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WEEK 10 BIOCHEMISTRY Biochemistry is the study of those compounds produced and used by living organisms. ELEMENTAL COMPOSITION Most organisms are composed of only 16 chemical elements. Hydrogen, oxygen, nitrogen and carbon comprise more than 99% of living matter. They are the smallest atoms that can attain stable electronic configurations by sharing one, two, three, and four electrons, respectively. All these elements form very stable covalent bonds. Oxygen, nitrogen and carbon form stable multiple bonds. Carbon can bond to oxygen by a double bond either within a biological compound or in the gaseous substance carbon dioxide, a waste product produced by many living organisms. Carbon also bonds very stably to hydrogen and to nitrogen. O2 is vital to many living organisms. Two oxygens are joined by a double bond. Oxygen is extremely electronegative and easily accepts electrons in energy yielding biochemical reactions. This property satisfies the energy needs of many cells. The action of phosphorous and sulfur in organisms is also related to cellular energy. In the presence of water, large amounts of energy are required for these elements to form bonds. Their formation is a means of carrying energy. When these bonds (high-energy bonds), are hydrolyzed, large quantities of energy are released. ATP and coenzyme A which contain sulfur act as energy carriers in living organisms. Sodium is the major positive ion in extracellular fluids. Within cells, potassium is the major positive ion. Both are involved in the actions of muscles and the nervous system. Magnesium is necessary for the actions of enzymes and is found in cells. Chloride is the major negative ion in the body. Approximately two-thirds of the negative ions in the body are chloride ions. They are mainly in extracellular fluids. Calcium ions in the forms of calcium phosphate and calcium hydroxide are important components of bone and teeth. Calcium initiates the blood-clotting process; it also functions in the actions of nerves and of muscles and is important to the activity of some enzymes and hormones. Manganese, copper, and zinc act as parts of enzyme systems. The proper functioning of nerves and the development of strong bones require manganese. Copper is important for the synthesis of collagen, a connective-tissue protein. Besides being present in some enzymes, zinc is also present in bone. The oxygen-carrying ability of blood is dependent on iron. Copper and iron also play an important role in respiration. They participate in a series of oxidation and reduction reactions known as the electron-transport chain. MOLECULAR COMPOSITION The most abundant molecule found in living organisms is water. It has two properties that qualify water as the basis of life: polarity and its formation of intermolecular hydrogen bonds. Because it is polar, water easily dissolves other polar substances; therefore, it provides an excellent transport system for nutrients. The extensive hydrogen bonding between molecules of water causes its specific heat, heat of vaporization, and heat of fusion to be unusually high in comparison to those of many other substances. Because approximately 70% of the mass of an adult human is water, a considerable amount of heat can be absorbed or released from a body without much change in body temperature. An animal loses small quantities of body fluids in ridding itself of large amounts of heat by surface evaporation. Organisms are protected against freezing, because large quantities of heat must be removed before freezing occurs. AMINO ACIDS Twenty amino acids are the building blocks of proteins. These may be divided into essential and nonessential amino acids. The nonessential ones can be synthesized by an adult human from intermediates of carbohydrate breakdown. Humans, however, cannot synthesize the essential amino acids, which therefore must be included in the diet. AROMATIC BASES The genetic code that governs growth and reproduction of cells is directed by nucleic acids. Two groups of nucleic acids are ribonucleic acids (RNA) and deoxyribonucleic acids (DNA). SUGARS Glucose is the building block for the carbohydrate compounds of cellulose, starch and glycogen. It is also the primary carbohydrate that circulates in the blood. Oxidation of glucose yields energy for cellular needs. The sugar ribose forms part of the structure of RNA. In the form of deoxyribose (ribose minus one oxygen) it is found in DNA. PHOSPHOLIPIDS Membranes that surround both procaryotic cells and eucaryotic