. ▪ Aromatic amino acids: e.g. phenylalanine, tyrosine and tryptophan ▪ Sulfur-containing amino acids:e.g. Methionine and Cysteine that gives cystine ▪ Heterocyclic amino acids: e.g. Histidine, Tryptophan, proline and hydroxyproline ✓ Histidine is basic in solution on account of the imidazole ring and often considered as basic amino acids. ✓ Tryptophan have indole ring and is often considered as aromatic amino acids since it has aromatic ring in its structure. ✓ Proline and hydroxyproline do not have a free -NH2 group but only imino group (NH) in a ring but can still function in the formation of peptides. Therefore, they are called as imino acids.  II) Acidic amino acids ▪ They contain 2 carboxyl groups and one amino group, e.g., glutamic acid and asparatic acid. ▪ These 2 acidic amino acids can produce 2 other amino acids (amide form) e.g. ✓ glutamic acid gives glutamine and ✓ asparatic acid gives asparagine.  III) Basic amino acids ▪ They contain 2 amino groups and one carboxyl group, e.g., Ornithine, citrulline, lysine, hydroxylysine and Arginine. ▪ Histidine heterocyclic amino acid is also considered basic O O  H2N CH C OH H2N CH C OH CH2  CH2 CH2  CH2 CH2  CHOH  CH2 CH2 NH2 NH2 Lysine Hydroxy lysine ▪ All amino acids are metabolized in human body to give bilogically active products. ▪ Ornithine & Citrulline do not enter in the synthesis of proteins. ▪ Lysine & Hydroxylysine participate in protein cross-linking. Nutritional classification of amino acids ▪ Essential amino acids are as follows (VITTAL LyMPH): ▪ Valine, ▪ Isoleucine, ▪ Threonine, ▪ Tryptophan, ▪ Arginine, ▪ Leucine, ▪ Lysine, ▪ Methionine, ▪ Phenylalanine & ▪ Histidine  Metabolic classification Ketogenic Ketogenic & Glucogenic glucogenic Leucine Lysine Rest of amino acids Isoleucine Tyrosine Tryptophan Phenylalanine A. Structural function: enter in the structure of: a) Body peptides and proteins: e.g. plasma proteins, tissue proteins, enzymes, etc. b) Hormone: some hormones are amino acid derivatives e.g. thyroxin. c) Amines: Some amino acid gives corresponding amines by decarboxylation e.g. histidine gives histamine which is vasodilator. d) Biologically active products. Heme, purines B. Neurotransmitters: Some amino acids as glycine and glutamate act as N.T C. Detoxication: Some amino acids are used in detoxication reactions Peptides  The reaction between –COOH group of an amino acid and –NH2 group of another amino acid leads to the formation of peptide bond. It is formed by removal of water. O O O O H2N CH C OH + H2N CH C OH H2N CH C HN CH C OH R R R R an amino acid an amino acid H2O Dipeptide ▪ Peptides are compounds, formed of less than 50 amino acids linked together by peptide bonds: - Dipeptide (2 amino acids and one peptide bond). - Tripeptide (3 amino acids and 2 peptide bonds). - Oligopeptide (3-10 amino acids). - Polypeptide (10-50 amino acids).  Glutathione (glutamyl, cysteinyl, Glycine) ▪ It is a tripeptide formed of 3 amino acids: glutamic, cysteine and glycine. ▪ Glutathione is commonly abbreviated as G-SH, where –SH indicates the sulfhydryl group of cysteine and it is the most active part of the molecule. Functions of glutathione: 1) Defence mechanism against certain toxic compounds (T):Glutathione combine with them to produce non-toxic compounds. T (toxic) + Glutathione -+ Non-toxic compound 2) Absorption of amino acids: glutathione has a role In transport of amino acids across intestinal cell membrane. 3) Protect against cell damage and hemolysis of RBCs: Glutathione break down the hydrogen peroxide (H 20 2 ) which causes cell damage and hemolysis. H20 2 +Glutathione------- H2O +oxidized glutathione 4) Activation of some enzymes. 