BIOCHEMISTRY LECTURE GUIDE AND LABORATORY MANUAL Compiled by: EVANGELINE A. LAYUG-MARTINEZTABLE OF CONTENTS PART I: LECTURE GUIDE Chapter 1 Introduction to Biochemistry Chapter 2 The Cell and Its Chemistry Chapter 3 Carbohydrates Chapter 4 Amino Acids and Proteins Chapter 5 Enzymes Chapter 6 Lipids Chapter 7 Nucleic Acids Chapter 8 Vitamins Chapter 9 Blood Chapter 10 Hormones Chapter 11 Urine Chapter 12 Bioenergetics Chapter 13 Chemistry of Digestion Chapter 14 Metabolism PART Il: LABORATORY MANUAL Laboratory Safety Rules Rules for the Students Experiment 1 pH Determination and Buffer Preparation Experiment 2 Buffer Capacity Experiment 3 Compound Microscope Experiment 4 The Cell and the Cell Membrane Experiment 5 Tests for Carbohydrates Experiment 6 Isolation of Polysaccharides Experiment 7 Isolation and Characterization of Casein Experiment 8 Identification of Amino Acid by Paper Chromatography Experiment 9 Action of Enzymes Experiment 10 Lipids Experiment 14 Nucleic Acids Experiment 12 Blood Analysis Experiment 13 Urinalysis Experiment 14 Metabolism Appendix Bibliography 20 30 41 50 63 76 85 93 103 108 111 114 126 127 128 132 136 140 146 150 155 162 167 173 180 185 189 194 199 201PREFACE This book has been designed for one-semester course in Biochemistry and especially Prepared for Nursing and Psychology students. This book covers the topics of Biochemistry so designed to be la students a better understanding of the subject matter. Topics are presented in simple and coherent manner. Theories and principles of aes a Presented in the first Part of the book, the lecture guide. At the end of Piha chapter in the first part, exercises are provided to test the understanding of rll students. And the second part contains laboratory experiments which emphasiz the development of analytical mind through questions provided during the Performance of the experiments.CHAPTER? INTRODUCTION TO BIOCHEMISTRY Objectives: At the end of this chapter, the students should be able to: 1, define Biochemistry 2. identify the objective as well as the scope of Biochemistry 3. know and understand the different chemical reactions occurring inside living organisms 4. know the importance of biochemistry 5. know the uses of biochemistry What is Biochemistry Biochemistry is the study of life processes, structures, mechanisms, reactions, at the molecular level. It had its roots in Chemistry, Biology and Genetics, which had been moving independent of each other. The first interrelationship of the disciplines came in the 19" century when Friedrich Wohler synthesized urea, an organic compound from the inorganic precursor — ammonium cyanate. In 1897, Edward Buchner and Hans Buchner found that dead yeast cells are still able to undergo fermentation, demonstrating that reactions can occur in vitro. This opened the doors for the study of enzymes and reaction mechanisms. In the mid-1900, Gregor Mendel started describing genes. However, it was not until the 20" century that genes were determined to be made up of deoxyribonucleic acid (DNA). James Watson and Francis Crick proposed the structure of the DNA in 1953, and from then on the intermarriage of the three disciplines was complete. Together with the rapid development in the emerging discipline, Biochemistry as a science came with the development of new and more powerful technologies. Among these were tools for sequencing proteins, ultracentrifuge, and electron microscope. The discovery of restriction endonucleases in the 21% century fused the three disciplines into a new branch of science - molecular biology (Figure 1.1). From this point on, interests in the molecular basis of life have been tremendous and new studies and new developments are now being continuously explored. Today, biochemistry stands at the crossroads of many life sciences. It is the foundation of other disciplines — molecular biology, health sciences, agriculture, nutrition, etc. from which it also derives knowledge. It will continue to grow with new developments and new technologies. Biochemistry 1 elmartinezMOLECULAR ‘BIOLOGY. (st onan seaimnces of higher onarieme Historealare priokg al Siscovrns tom ENA cr sar basis of carcer emma manor Daa R— Feet tnctaral geromm semanas HuranGenore Propet begins Gene reguiaton in eukarytes rare encase wal Scieprnmtatnintota retentions rere ssn ae ieee oe eee ARREARS NT Reg i =e very, Nec Leod. ae McCarty show DNA — Claude isolates fist mia hordtial factions Ser geesieneonnae apr efeenacekittide bealics sinensis wvboy coop nce poeple st frcsnnn Jedocoventes —— vena eral seceotinn ‘eeemecmaa tee grooves oateay Hrelaer Weber arthestes Figure 1-1; Interweavin biology, and genetics. Adapted from (San Francisco, CA: Addi Biochemistry Edunrc a Has Buctrer apa inthe laboratory BioceMsTRY woh |e one ceLL BICLOGY,; W. M. Becker, lorverrs esr tor On SQ lout trues, FecsscoveryofMendats tas by Corres, von Ticker rs in Seronaniterreraton eh aru a Fatteory of heredity _ | imu acrnetran NA r fmertonct ive —fpetrrovares cary =a microtome jgeretic intonation eee sig Omit ot once cicoas OMA me ‘Mendeldisc overs his a ree ‘urnrecta nas of get Saar Virchow every cet pr } corres troma cet ‘Snares: Seedionand caltheony Brown describes g of the historical traditions of biochemistry, cell ; L. J. Kleinsmitn, and J. Hardin, The World of the Cell bivees ison Wesley Longman, 2000). © Addison Wesley Longman, Inc elf Nez—— ee hae Slochemistry Living organisms are composed of molec all the familiar laws of chemistry. if janisms ai molecules, which are inanimate, how do these molecules posed a remarkable characteristics we call life? How te a that a living organi appears to be more than the sum of it ' The basic goal of the science of bioch the collections of inanimate molecules that Constitute living organisms interact with each other to maintain and perpetuate life. Although biochemistry yields 'mportant insights and Practical applications in medicine, agriculture, Nutrition, industry, it is ultimately c i A . 5 fi oncern\ wonder of life itself. 