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Chapter 2: Biomolecules: Amino Acids and Peptides

Genaro F. Alderite Jr.,MSERM Medical Biochemistry

At physiological pH’s (7.0-7.4), both the carboxyl and amino groups are charged

Phenylalanine hydroxylase

arginase

urea

Non-protein Amino Acids

Examples of Clinical Aminoacidurias
• Metabolic defects: Phenylketonuria (Phe), Tyrosinemias (Phe,Tyr), Maple Syrup Urine Disease (Leu, Val, Ile), Alcaptonuria (Tyr)

• Absorption/transport defects: cystinuria (Cys), Hartnup disease , Fanconi’s Syndrome • These diseases are generally diagnosed from indicators in the urine or plasma.

PEPTIDE BOND

Levels of Protein Structure

General Properties of Amino Acids:
I. Physical Properties: 1. White crystalline 2. Soluble in cold water, except cysteine and tyrosine. 3. Most are insoluble in alcohol. 4. Most are sweet like glycine, alanine, serine and proline. 5. Others like leucine are tasteless; while some are bitter like arginine.

II. Chemical Properties: 1. Amino acids are amphoteric. 2. They form esters with alcohol. 3. Amino acids can be acetylated, benzylated or methylated. 4. All amino acids except proline and hydroxyproline react with nitrous acid with the liberation of nitrogen gas.

Chapter 3: PEPTIDES AND PROTEINS
Genaro F. Alderite Jr.,MSERM Medical Biochemistry

Biomedical importance of proteins in The Body:
1. Enzymatic catalysis - almost all biological reactions are enzyme catalyzed. Enzymes are known to increase the rate of a biological reaction by a factor of 10 to the 6th power! There are several thousand enzymes which have been identified to date.

2. Binding, transport and storage - small molecules are often carried by proteins in the physiological setting (for example, the protein hemoglobin is responsible for the transport of oxygen to tissues). Many drug molecules are partially bound to serum albumins in the plasma.

3. Molecular switching - conformational changes in response to pH or ligand binding can be used to control cellular processes

4. Coordinated motion - muscle is mostly protein, and muscle contraction is mediated by the sliding motion of two protein filaments, actin and myosin.
5. Structural support - skin and bone are strengthened by the protein collagen

6. Immune protection - antibodies are protein structures that are responsible for reacting with specific foreign substances in the body. 7.Generation and transmission of nerve impulses some amino acids act as neurotransmitters, which transmit electrical signals from one nerve cell to another. In addition, receptors for neurotransmitters, drugs, etc. are protein in nature. An example of this is the acetylcholine receptor, which is a protein structure that is embedded in postsynaptic neurons.

8.Control of growth and differentiation -proteins can be critical to the control of growth,cell differentiation and expression of DNA. -For example, repressor proteins may bind to specific segments of DNA, preventing expression and thus the formation of the product of that DNA segment. -many hormones and growth factors that regulate cell function, such as insulin or thyroid stimulating hormone are proteins.

Classification of Proteins:
1. Simple proteins- true proteins found abundantly in both plants and animals. a. Albumins- are soluble in water and dilute neutral solutions. - Members include serum albumin, lactal albumin, and ovalbumin. b.Globulins- are soluble in neutral dilute salt solutions but not in water. - include legumin from peas, myosinogen from muscles.

c. Glutelins- soluble in dilute acids and alkalines but insoluble in neutral solvents. - examples are glutenin from wheat and oryzenin from rice. d. Prolamines- are insoluble in ordinary solvent but soluble in 70% alcohol at about neutral point. - Present in plants such as gliadin from wheat, zein from corn, and hordein from barley.

e. Histones- soluble in water, dilute acids and alkalines but not in dilute ammonia. - not readily coagulated by heat - strongly basic and occur in the tissues in the form of salt combinations. - examples are globin from hemoglobin, thymus histone and scobrone of mackerel.