5) Inactivation of insulin hormone by breaking its disulfide bonds. Insulin hormone ▪ It is formed from 2 polypeptide chains connected together by 2 disulfide linkages. Protein ▪ Proteins are organic compounds with a high molecular weight formed of carbon, oxygen, hydrogen and nitrogen ▪ They may also contain sulfur, phosphorus and metal ions. ▪ They are polymers formed of subunits called amino acids linked together by peptide linkage. ▪ The term protein is applied to describe molecules greater than 50 amino acids. Methods of precipitation of proteins 1. At the iso-electric point. 2. By various concentrations of salt solutions:, i.e., Salting out (sodium chloride, ammonium sulfate, magnesium sulfate),. 2. By heavy metals (mercury , silver) 3. By alkaloidal reagents (trichloroacetic acid and picric acid) 4. Alcohol precipitation 5. Heat coagulation 1. At the iso-electric point. ▪ Amino acids are amphoteric molecules; i.e. (They have both basic (NH2) & acidic ( ̶ COOH) groups.) ▪ Monoamino-monocarboxylic acids exist in aqueous solutions as zwitterions which means that they have both positive & negative charges: ✓ The α-carboxyl group is dissociated and negatively charged. ✓ The α -amino group is protonated and positively charged. ▪ Thus, the overall molecule is called zwitterion. It is elecctrically neutral (net charge is zero) and can't migrate in electric field. O O O + + H3N CH C OH H3N CH C O- H2N CH C O- R R R Cation Zwitterion Anion Separation techniques for proteins and amino acids Separation techniques for proteins and amino acids are 1. Electrophoresis. 2. Chromatography. 3. Precipitation. 4. Ultracentrifugation. 5. Dialysis. 6. Precipitation by antibodies.  1. Electrophoresis. ▪ It is movement of charged particles in an electric field towards the oppositely charged electrode.  2. Dialysis ▪ Dialysis means separation of colloids from crystalloids using a semi-permeable membrane. Dialysis membrane Crystalloids Colloids Amino acid Derived biologically important products Glycine (inhibitory a. Protein neurotransmitter) b. Heme (hemoglobin) c. Glycocholic acid (bile acid) d. Glutathione (antioxidant tripeptide) e. Creatine phosphate (Source of energy in muscles) f. Purines (nitrogenous base in nucleotides and nucleic acids) g. Choline (lipotropic factor) Glutamic acid (Excitatory a. Protein neurotransmitter) b. Gamma aminobutyric acid (GABA, inhibitory neurotransmitter) c. Glutamine (remove ammonia from brain) d. Glutathione. Amino acid Derived biologically important products Phenyl alanine which is a. Protein converted to Tyrosine b. Thyroid hormones c. Adrenaline and Noradrenaline d. Melanin pigments. Tryptophan a. Protein b.Serotonin (5-Hydroxytryptamine, stimulatory neurotransmitter) c. Melatonin d. Nicotinic acid Cysteine a. Proteins and polypeptide (Insulin) b. Glutathione c.in active site of enzymes d. Taurine (neurotransmitter) & Taurocholic acid (Bile acid) Proline & lysine They give hydroxyproline & hydroxylysine (activate collagen) 1. All proteins contain the (A) Same 20 amino acids (B) Different amino acids (C) 300 Amino acids occurring in nature (D) Only a few amino acids 3. Sulphur containing amino acid is (A)Methionine (B) Leucine (C) Valine (D) Asparagine 4. All the following are sulphur containing amino acids found in proteins except (A)Cysteine (B) Cystine (C) Methionine (D) Threonine 5. An aromatic amino acid is (A)Lysine (B) Tyrosine (C) Taurine (D) Arginine 6. An essential amino acid in man is (A)Aspartate (B) Tyrosine (C) Methionine (D) Serine 7. Which one of the following is semiessential amino acid for humans? (A)Valine (B)Arginine (C) Lysine (D) Tyrosine 8. A ketogenic amino acid is (A)Valine (B) Cysteine (C) Leucine (D) Threonine 2o. The basic amino acids are (A) Lysine (B) Bile acids (C) Glycine (D) Alanine 21. All the following are branched chain amino acids except (A) Isoleucine (B) Alanine (C) Leucine (D) Valine Chemistry of protein Lecture II By Dr. Nashwa Abdel-Ghaffar Abdel-Rahman Lecturer at Medical Biochemistry Department  Objectives ❑ Bonds Responsible For Protein Structure ❑ Structure of Proteins ❑ Denaturation of proteins ❑ Classification of Proteins ❑ Biological importance of Proteins  Bonds Responsible For Protein Structure Bonds Responsible For Protein Structure I- Strong bonds: ✓ Peptide bonds (primary bond). ✓ Disulfide bonds (secondary bond). II- Weak bonds: ✓ Hydrogen bond. ✓ Hydrophobic bond. ✓ Electrostatic bond.  