7 P — remistry is to determine how Scope of Biochemistry . Biochemistry is the chemistry of living matter in its different phases of activity, from the smallest microorganisms such as viruses to the most complex ones as humans, The relationship of the living beings to their environment; the Processes by which an exchange of chemical substances takes place between the living organism and its environment through digestion, absorption and excretion; the Processes by which the absorbed materials are utilized for anabolic reactions leading to growth and formation of tissues; the metabolic breakdown of the materials to supply energy for all the above processes; the mechanisms which regulate with precision all these processes by means of hormonal and neuro-regulatory stimuli _ all these fall under biochemistry. Chemical Reactions Occurring in Living Organism 1. Oxidation — most of the energy liberated by living matter is derived from the oxidation of organic substances such as carbohydrates, fats and proteins. It occurs when it gains oxygen or loses hydrogen. a. Aerobic Oxidation — takes place in the presence of oxygen b. Anaerobic Oxidation — occurs in the absence of oxygen 2. Reduction - it is the reverse of oxidation, a gain of hydrogen or loss ganic compound 3 Mae oes of a "substance with one or more water molecules forming an unstable “substance-water complex”, which is subsequently fragmented. Large molecules are broken down into smaller and simpler forms. elmartinez Biochemistry4. Condensation — simple fragments unite with one another to form @ more complex compound, and involves removal of water molecule 5. Decarboxylation — removal of carboxyl group of organic acids forming carbon dioxide 6. Transfer Reactions: ‘a. Phosphorylation — addition of a phosphate group to an organic molecule b. Transamination — transfer of an amino group from one compound to another c. Acetylation - addition of an acyl group to an organic molecule d. Transmethylation — transfer of a methyl group from a methyl donor to another compound All these reactions occur in the living organisms with the aid of enzymes. Importance of Biochemistry Biochemistry is important to physiology as both biochemistry and physiology overlap and merge. The physicians must have the knowledge of biochemistry and biochemical tests to diagnose the disease and for treating patients. The pharmacologist must know biochemistry, since the action of drugs involves some alterations in the biochemical reactions of body. Microbiologists and pathologists are concerned with chemical changes produced in various diseases. Microbiologists are also concerned with vaccines, sera, antitoxins. Health professionals who acquire a sound knowledge of biochemistry will be in a strong position to deal with two central concerns of the health sciences: 1) the understanding and maintenance of health 2) the understanding and effective treatment of disease Uses of Biochemistry The results of biochemical research are used extensively in the world outside the laboratory - in agriculture, medical sciences, nutrition, and many other fields. In clinical chemistry, biochemical measurements on peeenelp diagnose illnesses and monitor responses to treatment. Liver Biaea ieee routinely diagnosed and monitored by measurements of peso of enzymes called transaminases and of a hemoglobin Product called bilirubin. Biochemistry 4 elmartine!Pharmacology and toxicology are concerned with the effects of external chemical substances on metabolism. Drugs and poisons usually act by interfering with specific metabolic pathways. A good example is the antibiotic penicillin, which kills bacteria by inhibiting an enzyme that synthesizes an essential polysaccharide of the bacterial cell wall. Because animal cells do not synthesize these polysaccharides, they are not harmed by this inhibitor, which can therefore be used therapeutically. A particularly exciting prospect in contemporary biochemistry is that of creating so-called designer drugs. If the target site for action of a drug is a protein enzyme or receptor, determining the detailed molecular structure of that target allows us to design inhibitors that bind to it with great selectivity. Early products of this drug architecture are now being tested. In the mid-1980s the detailed molecular structures of viruses began to be determined, opening the possibility of rational design of antiviral agents as well. Herbicides and pesticides, in many instances, act in similar ways - by blocking enzymes or receptors in the target organism. The first generations of these toxic agents (such as DDT) were so nonspecific in their effects that organisms other than the target populations were often affected, resulting in unforeseen and often severe damage to the environment. Furthermore, the indiscriminate use of these agents gave rise to resistant target populations, so that an ever-increasing number of toxins needed to be used. Biochemistry is involved in understanding che actions of herbicides and pesticides, in increasing their selectivity, and in understanding and dealing with mechanisms by which target organisms become resistant to them. Thus, biochemistry has become an important component of environmental science. Biochemistry elmartinez; Exercise No. 4 introduction to Biochemistry CN: ———__ SCORE: DATE: NAME: ao SECTION: Test I. Simple recall, Identify the following: - 1. Itis referred to as the chemistry of life. tL 2. He synthesized urea from ammonium cyanate. Stang 3 eee el }rte roots of Biochemistry tiie einai 6, std tins 6. 1 et sS7. + They proposed the structure of DNA ——______ 8. Type of reaction that involves addition of water ———__________ 9. Type of reaction that involves transfer of amino phosphate group from one molecule to another Lian) 10. Type of reaction that involves a gain of hydrogen —__________ 11. Type of reaction that involves a loss of oxygen —fky) 12. 13. + They discovered that dead yeast cells still undergo fermentation 14, 15. ] What are the two concerns of health sciences? Test Il. Answer the following questions: 4. What is the major objective of Biochemistry? 2. What is the scope of Biochemistry? 3. Explain the two concerns of health sciences. 