cells are composed of phospholipids and proteins. The phospholipids are formed from the fatty acid, palmitic acid, the alcohol, glycerol and the amine choline. MACROMOLECULES Small precursor molecules join together to form macromolecules. They form 85% to 95% of the dry mass of most organisms. The major types of macromolecules are proteins, carbohydrates, and nucleic acids. The formation of all three is based on reactions between the precursor molecules, which involve the elimination of water between reacting sites in the molecules. These large macromolecules are broken apart by another reaction, hydrolysis. These synthesis and degradation reactions also require the presence of enzymes which help to properly align the molecules. Synthesis is an energy-requiring process and degradation is an energy releasing process. A general name for the former is ENDERGONIC and for the latter is EXERGONIC. PROTEINS – Polymerization of amino acids produces these macromolecules. They have many functions. They form structures within organisms, and exercise control over cellular reactions. POLYSACCHARIDES – Polymers of sugar molecules, mostly glucose, act as storage forms of energy in plant, animal, and human organisms. When energy is needed, polysaccharides are degraded back to the precursor molecules. NUCLEIC ACIDS DNA and RNA are responsible for the transmission of genetic information and the synthesis of protein by a cell. DNA is found in the cell nucleus and RNA in both the nucleus and the cytoplasm. Both are usually joined to proteins, forming nucleoproteins. Each individual strand of a nucleic acid is a polymer of units called nucleotides. Nucleotides are composed of purine or pyrimidine, ribose or deoxyribose and Phosphoric acid. An actual molecule of DNA consists of two chains of nucleotides coiled in a double-helix structure. Within each molecule, there is a 1:1 relationship between cytosine and guanine nucleotides and adenine and thymine nucleotides. RNA molecules generally are single-stranded. They can form helical structures because of intrastrand hydrogen bonding. The bases usually pair adenine-uracil and guanine-cytosine. METABOLISM The study of all the enzymatically controlled reactions in a living cell is METABOLISM. This process can be divided into two parts: catabolism and anabolism. CATABOLISM is a breaking-down process and produces the precursor molecules used by cells and chemical energy for cellular needs. ANABOLISM is a building-up process and forms the macromolecules of proteins, carbohydrates, and nucleic acids. Lipids are also produced by these synthetic processes, but are not considered macromolecules because they are not polymers. WEEK 10 PROTEINS Their functions are both structural and dynamic. They along with lipids form biological membranes. These proteins are responsible for many of the processes that the membranes perform. Two of these processes are transport and communication. They act as hormones and antibodies, form a portion of the oxygen-carrying molecules hemoglobin and myoglobin, and as enzymes serve as regulators of all biological reactions. Proteins are the focal point of the work of the embalmer. The embalming operation achieves preservation and disinfection of a body by cross-linking proteins – both the proteins of the human remains and the proteins present in microorganisms. PROTEIN COMPOSITION All proteins contain the elements carbon, hydrogen, oxygen, and nitrogen. Many also contain sulfur and some contain small amounts of phosphorus, iodine, copper, manganese, magnesium, and zinc. Proteins are very large molecules. They are polymers of amino acids. Upon hydrolysis, they form mixtures of amino acids. Hydrolyzing agents are acids, bases, and enzymes called proteases. AMINO ACIDS Twenty amino acids are the building blocks of proteins. Amino acids contain two functional groups. They are the carboxyl group (-COOH) and the amino group (-NH2). All amino acids contain another carbon, another hydrogen, and a side chain called and R group. R H2N C COOH H The amino group is always attached to the carbon next to the carboxyl group. In organic chemistry terminology, this carbon is the alpha carbon. Amino acids of this form are called alpha amino acids. A specific amino acid is identified by its R group. The simplest is glycine, in which the R group is a hydrogen. Structures for additional amino acids are written by changing the R group. Because all amino acids have both the carboxyl group and amino group, they may act as both acids and bases. Some amino acids have additional