Strong bonds Peptide bonds (primary bond): ▪ It is a covalent bond formed by a reaction between amino group of one amino acid and a carboxylic group of the next amino acid with the loss of H2O that required ATP. ▪ It is the strongest bond in the protein molecule that resists denaturation ▪ It is called primary because it is the only bond in the primary structure of the polypeptide. Disulfide bonds (secondary bond): ▪ The disulfide bond is formed between the SH groups of two cysteine residues within same (intra-chain) or two different polypeptide chains (inter-chain). ▪ It maintain secondary structure of a peptide chain or connects two polypeptide chains together in the tertiary structure. ▪ It follows the peptide bond in strength but liable to denaturation.  Weak bonds Hydrogen bond ▪ Hydrogen bond is a weak bond formed between the hydrogen atom of –NH of a peptide bond on one peptide chain and the oxygen of C=O of another peptide bond on an adjacent peptide chain or a loop belongs to same peptide chain. Hydrophobic bonds ▪ The non polar side chains of neutral amino acids tend to associate in hidden core of protein molecule away from solvent. Electrostatic bonds( Ionic bond) ▪ These are salt bonds formed between oppositely charged groups in the side chains of amino acids e.g. ε-amino group of lysine and the carboxyl group of asparatic acid  Structure of Proteins ▪ There are 4 levels or orders of organization of the structure protein molecule; ✓ primary, ✓ secondary, ✓ tertiary and ✓ quaternary structures. ▪ This complication gives the molecule its functional domain to explain its structure- function requirements that if changes due to mutation will give non-functional protein and, therefore, a disease  Primary Structure ▪ It is the arrangement and number of amino acids that enter in the structure of protein. ▪ Peptide bonds are covalent bonds responsible for primary structure.  Secondary Structure ▪ It is spatial relationship of adjacent amino acid residues (first and fourth). ▪ They are 2 forms : a-helix or P-pleated sheets ▪ Hydrogen bonds are responsible for secondary structure  Tertiary Structure ▪ It is the spatial relationship of more distant amino acid residues. ▪ Hydrogen, hydrophobic, electrostatic and disulfide bonds are responsible for tertiary structure There are 2 forms of tertiary structure : fibrous (extended) and globular (com pact) form  Quaternary structure ▪ It is the arrangement of proteins having more than one subunit. ▪ Hydrogen , electrostatic and hydrophobic bonds are responsible for quaternary structure. Definition of Denaturation ▪ Unfolding and loss of secondary, tertiary and quaternary structure. ▪ Does not affect primary structure i.e. not accompanied by hydrolysis of peptide bond. ▪ Denaturation may be reversible (in rare cases) Denaturating factors include: 1. Heat: causes coagulation and precipitation of certain proteins like albumin. 2. Strong acids or bases: : cause disruption of hydrogen and electrostatic bonds 3. Heavy metals: as lead and mercury salts: 4. Enzymes: e.g. Digestive enzymes. 5. Urea, ammonium sulphate and sodium chloride: cause precipitation of proteins. 6. Repeated freezing and thawing: cause disruption of hydrogen and other bonds. Effects (or fate) of Denaturation 1. Physical changes: Increase in viscosity, decreased solubility (insoluble). 2. Chemical changes: as loss of hydrogen, hydrophobic and electrostatic bonds but not of the peptide or disulfide bonds. This leads to loss of secondary tertiary and quaternary structures but not of the primary structure. 