4. Give one use of Biochemistry and explain. Biochemistry 6 elmartinezCHAPTER 2 THE CELL AND ITS CHEMISTRY Objectives: At the end of this chapter, the students should be able to: 1. define cell 2. identify the parts and understand the function of each part 3. identify the constituents of a cell | 4. have a general knowledge of the chemical and biological properties of water 5. describe how buffers function to maintain a constant physiological pH Cells are the structural and functional units of all living organisms. The smallest organisms consist of single cells and are microscopic, whereas larger organisms are multicellular (Figure 2.1). The human body, for example, contains at least 10" cells. Unicellular organisms are found in great variety throughout virtually every environment from cold to hot to the inner recesses of larger organisms. Multicellular organisms contain many different types of cells, which vary in size, shape and specialized function. Yet no matter how large and complex the organism, each of its cells retains some individuality and independence. In order to understand living processes, we must therefore understand cellular processes. And one of the basic methods of understanding cellular processes is to study the processes of the component structures. Types of Cells There are two types of cells: prokaryotic and eukaryotic cells. Prokaryotes are organisms composed of simple cells, with no membrane- enclosed organelle systems. Its genome consists of a single circular loop ONA; respiratory processes may or may not be present. If present, the respiratory processes occur and are found in the membrane while all other Structures necessary for its survival is found in the cytoplasm. Eukaryotes are organisms made up of complex cells with highly organized organelle systems. A membrane that separates it from its surroundings encloses each organelle. A double-membrane called the nuclear envelope encloses the genome. Respiratory processes occur in an organelle called the mitochondria. Other functions are assigned to other organelles such as the lysosomes, peroxisomes, golgi bodies, mitochondria, chloroplast, endoplasmic reticulum, cytoskeleton, etc. Biochemistry 7 elmartinez:|_*¢ e i we Oo . J— Frog cans: seo mi Ve soe SS sma membrane Cytoplasm, Cell all. re Faget Figure 2.2: 4 prokaryotic cell. Biochemistry 8 elmartine‘Nudeus Vi Noctelus ws ‘Mitochondria Smooth Plasmo- desmata reticulum Figure 2.4: A eukaryotic cell (Plant Cell). Biochemistry 9 elmartinezTable 2.1: Cell parts and their functions CELL PARTS _ STRUCTURE FUNCTION | Plasma membrane Lipid bilayer composed of — | Outer boundan phospholipids and of cells ¥ | | cholesterol with proteins that attach to either surface of the lipid bilayer Semipermeable; controls entry | and exit of substances | Receptor | molecules \ function in | intercellular communication | Marker molecules | enable cells to | recognize each other | | Nucleus Nuclear membrane —_| Double membrane | Separates enclosing the nucleus. The | nucleus from outer. membrane _ is, cytoplasm and continuous with the | regulates endoplasmic reticulum, | movement of nuclear pores —_extend | materials into and through the nuclear | out of the nucleus | envelope. Chromatin Thin strands of DNA, | DNAis the histones and other proteins | genetic material, condenses to form | DNA regulates Chromosomes during cell | protein synthesis | division and therefore the chemical | reactions | Nucleolus. One to four dense bodies | Assembly site of consisting of ribosomal RNA | large and small \ | and proteins. ribosomal \ subunits | | ie Biochemistry 10 elmartine!Cytoplasm [et Cytosol 2. Cytoskeleton- | Microtubules Microfilaments | or actin filaments 3. Cytoplasmic inclusions Intermediate fibers 4. Organelles | Ribosome | Rough ER Biochemistry Water with dissolved ions and molecules colloid with suspended proteins Hollow cylinders composed of the protein tubulin; 25nm_ | | in diameter ‘Small fibrils of protein; 88m in diameter Protein fibers, 10nm in diameter. Aggregates of molecules manufactured or ingested by the cell, may or may not be membrane bound Ribosomal RNA and proteins form large and small subunits; attached to ER or free. Membranous tubules and flattened sacs with attached ribosomes. Contains enzymes that catalyze catabolic | and anabolic | reactions. | cytoplasm.form Support the cytoplasm and form centrioles, spindle fibers, cilia and flagella; responsible for cell movements Support the microvilli, responsible for cell movements. Support the cytoplasm Function depends | on the molecules: energy storage (lipids, glycogen), oxygen transport (hemoglobin), skin color (melanin) | | and others. Site of protein synthesis Protein synthesis and transport to golgi apparatus 1 $e elmartinezSmooth ER Golgi apparatus Secretory vesicles Lysosomes Peroxisomes Mitochondria Centrioles ee Biochemistry — Membranous tubules and flattened sacs with no attached ribosomes. Flattened membrane sacs stacked on each other Membrane-bound sac pinched off Golgi apparatus | Membrane-bound sac pinched off Golgi apparatus | Membrane-bound vesicle Spherical, rod-shaped or thread like structures; enclosed by double membrane; inner membrane forms projections called cristae Pair of cylindrical organelles from the centrosome, | consisting of triplets of parallel microtubules 12 | peroxide Manufactures lipids and carbohydrates; detoxifies harmful | chemical. Modification, packaging and distribution of proteins and lipids for secretion or internal use. Carries proteins | and lipids to cell | surface for | secretion Contains digestive enzymes Detoxifies harmful molecules and breaks down hydrogen | Major site of ATP synthesis when oxygen is available Centers for microtubules formation; determine cell polarity during cell division; form the basal bodies of cilia and flagella elmartine!T [ Cilia Extensions of the plasma Move materials | membrane containing over the surface | | parallel microtubules; 10ym | of the cells | | | in length | Flagelia Extensions of the plasma —_| In humans, membrane containing responsible for | parallel microtubules; 55um | movement of in length spermatozoa | | | Microvii _ Extensions of the plasma _| Increase surface membrane containing area of the microfilaments | plasma | | membrane for absorption and secretion; modified to form sensory receptors Membrane Transport Mechanisms transported molecule oT 7 . © channel carrier protein protein / lipid [ concentration | gradient bilayer simple channel- carrier- diffusion mediated mediated Se ee PASSIVE TRANSPORT — ACTIVE TRANSPORT. Figure 2.5: Transport of materials across the cell membrane. One of the major parts of the cell is the plasma membrane and one of its functions is to transport materials in and out of the cell (Figure 2.5). Solutes cross cell membranes by passive or active transport. If uncharged solutes are small enough, they can move down their concentration Ls gradients directly across the lipid bilayer itself by simple diffusion Examples of such solutes are ethanol, carbon dioxide, and oxygen. Most solutes, however, can cross the membrane only if there is a membrane Biochemistry 13 elmartinezannel protein) to transfer th, transport protein (a carrier protein or & channe! % As indicated, pases transport, in the same direction a8 @ concentratg gradient occurs spontaneously, whereas ravetnr net @ concentra