f. Protamines- contain smaller number of amino acids. - strongly basic and form soluble salts with strong mineral acids. - e.g salmin from salmon sperm g. Scleroproteins- insluble in water and neutral solvents. - e.g keratin of the epidermal tissues, elastin from ligaments and collagen from hides, bones and cartilages

2. Conjugated Proteins a. Nucleoproteins- are combination of histones and protamines with nucleic acid. - soluble in dilute solutions of NaCl and can be extracted from the tissues by the use of this solvent. - typical examples are chromatin, and the products obtain from glandular tissues and germ of grains.

b. Glycoproteins- are compounds of proteins with a carbohydrate component. - they are utilized for lubricating purposes in view of their slimy nature. - mucin from saliva, tendomucoid from tendons and osseomucoid from bones belong to this group. - are not digested in the GI tract and used as protection.

c. Phosphoproteins- have the prosthetic group (H3PO4) joined in the protein molecule. - casein from milk and vitelline of the egg yolk are rich in this type of protein. d. Chromoproteins- are protein compounds with hematin or similar pigments in their molecule. - examples are hemoglobin, cytochromes and rhodopsin.

e. Lipoproteins- have fatty substances combined with their molecules like lecithin, cephalin, etc. - they are present in the blood serum, brain tissues, cell nuclei, egg yolk and milk.

3. Derived Proteins- substances formed from simple and conjugated proteins. a. Primary protein derivatives- are proteins which have undergone intramolecular rearrangement through the hydrolytic action of certain physical and chemical agents. - They are synonymous with denatured proteins.

a1. Proteans- are insoluble substances resulting from the preliminary action of water, dilute acids, or enzymes. - myosan from myosin and edestan from edestin are good examples. a2. Metaproteans- are product of further hydrolysis. - soluble in weak acids and alkalies, but insoluble in neutral salt solutions. -acid metaproteans(acid albuminate); alkali metaproteans (alkali albuminate

a3. Coagulated proteins- are insoluble products resulting from either the action of heat, alcohol, ultraviolet are or even simple mechanical shaking. -cooked egg albumin, cooked meat,etc.

b. Secondary protein derivatives- are products of more extensive hydrolysis. b1. Primary proteoses- are soluble in water, precipitated by concentrated nitric acid. - not coagulated by heat. b2. Secondary proteoses- precipitated only by complete saturation with ammonium sulfate but not with nitric or picric acid.

b3. Peptones- are insoluble in water, not coagulated by hear and not precipitated by ammonium sulfate but by certain alkaloidal reagents such as phosphotungstic and tannic acids. b4. Peptides- are combinations of two or more amino acids , the carboxyl group of one being united with the amino group of the other. -same properties with peptones. -ex: di,tri,tetra, penta,poly

Protein Structure Levels
• PRIMARY: the linear sequence of amino acids linked together by peptide bonds • SECONDARY: regions within polypeptide chains with regular, recurring, localized structure stabilized by H-bonding between constituent amino acid residues

Secondary Protein Structure: a-helix & b-sheet

Super-secondary structure examples

• TERTIARY: the overall three-dimensional conformation of a protein • QUATERNARY: the three-dimensional conformation of a protein composed of multiple polypeptide subunits

• THE PRIMARY AMINO ACID SEQUENCE IS THE ULTIMATE DETERMINANT OF FINAL PROTEIN STRUCTURE

Myoglobin

b-subunit Hemoglobin

Structure of Myoglobin and Hemoglobin
• The amino acid sequences of myoglobin and hemoglobin are similar (or, highly conserved) but not identical • Their polypeptide chains fold in a similar manner • Myoglobin is found in muscles as a monomeric protein; hemoglobins are found in mature erythrocytes as multi-subunit tetrameric proteins. Both are localized to the cytosol

Stabilizing Forces
1. Electrostatic/ionic 2. Hydrogen bonds 3. Hydrophobic interactions 4. Disulfide bonds

1. Electrostatic/ionic 2. Hydrogen bonds

3. Hydrophobic interactions 4. Disulfide bonds

Biochemical Methods to Analyze Proteins
• Electrophoresis • Chromatography: Gel filtration, ion exchange, affinity • Mass Spectrometry, X-ray Crystallography, NMR

Gel filtration
• Separation is based on protein size.Dextran or polyacrylamide beads of uniform diameter are manufactured with different pore sizes. • Depending on the sizes of the proteins to be separated, they will enter the pore if small enough, or be excluded if they are too large.