3. Biological changes: which include loss of enzymatic, hormonal and other biological properties of proteins.. Significance and Application of denaturation 1. Denatured proteins, e.g., cooked meat are easily digested 2. Blood samples to be analyzed for small molecules, e.g., uric acid and glucose are first treated with acid such as trichloroacetic acid or phosphotungestic acid to precipitate the plasma proteins (by denaturation). 3. Detection of albumin in urine by heat coagulation test is based on denaturation by heat. 4. Several approaches for stoppage of bleeding and treatment of burns is based on precipitation and denaturation of a superficial protein layer. 5. Avoidance of denaturation is important for biological samples used for determination of enzymatic, hormonal or protein contents. I. According to the biological importance of the protein: ✓ Proteins of high biological value ✓ Proteins of low biological value II. According to the axial ratio of the protein molecule: Axial ratio = Length/Width of the protein molecule ✓ Fibrous proteins ✓ Globular proteins III. According to the chemical composition of the Protein: ✓ Simple Proteins ✓ Conjugated proteins ✓ Derived Proteins  According to the biological importance of the protein Proteins of high biological value ▪ These are all proteins of animal origin (with a few exceptions) and some proteins of plant origin, that contain all the 10 essential amino acids in well balanced amounts and are easily digestible. ▪ Examples of animal proteins include; milks and its products, egg, liver, fishes, red and white meats. ▪ Examples of the few plant proteins of high biological value are lentils and broad beans  Proteins of low biological value ▪ These are proteins that ✓ are deficient in one or more of the essential amino acids or ✓ containing very little amount of one of them or ✓ are indigestible. ▪ Most of plant protein are of low biological value and a very few animal proteins are also of low biological value such are ✓ collagen because is deficient in tryptophan and cysteine and ✓ keratins because they are indigestible.  According to the axial ratio of the protein molecule I. Fibrous proteins ▪ They have an axial ratio of more than 10. Axial ratio = Length/Width of the protein molecule. ▪ They are ✓ fairly stable proteins in which the straight polypeptide chains lie parallel (or antiparallel) to one another along a single axis forming fibers or sheets. ✓ usually insoluble and non motile. ▪ Examples:- a. Keratin proteins in hairs, wool and skin. b. Myosin is the major protein of muscles  II. Globular proteins ▪ Their axial ratio is less than 10. ▪ Their one or more peptide chains are folded or coiled on themselves in a very compact manner. ▪ They are ✓ less stable than fibrous proteins ✓ usually soluble and motile. ▪ Examples are albumin, globulins, and insulin. According to the chemical composition of the Protein  I. Simple Proteins ▪ These are proteins which on hydrolysis produce amino acids only Albumins & Globulins Albumin Globulin - Soluble in water and salt solution - Soluble in salt solution. - M.W.: 68 KDa. - M.W.: 150 KDa. -Precipitated by full saturation with -Precipitated by half saturation with ammonium sulfate. ammonium sulfate. -They are present in: serum, egg, milk They are present in: serum,milk, eggs - It functions as transporting protein for - It functions in transport also but its elements, vitamins, and hormones other major function is being antibodies. than keeping blood osmosis.  