gradient (active transport) requires a input of energy. Only carrier proteing oon carry out active transport, but both carrie! pr chang, proteins can carry out passive transport Overview of Biomolecule: Molecules of Life h living things are called biomolecules i ic molecules always contai and are classified as organic molecules. Organic in garbon (C) and hydrogen (H). The chemistry of carbon accounts for the formation of the very large variety of organic molecules. The molecules associated wit! The molecules of life are divided into four classes: carbohydrates, proteins, lipids and nucleic acids. Carbohydrates, proteins and lipids are very familiar to you because certain foods are known to be rich in these molecules. These molecules of life are macromolecules. Just as atoms can join to form a molecule so molecules can join to form a macromolecule, The smaller molecules are called monomers, and the macromolecule is called a polymer. A polymer is a chain of monomers. Carbohydrates, proteins and nucleic acids are polymers, lipids are not. 4 Carbohydrates — are the most abundant members of the class of biomolecules, which are compounds that have aldehyde or ketone functional groups and multiple hydroxyl groups. The more important of these carbohydrates may be classified into: monosaccharides, compounds with a single aldehyde or ketone unit and multiple hydroxyl groups; oligosaccharides, which are defined by the identity of their monosaccharides and the type of glycosidic bond between the monosaccharides; and polysaccharides, which are mixtures of polymers with varying lengths and have different molecular weights. Carbohydrates first and foremost function for quick and short-term energy storage in all organisms, including humans. Figure 2.6: A carbohydrate. Biochemistry a elmartit ee— being one of the major constituents of living cell, are . Cees aeaes shat contain amino acids as building blocks joined together by peptide bonds. They have a variety of functions such as they catalyze biochemical reactions, regulate the activity of various organs in the body, counteract adverse effects of antigens, transport molecular oxygen and serve as structural materials of the muscle, skin and hair. Figure 2.7: A protein. ‘# Lipids — are water-insoluble organic substances found in cells but soluble in organic solvents such as chloroform, ether, benzene and alcohol. Lipids include fats and oil. They are classified into groups: simple lipids and compound lipids. Simple lipids are fats and oils that came from plants and animals. Compound lipids are those that yield hydrolytic products other than fatty acids and alcohols. They include phospholipids and the sphingolipids. Fatty acids and glycerol are the building blocks of lipids. 4 PHO OH HH HH HH A i De mt Ota tae oo H-e- OH. e—c-¢ -¢-¢-¢-¢~G—6-H ena Titel ete vgs HHH HHA A A HHH HH OH Ve lg tet 4 e-C-¢ ~C-C-C-C=-C—C—-H fe aaa tetera -C-C-C-c-¢-¢-C-c-H ao EGS FE GFE HH HHH HHH Glycerol Fatty Acids Figure 2.8: A lipid. Biochemistry 15 elmartinezthese are polyMers OF eee 4 Nucleic Acids — ural classes of i i Two struct hich the repeating unit is nucleotide s u nucleic acids occur in cell, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids are important for the growth and reproduction of cells and organisms. Human genes are composed of DNA. The RNA works in conjunction with DNA to bring about protein synthesis. Monomer. Nucleotide Polymer: DNA strand Figure 2.9: A nucleic acid. Some Important Inorganic Molecules Inorganic molecules are characterized by the presence of small number of atoms ionically bonded together. Water is an exception to this Statement because its atoms are covalently bonded. { Water - is the most abundant molecule in living organisms and it makes up about 60 to 70% of the total body weight of most organisms. The physical and chemical properties of water make life Possible as we know it. Because the water molecule has charged atoms, it is called a polar molecule. Hydrogen bonding occurs between water molecules because they are polar. Because of ils polarity and hydrogen bonding, water has many characteristics beneficial to life. Hydrogen bonding causes water molecules 10 be Cohesive and cling together. Without hydrogen bonding between molecules, water would boil much below 100°C and freeze Le lower than 0°C. Because water remains as a liquid between ! and 0°C, it is suitable for life. oimartt® Biochemistry 16Water molecules Unstable hydrogen bonds Figure 2.10: A liquid water. Properties of Water 1. Water is the universal solvent and facilitates chemical reactions both outside of and within living systems. . Water molecules are cohesive and fill vessels. . The temperature of liquid water rises and falls slowly, preventing sudden or drastic changes. 4. Water has a high heat of vaporization, keeping the body from heating up. 5. Frozen water is less dense than liquid water so that ice floats on water. wr @ Hydration shells crystal Figure 2.11: Water as the universal solvent. Biochemistry 17 elmartinezW Acids — based on the Bronsted-Lowry concept, an acid is a potential proton donor “® Bases — based on the Bronsted-Lowry concept, a base is a potential proton acceptor ‘# Buffers ~ are solutions that resist drastic changes in pH upon the addition of small amounts of acids or bases. Buffers help keep the PH within normal limits because they are chemicals or combinations of chemicals that take up excess hydrogen ions (H") or hydroxide ions (OH). For example, carbonic acid (H2COs) is a weak acid that Minimally dissociates and then reforms in the following manner: H2CO; H* + HCO; ton Examples o Concentration Eemane 11 atve 00 — fey ee vate 10°? —fi§ — 2 — stomach acid Lemon juice 10° — BF — 3 — Vinegar, cold beer bs Saliva water 107 — — Pure water Sage Blood or! 10*— —8 — Seawater ee —10 — Great Salt Lake Milk of magnesia — 11 — Household ammonia f 12 — Household bleach 10-'° — BER — 13 — Oven cleaner — , ie. 10°" — Bl! — 14 — Sodium hydroxide et ow Figure 2.12: The pH scale. elmatt® Biochemistry 18Exercise No.2 _ The Cell and Its Chemistry N: SCORE: DATES, 22a ci SECTION: Test I. Identify the following The cell theory states that cells arise only from cells. Ttis the basic unit of life. Nucleic acids are composed of __. Rough ER has __, but smooth ER does not have as much. Lysosomes contain enzymes. Vesicles derived from the ER make their way to the an organelle that functions in packaging, Storage and distribution. 