Carbohydrates - Laboratory Work

Carbohydrates A. Definition B. Roles C. Monosaccharides-Chemistry 1. Chirality a. One or more asymmetric carbons b. Linear and ring forms 2. Derivatives: the chemistry of carbohydrates a. Oxidation b. Reduction c. d. Ester formation Amino sugars i. ii. i. ii. C1 C6 C1/C2 Other carbons E. Polysaccharides 1. Polymers of glucose 3. Polymerization a. The Glycosidic Bond b. Non-covalent bonds in macro-molecular structure D. Oligosaccharides 1. 2. 3. 4. Glycoproteins & glycolipids O-linked N-linked Sequence determination-ABO a. b. Alpha polymers Beta polymers 2. Polymers of disaccharides a. b. c. Extracellular matrix Glycosaminoglycans i. Hyaluronic acid ii. Chondroitin/Dermatan sulfate iii. Heparin sulfate iv. Keratan sulfate Proteoglycans Carbohydrates Oligosaccharides: Determination of Sequence – In vertebrates, ganglioside carbohydrate composition determines blood groups. CH2OH OH 6CH OH 2 O–Cer OH OH OH OH OH OH OH OH L-Fuc a(1à2)-D-Gal b(1à4) D-GlcNAc b-Ceramide O blood group D-Gal a(1à3) D-GalNAc a(1à3) B blood group A blood group Carbohydrates Oligosaccharides: Determination of Sequence – In vertebrates, ganglioside carbohydrate composition determines blood groups. CH2OH OH 6CH OH 2 O–Cer OH OH OH OH OH OH OH OH L-Fuc a(1à2)-D-Gal b(1à4) D-GlcNAc b-Ceramide O blood group D-Gal a(1à3) D-GalNAc a(1à3) B blood group A blood group Carbohydrates Polysaccharides Carbohydrates Polysaccharides • The majority of natural carbohydrates are usually found as large polymers. • These polysaccharides can be: – homopolysaccharides (one type of monomer unit*) – heteropolysaccharides (multiple types of monomer units) – linear (one type of glycosidic bond) – branched (multiple types of glycosidic bonds) • Polysaccharides do not have a defined molecular weight. – This is in contrast to proteins because, unlike proteins, no template is used to make polysaccharides. – Polysaccharides are often in a state of flux; monomer units are added and removed as needed by the organism. Carbohydrates Polysaccharides: Polymers of Glucose Homopolymers of Glucose: - Starch - Glycogen - Cellulose - Chitin • Glycogen and Starch are the main storage polysaccharides for energy. – Glycogen and starch are insoluble due to their high molecular weight and often form granules in cells. – Molecular weight reaches several millions (~200 x 106)(can see in microscope). • Glycogen is a branched homopolysaccharide of glucose. – Glucose monomers form a(1 ® 4) linked chains. – There are branch points with a(1 ® 6) linkers every 8–12 residues. • Starch is a mixture of two homopolysaccharides of glucose. – Amylopectin is like glycogen, but the branch points (a(1 ® 6) linkages) occur every 24–30 residues. – Amylose is an unbranched polymer of a(1 ® 4) linked residues. Carbohydrates Polysaccharides: Glycogen & Starch Glycosidic Linkages in Glycogen and Starch (Glc-a(1à4)Glc)n with a(1à6) branching, where n≈106 Non-reducing à reducing Haworth projections are not good to show the actual shape, which is helical! Carbohydrates Polysaccharides: Glycogen & Starch n≈150 I2 2 3 5 1 4 6 Axial = a-OH 4 6 3 5 2 1 • Granules contain enzymes that synthesize and degrade these polymers. • Glycogen and amylopectin have one reducing end but many nonreducing ends. • Enzymatic processing occurs simultaneously in many nonreducing ends. Carbohydrates Polysaccharides: Glycogen & Starch n≈150 I2 2 3 5 1 4 6 Axial = a-OH 4 6 3 5 2 1 Carbohydrates Polysaccharides: Glycogen & Starch Mixture of Amylose and Amylopectin in Starch Carbohydrates Polysaccharides: Homopolymers of Glucose: - Starch - Glycogen • Cellulose is a linear homopolysaccharide of glucose. – Glucose monomers form b(1 ® 4) linked chains. - Cellulose – Hydrogen bonds form between adjacent monomers. – There are additional H-bonds between chains. - Chitin – Structure is now tough and water insoluble. – It makes up almost 50% of plant mass; in cell walls. – Cotton is nearly pure fibrous cellulose. • Chitin is a linear homopolysaccharide of Nacetylglucosamine (GlcNAc). – N-acetylglucosamine