Proteins - Learning Material PDF

IV. PROTEINS Next to water, PROTEINS are the most abundant substances in most cells. They are particularly important class of food molecules because they provide an organism not only with C and H, but also with N and S, which elements are unavailable from fats and carbohydrates. Other elements, suc...

IV. PROTEINS Next to water, PROTEINS are the most abundant substances in most cells. They are particularly important class of food molecules because they provide an organism not only with C and H, but also with N and S, which elements are unavailable from fats and carbohydrates. Other elements, such as phosphorus and iron, are essential constituents of certain specialized proteins (casein & hemoglobin). They are the most abundant macromolecules in cells, and they typically account for the majority of the dry weight of cells. PROTEINS are high molar mass compounds consisting largely or entirely of chains of amino acids. They are utilized in the building of new tissues and in the maintenance of tissues already developed. CLASSIFICATION of PROTEINS A. Based on Composition 1. Simple proteins – contain purely amino acid residues eg. Albuminoids - keratin in skin, hair, nails; collagen in cartilage Albumins - egg albumin, serum albumin Globulins - antibodies Histones - chromatin in chromosomes 2. Conjugated proteins – protein + nonprotein moiety (prosthetic group) Type Example Prosthetic group Function of example Hemoproteins hemoglobin heme unit carrier of O2 in blood myoglobin oxygen binder in muscles Glycoproteins Interferon carbohydrates antiviral protection Mucin lubricant in mucous secretions Gamma globulin antibody Lipoproteins Lipoproteins TAG, phospholipids, cholesterol lipid carrier Metalloproteins Iron - ferritin Metal ion storage complex for iron Zinc – alcohol dehydrogenase enzyme in alcohol oxidation Nucleoproteins Nucleic acids Ribosomes site for protein synthesis Viruses self-replicating, infectious complex Phosphoproteins Casein Phosphate Glycogen phosphorylase enzyme in glycogen phosphorylation B. Based on structural shape 1. fibrous protein – a protein in which polypeptide chains are arranged in long strands or sheets; have long, rod-shaped or string-like molecules that can intertwine with one another and form strong fibers. Keratins in wool, feathers, hooves, silk, and fingernails Collagens in tendons, bone, and other connective tissues Elastins in blood vessels and ligaments Myosins in muscle tissue Fibrin in blood clots 2. globular protein – a protein in which polypeptide chains are folded into spherical or globular shapes; either dissolve in water or form stable suspensions in water, which allows them to travel through the blood and other body fluids to sites where their activity is needed. Insulin regulatory hormone for controlling glucose metabolism Myoglobin involved in oxygen transport in muscles Hemoglobin involved in oxygen transport in blood Transferrin involved in iron transport in blood Immunoglobulins involved in immune response 1 C. Based on Function C.1. Dynamic functions 1. Enzymes  catalyze chemical reactions, converting a substrate to a product at the enzyme‟s active site; genetic traits are expressed through synthesis of enzymes, which catalyze reactions that establish the phenotype  many genetic diseases result from altered levels of enzyme products or specific alterations to their amino acid sequence. 2. Transport  Hb and myoglobin transport O2 in blood and muscles respectively  Transferrin transports iron from the liver to the bone marrow, where it is used to synthesize the heme group for hemoglobin  Transport proteins are made used of in transport of TAG, cholesterol, etc.; drugs and toxic compounds are transported bound to proteins 3. Contractile mechanisms (movement)  myosin and actin are used in muscle contractions; eg. the heart contract and expand through the interaction of actin and myosin proteins  sperm cells are motile because they have long flagella that are an intricate aggregation of the protein tubulin  bacterial motility also occurs by means of flagella, which are an assembly of the protein flagellin 4. Protective role through a combination of dynamic functions  immunoglobulins or antibodies and interferon protect humans against bacterial or viral infection  fibrin stops the loss of blood on injury to the vascular system 5. Regulatory function by hormones  eg. are insulin, growth hormones, ACTH, etc. 6. Proteins control and regulate gene transcription and translation  histones are closely associated with DNA  repressors and enhancers are transcription factors 7. Storage * casein stores protein as milk * ferritin stores iron in liver for production of rbc C. 2. Structural functions  proteins provide the matrix for bone and connective tissue, giving structure and form to the organism  structural proteins provide mechanical support to large animals and provide them with their outer covering.  