ability to act as bases, because they contain nitrogen atoms in the side chains. Two amino acids have a carboxyl group in their side chains and are therefore, acidic amino acids. The major properties of the amino acids are inherent in the properties of the carboxyl and amino groups. Two of these properties are amphoterism and peptide bond formation. AMPHOTERISM Amino acids contain the carboxyl group and the amino group, so they act as both acids and bases. Carboxyl groups cause a compound to act as a weak acid because of the following ionization: O O C OH O- C + H2O + H3O+ Similarly, the presence of the amino group in a compound gives it basic properties. NH2 + H+ NH3+ Because both of these groups are found in each amino acid, an acid-base reaction may occur within each molecule. R R H2N C + H3N COOH C COO- H H The product of this reaction is a dipolar ion. Another name for this ion is the Zwitterion. In a neutral solution, the predominant form of amino acids has the carboxyl and amino groups ionized. In a very acid solution (pH=1), the major form of amino acids is a positively charge ion. R + H3N C COOH H In a very basic solution (pH=12), the predominant form is a negatively charge ion. R H2N C H COO- Any compound that can act as both an acid and a base is called amphoteric. Amphoterism is a property of all amino acids. Because of their amphoteric nature, amino acids are buffers in solution. To buffer means “to protect”. BUFFERS are compounds that protect a solution against changes in pH. By neutralizing either acids or bases that enter a solution, amino acids keep the pH of a solution relatively constant within limits of the added acid or base. Proteins are polymers of amino acids, so they can also function as buffers. PEPTIDE LINKAGE By definition, a protein is a chain of alpha amino acids joined together by the peptide linkage. If we draw the structures for any two amino acids with the carboxyl group of one next to the amino group of another, their chemical linkage can be demonstrated. For example: H2N R O C C OH H R O + H N C H C OH R O R O H2N C C N C C OH H H + H2O H H The group that joins the two amino acids is the peptide linkage. It contains the carbonyl group and the imide group: O H C N Carbonyl Imide The product of joining together two amino acids is a dipeptide. Two more amino acids can be joined to it at its ends. We can continue to do this until we have formed a large chain of amino acids, or a protein. It is important to see that no matter how large the protein, on one end will always be a free carboxyl group and on the other a free amino group. Like amino acids, proteins are amphoteric and act as buffers. The buffering action of proteins is enhanced by acidic and basic groups present in the R groups of the amino acids. Proteins buffer the blood by donating protons if it becomes too basic and accepting protons if it becomes too acidic. CROSS-LINKING PROTEINS During the embalming process, proteins are cross-linked to one another. This crosslinking results in the firmness of embalmed tissue. The most commonly used crosslinking agent is formaldehyde. The basis of cross-linking is the chemical reactivity of aldehydes with different forms of nitrogen. Three forms of nitrogen found in proteins are imide groups, amino groups, and the peptide linkage: C N N N H H O H Amino group Peptide linkage Imide H The reaction that occurs is basically the same for all three groups. Two protein chains are linked together and a methylene group is inserted between the nitrogens. A molecule of water is also formed. N N H H N + CH2O CH2 + H2O N The formation of water in these reactions has the implication that embalming with formaldehyde is dehydrating to the embalmed tissue. The peptide linkage is the most important cross-linking site in a protein. Between every two amino acids in the chain is a peptide linkage. Reaction with formaldehyde at these linkages joins many protein chains together. Another cross-linking agent is the dialdehyde glutaraldehyde. Unlike formaldehyde, glutaraldehyde does not join every peptide linkage, as is possible with formaldehyde. The size of the five-carbon glutaraldehyde sterically prevents a reaction at every peptide site. Tissue embalmed with glutaraldehyde is not as hard as that embalmed with formaldehyde. A possible explanation for this is the lesser number of cross-links that result from cross-linking with glutaraldehyde. PROTEIN STRUCTURE To understand protein structure, we discuss it at different levels, referred to as primary, secondary, tertiary, and quaternary. Only