7. are a part of the cytoskeleton, and interact with myosin molecules. Water boils at a much higher temperature and freezes at a much lower temperature than other liquids because of between the molecules. 9. take up either hydrogen ions or hydroxide ions and therefore act to stabilize the pH. 10. When an acid is added to water, the number of hydrogen ions and the pH z RON On @ Test Il. Critical Thinking 1. Describe the structure of a prokaryotic cell and compare it to eukaryotic cell. v 2. Relate the characteristics of water to its polarity and hydrogen bonding between water molecules. 3. What are buffers, and why are they important to life? Bi jiochemistry 19 elmartinezSHAPTER 3 CARBOHYDRATES Objectives: At the end of this chapter, the students should be able to 1. define carbohydrate 2. describe the difference belween Monosaccharides, oligosaccharides, and polysaccharides and explain the system used to categorize monosaccharides 3. identify four important di arides and describe how the Feesaccharide residues in them are joined to one another 4. distinguish homopolysaccharides from heteropolysaccharides On the earth, more than half of the carbon atoms tied up in organic compounds is found in carbohydrates, Plants Produce most of the Carbohydrates play many important biological roles. Some, such as starch and glycogen, are used to store energy. Others, including glucose Carbohydrates are defined chemically as aldehyde or ketone derivatives of higher polyhydroxy alcohols. Chemically they contain the elements such as carbon, hydrogen, and oxygen. The hydrogen and oxygen are usually present in the same ratio as that found in water (H,0), suggesting that they are carbon hydrates Classification Carbohydrate molecules can be placed into one of the ee categories: monosaccharides, oligosaccharides, and ae Monosaccharides are used as building blocks to produce Oe ea which contain 2 to 10 monosaccharide residues, and poe term which contain more than 10 monosaccharide residues. Saccharide came from the Greek word sakcharon (sugar). elmartsé Biochemistry 20Monosaccharides These are polyhydroxy aldehydes or ketones containing three = carbon atoms. In simpler terms, this means that ea aldehyde or a ketone and that each OH) group (Figure more monosaccharide molecule is either an of the other carbon atoms usually carries a hydroxyl (- 3.1). HHH 4 H re tetby oft tty oududndy buon On ‘An aidopentooe A ketotstrove Figure 3.1: Monosaccharides. Monosaccharides can be classified based on functional group, on number of carbon atoms, or both (Table 3.1). Those that contain an aldehyde group are aldoses and those that contain a ketone group are ketoses. Trioses have three carbon atoms, tetroses have four carbon atoms, pentoses have five, and so on. An aldohexose is an aldehyde sugar with six carbon atoms. The “ose” ending indicates that the molecule being named is a carbohydrate. Table 3.1 MONOSACCHARIDES “Type __| Functional Group | Number of Carbon Atoms Aldose Aldehyde Ketose Ketone = | Triose 3 Tetrose 4 Pentose z 5 | Hexose | 6 _Heptose 7 _Octose 8 9 Aldehyde 3 Aldopentose | Aldehyde _ 5 Ketoheptose Ketone I rls As a family, monosaccharides have a sweet taste, high melting points, and are hydrophilic. The latter two properties are primarily due to the ability of monosaccharides to form hydrogen bonds. This class of carbohydrate has an asymmetric carbon atom or chiral carbon atom, a carbon atom to which four different atoms or groups of atoms are attached to it. The presence of asymmetric carbon atoms in a compound gives rise to the formation of isomers of that compound. Such compounds, which are Biochemistry 21 elmartinezstercoisome se position and differ only in spatial configurati stereoisomers (Fig. 3.2). ry) eee HO. HERO met MONE Cy CH.OH D-Glyceraldehyde L-Glyceraldehyde Figure 3.2: The glyceraldehydes has one chiral carbon atom and exist in two Stereoisomeric forms. Monosaccharides can either be D- or L-sugars. In D-sugars, the -OH attached to the chiral carbon atom farthest from the C=O Points to the right, and in L-sugars the -OH points to the left. Monosaccharides have the same name, except for the D or L designation, In nature, most monosaccharides are D-sugars. {8 Important Monosaccharides 1, Pentose a. Ribose — sugar found in RNA b. Deoxyribose — sugar found in DNA 2. Hexose a. Glucose — dextrose or blood sugar b. Galactose ~ combined with glucose to produce lactose ¢. Fructose — fruit sugar, combined with glucose to produce sucrose i Monosaccharide Derivatives 1. Deoxy sugars — a hydrogen atom replaces one or more of the - OH groups in a monosaccharide | 2, Amino sugars ~— an -OH group of a monosaccharide has been replaced by an amino (-NH2) group 3. ‘Alcohol sugars — the carbonyl group of a monosaccharide has been reduced to an alcohol group ra 4. Carboxylic acid sugars - an aldehyde or an Seer erate monosaccharide has been oxidized to form a carboxy! g! mh Monosaccharides in their Cyclic Form tes with 7 The aldehyde and ketone moieties of the uri (gfouPs five and six carbons will spontaneously react wi matt of Biochemistry 22present in neighboring carbons to produce intramolecular hemiacetals or hemiketals, respectively. This results in the formation of five- or six-membered rings. Because the five- membered ring structure resembles the organic molecule furan, derivatives with this structure are termed furanoses. Those with sixmembered rings resemble the organic molecule pyran and are termed pyranoses. HOH H-C-OH rom CH;OH Figure 3.3: Cyclic hemiacetal, Oligosaccharides Oligosaccharides are formed when between 2 and 10 monosaccharide residues are joined to one another by glycosidic bonds. Glycosidic bond is the bond that connects the acetal carbon to the newly added -OC group. Disaccharides, which contain two monosaccharide residues, are the oligosaccharides found most widely in nature. Maltose, cellobiose, lactose and sucrose are four important disaccharides. Maltose is the major degradation product of starch, it is composed of two glucose monomers in an a-(1—+4) glycosidic bond. CH;OH ——CH,OH H H fi % 4 NM HO ey OH H OH Figure 3.4: Maltose. Cellobiose contains two glucose residues and differs from maltose only in that its glycosidic bond is B-(1, 4). The oxygen atom that connects the two residues points up from the glucose on the left (B orientation) and down from the glucose on the right (carbon atom 4). Biochemistry “ 23 elmartinezCellobiose is formed during the breakdown of cellulose, and when hydrolyzed cellobiose yields two glucoses. While cellobiose gives the same hydrolysis products as maltose, humans are unable