Ion Exchange Chromatography
• Separation of proteins based on the net charge of their constituent amino acids.
• Different salt concentrations can be used to elute the bound proteins into tubes in a fraction collector. Resins for binding (+) or (-) charged proteins can be used

Affinity Chromatography
• Based on the target proteins ability to bind a specific ligand, only proteins that bind to this ligand will be retained on the column bead. • This is especially useful for immunoaffinity purification of proteins using specific antibodies for them.

Physical and Chemical Properties of Proteins:
1. When pure, proteins are generally tasteless except with hydrolates. 2. Mostly colorless. 3. Insoluble in fat solvents and present varied degrees of solubility in water, salt solution, dilute acids and alkalies. 4. Proteins are amphoteric. 5. Proteins are very reactive and highly specific.

Solubility of Proteins: (Major influences)
1. The effect of neutral salt. 2. The effect of pH. 3. The effect of organic solvents.

Actions of Heat
- when burned, proteins decompose and liberate a characteristic odor of burned hair or feather.
- solutions of proteins when heated between 3860 degrees centigrade, undergo slight intramolecular rearrangements. ( DENATURATION)

Precipitations:
1. By acids 2. By salts of Heavy metals 3. By alcohol
Hydrolysis: 1. Dilute acids; alkalies or enzymes - due to addition of elements in the peptide bond/ linkage.

CLINICAL SIGNIFICANCE OF PROTEIN:
1. The substitution of a hydrophobic amino acid (V) for an acidic amino acid (E) in the β-chain of hemoglobin results in sickle cell anemia (HbS). This change of a single amino acid alters the structure of hemoglobin molecules in such a way that the deoxygenated proteins polymerize and precipitate within the erythrocyte, leading to their characteristic sickle shape.

2. Collagens are the most abundant proteins in the body. Alterations in collagen structure arising from abnormal genes or abnormal processing of collagen proteins results in numerous diseases, including Larsen syndrome, scurvy, osteogenesis imperfecta and Ehlers-Danlos syndrome.
a. Ehlers-Danlos syndrome is actually the name associated with at least ten distinct disorders that are biochemically and clinically distinct yet all manifest structural weakness in connective tissue as a result of defective collagen structure.

b. Osteogenesis imperfecta also encompasses more than one disorder. At least four biochemically and clinically distinguishable maladies have been identified as osteogenesis imperfecta, all of which are characterized by multiple fractures and resultant bone deformities. c. Marfan syndrome manifests itself as a disorder of the connective tissue and was originally believed to be the result of abnormal collagens. However, recent evidence has shown that Marfan syndrome results from mutations in the extracellular protein, fibrillin, which is an integral constituent of the non-collagenous microfibrils of the extracellular matrix.

3. Several forms of familial hypercholesterolemia are the result of genetic defects in the gene encoding the receptor for low-density lipoprotein (LDL). These defects result in the synthesis of abnormal LDL receptors that are incapable of binding to LDLs, or that bind LDLs but the receptor/LDL complexes are not properly internalized and degraded. The outcome is an elevation in serum cholesterol levels and increased propensity toward the development of atherosclerosis.

4. A number of proteins can contribute to cellular transformation and carcinogenesis when their basic structure is disrupted by mutations in their genes. These genes are termed proto-oncogenes. For some of these proteins, all that is required to convert them to the oncogenic form is a single amino acid substitution. The cellular gene, RAS, is observed to sustain single amino acid substitutions at positions 12 or 61 with high frequency in colon carcinomas. Mutations in RAS are most frequently observed genetic alterations in colon cancer.

MARAMING SALAMAT!