proteins with more than one chain have a quaternary structure. Primary Structure This is the sequence of amino acids in a protein. For example, are the amino acids arranged glycine, glycine, alanine or are they arranged alanine, glycine, glycine. The primary structure also tells if there are disulfide bridges in the protein. These are covalent bonds between sulfur atoms in two different cysteine amino acids. The –SH groups on the cysteines can be oxidized to form a covalent bond called a disulfide bridge. The biological activity of a protein is related to its primary structure. A change of one amino acid in the sequence can alter biological function. Sickle cell anemia is caused by glutamic acid being replace by valine in one of the protein chains that make up hemoglobin. Secondary structure Proteins are chains of alpha amino acids. The chains take certain shapes, because of electrostatic interactions between atoms within one chain or between chains. Three common shapes of proteins are the alpha helix, the beta pleated sheet, and the triplestranded helix of collagen. Fundamental to these shapes is hydrogen bonding. A hydrogen atom attached to a nitrogen in one peptide linkage is attracted to an oxygen atom in another peptide linkage. This attraction or hydrogen bond holds the protein in a definite shape. Therefore, the secondary structure of a protein is its shape due to hydrogen bonding between the hydrogen on a nitrogen of one peptide linkage and the oxygen on the carbon of another peptide. In the alpha helix, hydrogen bonding occurs between the carbonyl group of each amino acid and the –NH group of the amino acid that is four amino acids ahead in the same chain. In a beta pleated sheet hydrogen bonding occurs between the carbonyl groups and the –NH groups in adjacent polypeptide chains. The triple-stranded helix of collagen is due to the coiling of the polypeptide chains to minimize repulsions. This causes each chain to form a helix to minimize repulsions on the side chains. The three chains then wrap around each other and form hydrogen bonds between the different chains. Tertiary structure Interactions between the R groups on the amino acids are responsible for folding of the protein molecules, often into very compact shapes. This folding is referred to as the tertiary structure. Several types of bonds that influence the tertiary structure of a protein are hydrogen bonds, electrostatic interactions often referred to as salt bridges, disulfide bridges, and hydrophobic-hydrophilic interactions. Hydrophobic literally means “water fearing” and hydrophilic means “water loving.” Those amino acids with nonpolar hydrocarbon side chains are hydrophobic. Hydrophilic amino acids have polar regions in their side chains. Proteins in an aqueous environment will assume a shape that minimizes the contact between the hydrophobic side chains and water, and maximizes the contact of the hydrophilic groups with water. Quaternary structure Those proteins with more than one polypeptide chain have another level of structure called quaternary. The polypeptide chains in these proteins are called subunits. A protein’s quaternary structure tells how the subunits are arranged. Hemoglobin has four polypeptide chains. These four chains arrange themselves tetrahedrally in one hemoglobin molecule. The quaternary structure of hemoglobin, therefore, is four subunits in a tetrahedral shape. HEMOGLOBIN One molecule of hemoglobin contains a protein part, globin, and a nonprotein part, heme. Every molecule of hemoglobin contains four polypeptide chains and four hemes. Two of the subunits, designated as alpha chains, each contain 141 amino acids. The other two, which are beta chains, each contain 146 amino acids. Nestled within the folds of each subunit is a molecule of heme. Each heme portion contains an atom of iron. The iron is responsible for carrying oxygen. Hemoglobin carries oxygen in blood. A similar function is performed by myoglobin in muscle. BREAKDOWN OF HEMOGLOBIN Red blood cells have a lifespan of about 120 days. Their degradation occurs in the spleen. The protein portion of hemoglobin is metabolized to amino acids. Heme is degraded in a stepwise procedure to several compounds of interest to the embalmer. In the first step, heme is converted to biliverdin, a green compound. A molecule of carbon dioxide is also formed. The second step is the reduction of biliverdin to bilirubin, a yellow compound. Both reactions require certain enzymes and coenzymes present in a living organisms. The bilirubin is then carried by serum