collagen and elastin form the matrix of bone and ligaments and provide structural strength and elasticity to organs and the vascular system  -keratin forms structure of epidermal tissues -Amino acids - proteins are polymers of -amino acids * common amino acids (~ 20 aa‟s)  “standard” * derived amino acids  “nonstandard” L--amino acid 2 - common, or “standard” amino acids contain in common a central -carbon to which a carboxylic acid group, an amino group, and a H atom are covalently bonded. Also, the -carbon is bound to a specific chemical group, called the side-chain R-group, that uniquely defines each of the 20 amino acids - side chains define chemical nature and structures of different amino acids - amino acids are polymerized into peptides and proteins; in general, protein is used for molecules composed of over 50 amino acids; peptide is used for molecules of less than 50 amino acids 1. Nonpolar amino acids - hydrophobic side chains; these amino acids are generally found buried in the interior of proteins, where they can associate with one another and remain isolated from water. - 9 amino acids: Gly (G), Ala (A), Val (V), Leu (L), Ile (I), Phe (F), Pro (P), Trp (W), Met (M) 2. Polar amino acids - have high affinity for water and are said to be hydrophilic; often found on the surfaces of proteins - subdivided into 3 classes: a) Polar, neutral amino acids  have side chains that have a high affinity for water but are not ionic  these aa‟s can associate with one another by H-bonding  6 amino acids: Ser (S), Thr (T), Tyr (Y), Cys (C), Asn (N), Gln Q) b) Acidic amino acids  negatively charged amino acids; have ionized carboxylate groups in their side chains  have a net charge of -1 at pH 7  2 amino acids: Asp (D), Glu (E) c) Basic amino acids  positively charged amino acids  have a net positive charge because their side chain contain positive groups  these aa‟s are basic because the side chain reacts with water, picking up a proton and releasing hydroxide ion  3 amino acids: Lys (K), Arg (R), His (H) 3 Derived amino acids (“nonstandard” amino acids) - usually formed by an enzyme-facilitated reaction on a common amino acid after that amino acid has been incorporated into a protein structure - examples are cystine, desmosine, and isodesmosine found in elastin hydroxyproline, and hydroxylysine found in collagen -carboxyglutamate found in prothrombin 4 Essential amino acids:  all amino acids are essential for normal tissue growth and development  the term is reserved for those amino acids that must be supplied in the diet for proper growth and development. The other amino acids can be synthesized from carbohydrates and lipids in the body by enzyme-catalyzed reactions if a source of nitrogen is also available.  must be supplied in the diet because either that there are no biochemical pathways available for their synthesis or the available pathway do not provide adequate amounts for proper nutrition.  His, Met, Arg, Thr, Trp, Ileu, Leu, Lys, Val, Phe (semi-essential; only on infancy stage) Some of the first informations on the biological value of dietary proteins came from studies on rats. In one series of experiments, young rats were fed diets containing 18% protein in the form of either casein (a milk protein), gliadin (a wheat protein), or zein (a corn protein) Results: Casein: rats remained healthy and grow normally Gliadin: rats maintained their weight but did not grow much Zein: rats not only failed to grow but began to lose weight, & eventually died if kept on this diet Since casein evidently supplies all the required amino acids in the correct proportions needed for growth, it is called a complete protein. Analysis of gliadin revealed that it contains too little lysine and that