monomers form b(1 ® 4)linked chains. – forms extended fibers that are similar to those of cellulose – hard, insoluble, cannot be digested by vertebrates – structure is tough but flexible, and water insoluble – found in cell walls in mushrooms and in exoskeletons of insects, spiders, crabs, and other arthropods Carbohydrates Polysaccharides: Cellulose & Chitin Glc b 1 OH 4 Glc OH Glycosidic Linkages in Cellulose & Chitin (Glc-b(1à4)Glc)n with NO branching, n ≈ 104 Notice that each chair is alternatively flipped 3 Equitorial = b-OH 4 THE most abundant biological molecule in the world: ~300 trillion kg 2 5 6 1 Carbohydrates Polysaccharides: Cellulose & Chitin • The fibrous structure and water insolubility make cellulose a difficult substrate to act upon. • Most animals cannot use cellulose as a fuel source because they lack the enzyme to hydrolyze b(1®4) linkages (b-Amylase or cellulase). • Fungi, bacteria, and protozoa secrete cellulase, which allows them to use wood as source of glucose. • Ruminants and termites live symbiotically with microorganisms that produce cellulase and are able to absorb the freed glucose into their bloodstreams. • Cellulases hold promise in the fermentation of biomass into biofuels. Carbohydrates Polysaccharides: Cellulose & Chitin b b 4 1 4 (GlcNAc-b(1à4)-GlcNAc)n Carbohydrates Polysaccharides: Polymers of Disaccharides First, need to describe the Extracellular Matrix (ECM) • • • Material outside the cell Strength, elasticity, and physical barrier in tissues (varies tremendously) Main components – proteoglycans – collagen & elastin fibers • Proteoglycans – Different glycosaminoglycans are O-linked to the “core protein.” – Linkage from anomeric carbon of xylose to serine hydroxyl – Our tissues have many different core proteins; aggrecan is the best studied. ECM is a barrier for tumor cells seeking to invade new tissues. Carbohydrates Polysaccharides: Polymers of Disaccharides Extracellular Matrix (ECM) Carbohydrates Polysaccharides: Polymers of Disaccharides Extracellular Matrix (ECM) • Main components – proteoglycans – collagen & elastin fibers • Proteoglycans – Different glycosaminoglycans are O-linked to the “core protein.” • ECM is a barrier for tumor cells seeking to invade new tissues. – Some tumor cells secrete heparinase that degrades ECM. • Cosmetics Carbohydrates Polysaccharides: Polymers of Disaccharides Glycosaminoglycans (the carbohydrate part of proteoglycans) • • Linear polymers of repeating disaccharide units (sugarX-sugarY)n One monomer (sugarX) is either sugar acid or Gal – uronic acids (C6 oxidation) – Most have sulfate esters • One monomer (sugarY) is either: – N-acetyl-glucosamine (GlcNAc) or N-acetyl-galactosamine (GalNAc) – Also sulfate esters • Extended hydrated molecule – Negatively charged – minimizes charge repulsion • Forms meshwork with fibrous proteins to form extracellular matrix – connective tissue – lubrication of joints • Form huge (Mr > 2 • 108) noncovalent aggregates (Hyaluronan and Aggrecan). – They hold a lot of water (1000´ its weight) and provide lubrication. – Very low friction material – Covers joint surfaces: articular cartilage o reduced friction & load balancing Carbohydrates Polysaccharides: Polymers of Disaccharides Glycosaminoglycans Carbohydrates Polysaccharides: Polymers of Disaccharides Glycosaminoglycans Carbohydrates Polysaccharides: Polymers of Disaccharides Glycosaminoglycans Carbohydrates Polysaccharides: Polymers of Disaccharides Glycosaminoglycans Carbohydrates Polysaccharides: Polymers of Disaccharides Glycosaminoglycans Carbohydrates Polysaccharides: Polymers of Disaccharides Glycosaminoglycans Linkages all b(1à3) Linkages all b(1à4) (COO–) GlcUA – GalNAc GlcUA – GlcNAc L-IdoUA (C5 epimer C4sulfate only C6/C4-sulfate of D-Glc) & – (SugarX – SugarY)n – Linkages all b(1à4) GlcUA(IdoUA) or Gal GlcNAc or GalNAc or GlcN b(1à3) a(1à4) (COO–) GlcUA – GlcN Gal – L-IdoUA GlcNAc Sulfate everywhere, including GlcN GlcNAc C6-sulfate Glycosaminoglycans – (SugarX – SugarY)n – GlcUA(IdoUA) or Gal GlcNAc or GalNAc