albumin to the liver, from which it is secreted as bile. Nondegraded hemoglobin is the cause of postmortem stain. Approximately 6 hours after death, because of hemolysis of red blood cells, hemoglobin may seep from the capillaries to the tissues. The pigment discolors the tissues, a condition known as postmortem stain. The breakdown products of hemoglobin, bilirubin and biliverdin, cause another cosmetic problem for the embalmer. This situation occurs in the embalming of jaundiced remains. Jaundice is caused by an elevated level of bilirubin in the blood. The resulting yellow color is known as jaundice. At one time, it was thought that reaction of formaldehyde with the excess bilirubin converted it to biliverdin, resulting in a greenish discoloration of the embalmed body. However, because the conversion of bilirubin to biliverdin is an oxidation reaction and formaldehyde is a reducing agent, formaldehyde cannot be the cause of these color changes. Some other chemical reaction must be responsible. DENATURATION A protein is denatured when its structure is changed in a way that modifies its properties. The extreme result of denaturation of a protein is loss of biological activity. In an aqueous solution, denaturation is generally observed by precipitation of the protein. Denaturing agents interfere with the secondary and tertiary structures of a protein. Coagulation is a type of denaturation. Coagulation is the process of converting soluble protein to insoluble protein by heating or by contact with a chemical such as an alcohol or an aldehyde. Denaturation may have both bad and good effects. Loss of biological activity by enzymes that have been denatured may seriously affect an organism. On the other hand, blood clotting, which is the result of protein precipitation, is a normal biological function. PROTEIN BREAKDOWN Protein breakdown into smaller molecules begins in the stomach. The reactions are all catalyzed by enzymes. Here in an acid environment, the enzyme pepsin catalyzes the hydrolysis of peptide linkages. The acid pH of the stomach also aids in the digestion by denaturation of the protein. The partially digested proteins, which are now smaller polypeptides, move to the small intestine, where enzymes that have been secreted by the pancreas complete digestion. From the small intestine amino acids, the final products of protein digestion are absorbed into the bloodstream. In the blood, the amino acids are a source of protein material called the amino acid pool. They may be removed from the pool to build new proteins for the body’s use or may undergo oxidations. The final oxidation products of protein breakdown are carbon dioxide, water, urea, and energy. PUTREFACTION The anaerobic decomposition of proteins brought about by the action of enzymes is putrefaction. It begins after cellular death. The rate at which it occurs depends on several factors, including temperature, humidity, moisture content of the body, and cause of death. It is favored by moisture and the presence of bacteria and increases with temperature. IMBIBITION may increase the rate of putrefaction. It is the swelling and softening of tissues and organs as a result of absorbing moisture from adjacent sources. Putrefaction involves three major chemical reactions: hydrolysis, deamination, and decarboxylation. Hydrolysis This chemical reaction initiates the putrefactive process. Hydrolysis is a chemical property of water. In this process, biological compounds are broken apart and the constituents of water are incorporated into their structure. Enzymes are necessary for the process to occur. As in digestion, amino acids are the final hydrolysis products. Deamination This reaction is the removal of the amino group from an amino acid. Each amino acid undergoes a specific reaction, but in general ammonia and an organic acid are the products. The formation of ammonia as a product of putrefaction is significant to the embalming process. Formaldehyde reacts with ammonia, producing urotropin. If extensive putrefaction has occurred before embalming, there will be a higher-than-normal formaldehyde demand due to this production of ammonia. Decarboxylation By this reaction, the carboxyl group of an amino acid is removed. The products are carbon dioxide, water, and an amine. The amines may be further broken down into ammonia and various hydrocarbons. Actually, during putrefaction, both deamination and decarboxylation occur simultaneously. Overall, the decomposition of proteins produces hydrocarbons, organic acids, amines, ammonia, and carbon dioxide.