to use cellobiose as a fuel source because humans do not produce the enzyme necessary to catalyze this particular reaction. Lactose is found exclusively in the milk of mammals and consists of galactose and glucose in a B-(1, 4) glycosidic bond. It is an important fuel source for infants. Figure 3.5: Lactose When hydrolyzed in the intestines with the assistance of the enzyme lactase, lactose yields one galactose and one glucose molecule. Several problems are associated with lactose and its digestion. Lactose intolerance, which is the inability to hydrolyze lactose, is a common ailment. Sucrose, also known as table sugar, is composed of glucose and fructose through an a, B-(1, 2) glycosidic bond CH;OH —-CH;OH °. Figure 3.6: Sucrose Sucrose is an energy and carbon storage molecule for many plants, including the sugar cane and sugar beets. elmat™ Biochemistry 24Polysaccharides nature occur in the form of high he carbohydrates found in page aS saccharides. The monomeric weight polymers called polys n Te becks: used fo generate polysaccharides can be varied. iin all tases. however, the predominant monosaccharide fount 7 polysaccharides is D-glucose. When polysaccharides are composed of a Single monosaccharide building block, they —_ are termed homopolysaccharides. Polysaccharides composed of more than one type of monosaccharide are termed heteropolysaccharides. # Homopolysaccharides 1. Cellulose — is of major importance in the structure of plants, consists of a long chain of B-glucose residues joined by B -(1.4) glycosidic bond 2. Starch - is the major form of stored carbohydrate in plant cells. Its structure is identical to glycogen, except for a much lower degree of branching (about every 20-30 residues). Unbranched starch is called amylose; branched starch is called amylopectin. 3. Glycogen - is the major form of stored carbohydrate in animals. This crucial molecule is a homopolymer of glucose in a-(1,4) linkage; it is also highly branched, with a—(1,6) branch linkages occurring every 8-10 residues. Glycogen is a very compact structure that results from the coiling of the polymer chains. CH)OH o” \OH 2-1 glycosidic linkages Figure 3.7: Section of glycogen showing glycosidic linkages. Biochemistry 25 elmartinez}y SY, Sas A SAS = SRS reopen tops Figure 3.8: Comparison of glycogen and starch. ae # Heteropolyssaccharides 1. Hyaluronic acid — found in the lubricating fluid that surrounds joints and in the vitreous humor present in the eye; made up of alternating residues of glucosamine and glucuronate connected to one another 2. Chondroitin sulfate — consists of galactosamine sulfate residues alternating with glucuronate residues; present in connective tissue 3. Peptidoglycan — found in the cell wall of bacterial cell General Properties t# Physical Properties 1. Monosaccharides and disaccharides are white crystalline substance. Starches are amorphous powder. Cellulose is fibrous. 2. Solubility to ordinary solvents is inversely proportional to the complexity of their structures. 3. Monosaccharides and disaccharides are sweet. Starch and cellulose are tasteless. # Chemical Properties 1. Reducing power — all monosaccharides and disaccharides containing the potentially free aldehyde/ketone group possess reducing properties 2. Osazone formation — reducing sugars form ciarecerete osazone crystals when heated with an excess of phenylhydrazine 3. Action of alkalies — reducing sugar heated with an alkali turns to yellow to orange and finally dark brown 4. Action of acids ~ disaccharides and other higher carbohydrates are readily decomposed elmarti® Biochemistry 26Denaturation can be caused by a variety of factors, including changes in temperature or pH, agitation, use of soaps or detergents. An increase in temperature is associated with an increase in kinetic energy, and this increased motion can be enough to disrupt hydrogen bonds and other noncovalent interactions. Varying pH affects protein shape because the charges on amino acid side chains involved in salt bridges or ion-dipole interactions may disappear. Detergents or soaps are amphipathic and they interfere with hydrophobic interactions. Itis important to understand the following about proteins: 1, Proteins are biopolymers (called polypeptides) of L-amino acids. 2. Amino acids in proteins are joined to each other via peptide bonds. 3. Only L-amino acids are used to make proteins (rare exceptions of proteins in bacterial cell wall, which contain some D-amino acids). 4. The process of putting amino acids together to make proteins is called translation. 5. Translation relies on the genetic code, in which three nucleotides in mRNA specify one amino acid in protein. 6. The order or sequence of amino acids distinguishes different proteins from each other. The sequence of amino acids determines the three-dimensional shape of the protein. Alterations to the amino. acid sequence of a protein changes its 3D shape. 7. The difference between a polypeptide and a protein is that the term polypeptide refers simply to a chain of amino acids. The term protein refers to the chain of amino acids after it folds properly and is modified, in some cases. Proteins may consist of more than one polypeptide chain. 8. Proteins are sometimes described as the workhorses of the cell because they do so many things — catalyze reactions, provide structural integrity, transport molecules, provide movement, bind molecules, and others. 9. Proteins give various color reactions. # Millon’s test ~ Million’s reagent + protein solution will give protein precipitate (mercury salt), on heating, precipitate turns flesh to red color if proteins-containing-tyrosine are present # Biuret test - CuSO, solution + alkaline solution of proteins will give rose pink to violet to purple color. #8 Hopkin’s Cole reaction — protein (mixed with glyocalyx acid) + H2SO, will give violet ring at the point of contact of the two solutions “ Xanthoproteic reaction — protein + HNO; will give yellow to orange color when neutralized with NaOH ‘# Test for the SH group — proteins containing cysteine and methionine when heated with NaOH will give NazS; this Biochemistry 37 elmartinezExercise No. 3 Carbohydrates name; OE Setion; —— NAME: ; Test |. True or False. Write letter T if the statement is correct and write F the statement is incorrect. 