or GlcN GlcUA – GlcNAc GlcUA GlcUA – GalNAc (C6/C4-sulfate) Gal – GlcNAc C6-sulfate Linkages all b, and opposite of Hyl & CS alternate (1à4) & (1à3) GlcUA GlcUA or IdoUA – GlcN Sulfate everywhere, including GlcN Linkages all (1à4), alternate a & b IdoUA Hyl & Aggrecan Hyl & Aggrecan Carbohydrates Polysaccharides: Proteoglycans = Polymers of Disaccharides (glycosaminoglycans) + proteins a.k.a. - mucopolysaccharides Aggrecan Carbohydrates Polysaccharides: Proteoglycans = Polymers of Disaccharides (glycosaminoglycans) + proteins a.k.a. - mucopolysaccharides Sulfated glycosaminoglycans attached to a large rod-shaped protein in cell membrane • syndecans: protein has a single transmembrane domain • glypicans: protein is anchored to a lipid membrane Interact with a variety of receptors from neighboring cells and regulate cell growth Carbohydrates Polysaccharides: Proteoglycans = Polymers of Disaccharides (glycosaminoglycans) + proteins a.k.a. - mucopolysaccharides Sulfated glycosaminoglycans attached to a large rod-shaped protein in cell membrane • syndecans: protein has a single transmembrane domain • glypicans: protein is anchored to a lipid membrane Interact with a variety of receptors from neighboring cells and regulate cell growth Carbohydrates Polysaccharides: Proteoglycans = Polymers of Disaccharides (glycosaminoglycans) + proteins a.k.a. - mucopolysaccharides • Some integral membrane proteins are proteoglycans. • Other integral membrane proteins are receptors for extracellular proteoglycans. • These proteins link cellular cytoskeleton to the ECM and transmit signals into the cell to regulate: – – – – – – • Integrins (which then interact with Actin) cell growth cell mobility apoptosis wound healing In chondrocytes, huge amounts of cartilage proteoglycans are produced (Aggrecan) – – – – • syndecans Resilience Plasticity Cushioning Due to reversible hydration Heparin is linear polymer, 3–40 kDa. – – – – – – – Heparan sulfate is heparin-like polysaccharide but attached to proteins. Highest density of negative-charge of any biomolecule Prevent blood clotting by activating protease inhibitor antithrombin III Binding to various cells regulates development and formation of blood vessels. Modulates growth and development by binding growth-factor receptors Important role in cancer metastasis Can also bind to viruses and bacteria and decrease their virulence Carbohydrates Polysaccharides: Proteoglycans = Polymers of Disaccharides (glycosaminoglycans) + proteins a.k.a. - mucopolysaccharides Thrombin activates the blood-clotting cascade by proteolysis Anti-thrombin keeps thrombin inactive, preventing proteolysis Prevent blood clotting by activating protease inhibitor anti-thrombin III Role of Heparin Thrombin + Anti-thrombin ⇌ Thrombin•Anti-thrombin Ka is 1000 larger in presence of Heparin TABLE 7-2 Structures and Roles of Some Polysaccharides Size (number of monosaccharide units) Typea Repeating unitb Amylose Amylopectin HomoHomo- (α1èS4) Glc, linear (α1èS4) Glc, with (α1èS6) Glc branches every 24–30 residues 50–5,000 Up to 106 Glycogen Homo- (α1èS4) Glc, with (α1èS6) Glc branches every 8–12 residues Up to 50,000 Energy storage: in bacteria and animal cells Cellulose Homo- (β1èS4) Glc Up to 15,000 Structural: in plants, gives rigidity and strength to cell walls Chitin Homo- (β1èS4) GlcNAc Very large Structural: in insects, spiders, crustaceans, gives rigidity and strength to exoskeletons Dextran Homo- (α1èS6) Glc, with (α1èS3) branches Wide range Structural: in bacteria, extracellular adhesive Peptidoglycan Hetero-; peptides attached 4)Mur2Ac(β1èS4) GlcNAc(β1 Very large Structural: in bacteria, gives rigidity and strength to cell envelope Agarose Hetero- 3)D-Gal (β1èS4)3,6- anhydroL-Gal(α1 1,000 Structural: in algae, cell wall material Hyaluronan (a glycosaminoglycan) Hetero-; acidic 4)GlcA (β1èS3) GlcNAc(β1 Up to 100,000 Structural: in vertebrates, extracellular matrix of skin and connective tissue; viscosity and lubrication in joints Primer Starch Roles/significance Energy storage: in plants Starch