Scleroproteins (Albuminoids) ▪ Scleroproteins are characterized by their extreme insolubility in water, dilute acids and the most common reagents. ▪ They are extracellular fibrous proteins and never present inside the cells. ▪ Their main function is the protection of the body. ▪ Hairs, nails, natural silk and connective tissues, contain scleroproteins. ▪ They are never present in plants. ▪ The main important groups of scleroproteins are: ✓ Keratins ✓ Collagens ✓ Elastins ✓ Reticulins  Keratins ▪ Keratin is a typical fibrous protein. ▪ It consists of long polypeptide chains. ▪ Keratins are present in ✓ hairs, nails and superficial layer of the skin. ✓ They form the intermediate filaments of the cytoskeleton in the epithelial cells. ▪ Keratins are highly insoluble compounds. ✓ They are insoluble in all protein solvents, and are not digestible by proteolytic enzymes. ▪ The sulfur content of keratin is high. It is present in the form of cystine, which is responsible for the stability and insolubility of keratins.  Collagens ▪ They are present in white fibrous connective tissues, tendons and bones. ▪ They form about 30% of total body protein. ▪ Collagen molecules consist of 3 polypeptide chains, ✓They are twisted around each other forming triple helix molecule. ✓They are held together by hydrogen bonds. ✓Each helical turn contains only 3 amino acids. ▪ There are 19 types of collagen formed of different combinations of 30 type of subunits (polypeptides). ▪ Collagen is rich in glycine, proline and hydroxy proline but low in sulfur containing amino acids. ▪ Glycine, proline and hydroxy proline form about 2/3 of the total amount of amino acids present in the collagen molecule. ▪ Collagen is low biological value protein, because of ✓its high content of glycine and ✓it is deficient in tryptophan. ▪ When Collagen is boiled for a long time with water, it changes to gelatin. Thus, gelatin is a derived protein obtained from the partial hydrolysis of collagen  II. Conjugated proteins ▪ They are simple proteins combined with a non-protein group called prosthetic group. So on hydrolysis, they give amino acids and prosthetic group. ▪ They include: ✓ Phosphoproteins: as in casein (milk), vitellin (egg yolk). ✓ Lipoproteins. As in VLDL, HDL. ✓ Glycoproteins. ✓ Metalloproteins. As in Trasnferrin & ferritin (Iron binding proteins) ✓ Chromoproteins. As in hemoglobin  III. Derived Proteins ▪ They include: ✓ Primary derived proteins: Denatured protein: e.g., coagulated albumin or globulin or gelatin ✓ Secondary derived proteins : Hydrolytic product of protein: e.g. Protein  Proteoses  Peptone  Polypeptide. 1. Nutritional role: Provide the body with essential amino acids, nitrogen and sulfur. 2. Catalytic role: All enzymes are proteins in nature. 3. Hormonal role: Most of hormones and all cellular receptors are protein in nature. 4. Defensive role: The antibodies (immunoglobulins) that play an important role in the body’s defensive mechanisms are proteins in nature. 5. Osmotic pressure Plasma proteins are responsible for most effective osmotic pressure of the blood. 6. Transport role: Proteins carry lipids in the blood, hormones, e.g., thyroid hormones and minerals, e.g., calcium, iron and copper. Hemoglobin (a chromo- protein) carries O2 from the lung to tissues is a protein. 7. Structural role: Proteins are the main structural component in bone, muscles cyto-skeleton and cell membrane. 8. Blood clotting: coagulation factors are proteins. 9. Control of gene expression: Most factors required for DNA replication transcription and mRNA translation are protein in nature Which amino acid can form disulfide bonds? a- Glycine b- Proline c- Glutamate d- Cysteine Which of the following pairs of amino acids might contribute to protein conformation by forming electrostatic interactions? a- Glycine and leucine b- Glutamate and lysine c- Phenylalanine and tyrosine d- Lysine and arginine The highest concentration of cystine can be found in: a- Melanin b- Keratin c- Collagen d- Myosin Glycine and proline are the most abundant amino acids in the structure of: a- Hemoglobin b- Myoglobin c- Insulin d- Collagen Keratin is a derived protein a- True b- False