1. Simple sugar or monosaccharide is the building block of carbohydrates. All classes of carbohydrates readily dissolve in water, Monosaccharides can occur in cyclic forms, Monosaccharides, disaccharides and polysaccharides are the different classes of carbohydrates. Carbohydrates can be classified according to the number of saccaharide groups contained in their molecules All carbohydrates are sweet. One important characteristic of carbohydrates is that they contain chiral atoms and have stereoisomers. 8. Carbohydrates containing 10 or more monosaccharide residues are called oligosaccharides. 9. Fructose is the sweetest of all sugars. 10. Cellulose is an example of heteropolysaccharide. 2 soN No Test Il. Critical Thinking 1. How do oligosaccharides differ from polysaccharides? How are oligosaccharides similar to polysaccharides? 2. What are disaccharides? Describe how the monosaccharide residues in them are joined to one another? amet” Biochemistry 283. Explain what happens when a reducing sugar is reacted with Benedict's reagent. 4. Distinguish homopolysaccharides from heteropolysaccharides and give examples of each. 5. Both cellulose and amylose consist of (1+4)-linked D-glucose units and can be extensively hydrated. Despite the similarity, a person on a diet consisting predominantly of a-amylose (starch) will gain weight, whereas a person on a diet of cellulose (wood) will starve. Why? Biochemistry 29 elmartinezAMINO ACIDS AND PROTEINS. Objectives: At the end of this chapter, the students should be able to: 4. define Amino Acid and Proteins 2. describe the structure of amino acids and the system used to classify amino acids 3. distinguish between oligopeptides, Polypeptides and proteins and describe the bond that joins amino acid residues in these compounds 4. define primary, secondary, tertiary and quarternary structure 5. explain what is meant by the term denaturation, and list some ways to denature a protein Amino Acid Amino acids are organic acids containing an amine group (Figure 4.1). The most common amino acids are a-amino acids and the most common a-amino acids are the L-a-amino acids. H oy H-N—C— C—Ou HOR Ne ee ‘side chain a 5 ‘An camino cid Fig. 4.1: General structure of a-amino acid It is important to understand the following about amino acid structure 1. Only 20 L-a-amino acids are used to make proteins (Figure 4.2 and 4.3). Rare exceptions are bacterial membrane proteins which contain a few D-amino acids 2. Side groups (labeled R in Figure 4.1) are what distinguish the a- amino acids from each other. . 3. Amino acids can exist as 2witterions (Figure 4.4). Zwitterions are Substances containing equal numbers of positive and neve charge - due to their carboxyl and amine groups, which can Negatively and positively charc ed, respectively. 4. Several amino acids feud in cells, uch as ornithine and citrulline, are not used to make proteins, Ss. 5. Amino acids are the rangers units or building blocks os Amino acids are joined together covalently in peptide bonds: elmer Biochemistry 306. Amino acids are built into proteins by the process of translation using the genetic code. Gere (GG Alanine (a) A renee (16) ‘Sere (Ser1S Thesort%e Th)T — eemenne aM Aignita al ve oo a e Santi gee Gh)E Asparagine As) Figure 4.2: The amino acids found in proteins. CH, Pilani Alanine Figure 4.3: Stereoisomers of a-amino acids. Biochemistry 31 elmartinez5 — é \ ‘OH rea form 2wattenon form, (©) Seneraiz0d ming ac, forming zwiter on at ret oh Figure 4.4: Zwitterion, Classification of Amino Acids Amino acids are non-polar, polar acidic, polar basic or polar net * a sutral, depending on the nature of their side chain (Figure 4.2). 7 7 1. Non-polar ~ the side chain of non-polar amino is usually an alky! group, an aromatic ring, or a non-polar collection of atoms. Non- polar amino acids are glycine, alanine, valine, leucine, isoleucine, methionine, praline, phenylalanine and tryptophan. 2. Polar acidic — the side chain of a polar acid amino acid contains a carboxyl group. At pH 7, the carboxyl group is found in its conjugate base form (-CO;’), which means that the side chains on the two polar acidic amino acids, aspartic acid and glutamic acid, carry a negative charge at this pH 3. Polar basic ~ the side chain of polar basic amino acids contains an amino group. At pH 7 the amines exist in their conjugate acid form. So polar basic amino acids carry a positive charge at this pH. Polar basic amino acids are lysine, arginine, histidine a 4. Polar neutral - the side chain of polar neutral amino acids is usually an alcohol, a phenol. None of these functional groups is acidic or basic enough to carry a charge at pH 7. Polar neutral amino acids are serine, threonine, cysteine, tyrosine, asparagine and glutamine. Stereoisomers Js in Figure 4.2 rent groups). nits. mirror tions of the group and drawn With the exception of glycine, alll of the a-amino acid are chiral (the a-carbon atom in each is attached to four diffe This means that each amino acid is nonsuperimposable © 'mage, as shown in Figure 4.3 for alanine. The D and L designa! {wo enantiomers are based on the relative position of the amino ST the hydrogen atom attached to the a-carbon. When amino acids @ a elmati! Biochemistry 32up and the side OUP ota on the right side | gr jections with the carboxy! group polnnns cher proj t San panting down, a D-amino acid has the $nd an L-amino acid has it on the left side. The Peptide Bond Peptide bond is another name for the amide bond that eiasione amino acid residue to another. Amide bond is between a-amino She oe carboxyl groups. Two individual amino acids can be linked to form a Arg molecule, with the loss of a water molecule as a by-product of the reaction. Figure 4.5: The peptide bond. The newly created C-N bond between the two separate amino acids is called a peptide bond. The term ‘peptide bond’ implies the existence of the peptide group which is commonly written in text as -CONH- . Two molecules linked by a peptide bond become what is called a dipeptide. Similar to the system used for carbohydrates, a combination of three amino acid residues is a tripeptide, a combination of four is a tetrapeptide, and so on. Ologipeptides contain between 2 and 10 amino acid residues and polypeptides, more than 10 residues. It is customary to draw peptides and proteins with the N-terminus (the end of the peptide chain with the unreacted amino group) on the left and the C-terminus (the end of the peptide chain with the free carboxyl group) on the right. Biochemistry 33 elmartinezler fram the Néerine te eng ine amino acid residues, in order, from the N-terminus {0 the C-terminus. Names of larger oligopeptides, polypeptides, and proteins arrived at using this method can be quite lengthy, ‘so these molecules are sometimes known by common names Proteins A protein is made up of one or more polypeptide chains, each of which consists of amino acids which have been mentioned earlier. In general terms, a protein can be classified as being either fibrous or globular. Fibrous proteins, as their name implies, exist as long fibers or strings. These proteins are usually tough and water insoluble, such as collagen and keratin. Globular proteins are spherical in shape, highly folded and tend to be water soluble. The structure of proteins is understood in terms of four levels of organization (Figure 4.6): Primary protein structure re pequence of a chain of amino aciae Pleated aneet ‘Alpha helix -_ Secondary protein structure = Sccure wned fhe anqperce st ermno acide = [are linked by hydrogen bongs Quaternary protein structure | ip a protein conmsbng of more ‘amino ecrs chain Figure 4.6: Levels of Organization: Protein structure. einai Biochemistry 34ino acids. primary Structure ~ refers to the linear sequence of erous to the The primary structure of peptides and proteins | the sequence of arrangement of letters in a word. By rearranging 1h S°0 ST, fetters in @ word, you can entirely change its ™ a rece becomes vile, veil or evil. Similarly, rearranging the am residues in a peptide or protein changes its function a 2. Secondary Structure — regular patterns formed by pr! ry structure folding; results from hydrogen bonding that takes place between amide N-H and carbonyl C=O groups from different parts of a polypeptide chain. There are two types of secondary structure: ‘8 orhelix — assumes a shape that looks much like a coiled spring # B-sheet - or B-pleated sheet refers to a sheet-like arrangement that forms when different segments of a Polypeptide chain align side by side 3. Tertiary Structure — completely folded polypeptide with one or more domains; refers to the overall three-dimensional shape of a protein. Of the many folding patterns possible for a protein, there is usually only one that leads to a native or biologically active molecule 4. Quaternary Structure — association of multiple polypeptides; not found in all proteins. This structure is maintained through Noncovalent attractions such as hydrogen bonding, the hydrophobic effect, and salt bridges. Quartemary structure is important because cooperation between subunits is often a factor in the regulation of protein activity. 1 Properties of Proteins 4. General Properties Very large molecules ® Have characteristic amino acid composition # Some proteins contain chemical groups other than amino acids and they are called conjugated proteins (amino acid + prosthetic group) like lipoproteins, —_ glycoproteins, metalloproteins 2. Physical and Chemical Properties Generally tasteless Mostly colorless Insoluble in fat solvents, but varied degrees of solubility in water, salt solution, dilute acids and bases Amphoteric Very reactive and highly specific FE HEE Biochemistry 35 elmartinezClasses of Proteins 1. Enzymes — most varied catalytic activity 2. Transport ~ bind and carry specific molecules or ions from one organ to another 3. Nutrient and Stora the seedling 4. Contractile or Motile — endow cells and organisms with the ability fo contract, to change shape or move about 5. Structural — serve as supporting filaments to give biological structures strength Defense — defend organisms against invasion by other species Regulatory — help regulate cellular or physiological activity and most highly specialized proteins with ge — store nutrients required for the growth of N@ Denaturation The native shape of a protein is the biologically active one. Denaturation is any change in protein conformation caused by disruption of the noncovalent forces responsible for maintaining the native conformation. A loss of biological activity normally accompanies denaturation, and this process is reversible only if minor changes take place. Folded protein’ Denatured protein Figure 4.7: Denaturation. elnatt® Biochemistry 38Denaturation can be caused by a variety of factors, including changes in temperature or pH, agitation, use of soaps or detergents. An increase in temperature is associated with an increase in kinetic energy, and this increased motion can be enough to disrupt hydrogen bonds and other noncovalent interactions. Varying pH affects protein shape because the charges on amino acid side chains involved in salt bridges or ion-dipole interactions may disappear. Detergents or soaps are amphipathic and they interfere with hydrophobic interactions. Itis important to understand the following about proteins: 1, Proteins are biopolymers (called polypeptides) of L-amino acids. 2. Amino acids in proteins are joined to each other via peptide bonds. 3. Only L-amino acids are used to make proteins (rare exceptions of proteins in bacterial cell wall, which contain some D-amino acids). 4. The process of putting amino acids together to make proteins is called translation. 5. Translation relies on the genetic code, in which three nucleotides in mRNA specify one amino acid in protein. 6. The order or sequence of amino acids distinguishes different proteins from each other. The sequence of amino acids determines the three-dimensional shape of the protein. Alterations to the amino. acid sequence of a protein changes its 3D shape. 7. The difference between a polypeptide and a protein is that the term polypeptide refers simply to a chain of amino acids. The term protein refers to the chain of amino acids after it folds properly and is modified, in some cases. Proteins may consist of more than one polypeptide chain. 8. Proteins are sometimes described as the workhorses of the cell because they do so many things — catalyze reactions, provide structural integrity, transport molecules, provide movement, bind molecules, and others. 9. Proteins give various color reactions. # Millon’s test ~ Million’s reagent + protein solution will give protein precipitate (mercury salt), on heating, precipitate turns flesh to red color if proteins-containing-tyrosine are present # Biuret test - CuSO, solution + alkaline solution of proteins will give rose pink to violet to purple color. #8 Hopkin’s Cole reaction — protein (mixed with glyocalyx acid) + H2SO, will give violet ring at the point of contact of the two solutions “ Xanthoproteic reaction — protein + HNO; will give yellow to orange color when neutralized with NaOH ‘# Test for the SH group — proteins containing cysteine and methionine when heated with NaOH will give NazS; this Biochemistry 37 elmartinez