zein is low in both lysine and tryptophan. When gliadin diet was supplemented with lysine and zein with lysine and tryptophan, the test animals grew normally and thrived. Animal Protein Egg Complete None Milk Complete None Meat, fish, poultry Complete None Gelatin (denatured collagen) Incomplete Trp Vegetable Protein Grains Wheat Incomplete Lys Corn Incomplete Lys, Trp Rice (brown, white) Incomplete Lys Oats Incomplete Lys Legumes Beans Incomplete Met, Trp Peas Incomplete Met Nuts Almonds Incomplete Lys, Trp Walnuts Incomplete Lys, Trp A complete protein contains all the essential amino acids in the proper amounts. An incomplete protein is low in one or more of the essential amino acids, usually lysine, tryptophan, or methionine. Except for gelatin, proteins from animal sources are complete, whereas proteins from vegetable sources are incomplete. In a diet that includes animal protein, such as meat, milk, eggs, or cheese, all the essential amino acids are supplied. However, because these foods also contain saturated fats and cholesterol, many people obtain their protein from a diet of grains and vegetables. Such a diet must combine foods that have complementary proteins to ensure that all the essential amino acids are provided. For example, rice is deficient in lysine, and beans are deficient in methionine and tryptophan. However, when they are served together, they complement each other and provide all the essential amino acids General Properties of amino acids: 1. Configuration - only glycine has -C not asymmetric, hence, except glycine all amino acids are optically active and may exist either in the D- or L- enantiomeric forms - with few exceptions, the amino acids found in biological systems belong to the L-family (L-amino acids) 2. Dipolar nature of amino acids - when an amino acid is in aqueous solution, it exists in various ionic forms, the predominant form depends on the pH of the medium 5 - the COOH group can easily lose H+ in solution leaving COO- - the NH2 group can easily accept H+ in solution forming +NH3 - so the acidic and basic groups of amino acid react to form an internal salt or “zwitterion” which has no net charge for it contains one positive and one negative charge. R – CH – COOH R – CH – COO- |  | + NH2 NH3 “zwitterion” (German, „double ion‟) - the zwitterion form exists at a pH value called the isoelectric pH. At this pH the molecule is electrically neutral and would not move toward positive or negative electrode. - any amino acid in which the positive and negative charges are balanced is at its isoelectric point and the pH at which this balancing occurs is the isoelectric pH (IpH), a characteristic of each amino acid - nonpolar amino acids have isoelectric pH at values close to 7; basic amino acids reach their isoelectric point at much higher pH; acidic amino acids become electrically neutral at lower pH values. Amino acid Isoelectric pH e.g. Neutral 4.8 – 6.3 Ala (6.1) Acidic 2.8 – 3.2 Asp (2.8) Basic 7.8 – 10.8 Lys (9.7) - an amino acid is amphoteric and tends to be least soluble at its IpH because of the net zero charge - at pH values lower than IpH, the solubility of the amino acid increases because the carboxylate end of the zwitterion picks up a proton from the solution, and the amino ac.id acquires a net positive charge, which, if placed in an electric field, will migrate to the negative electrode R – CH – COO- R – CH - COOH | + H+  |  cathode + + NH3 NH3 - at pH values higher than IpH, the solubility of amino acid also increases because a proton is removed from the positively charged ammonium ion of the zwitterion & the net charge of the amino acid is negative, & will migrate to the positive electrode R – CH – COO- R – CH – COO- | + OH -  |  anode + NH3 NH2 - the migration of amino acid in electric field is called electrophoresis; it provides a useful technique for identifying and separating amino acid because the distance and the rate by which an amino acid moves at a specific pH is a characteristic for each individual amino acid Drill: For each amino acid: ala (pK1= 2.19; pK2= 9.67), asp (pK1= 2.09; pK2= 9.82; pKR= 3.86) & lys (pK1= 2.18; pK2= 8.95; pKR= 10.79) a) Draw the structure of the amino acid as the pH of the solution changes from highly acidic to strongly basic. b) Which form of the amino acid is present at isoelectric point? c) Calculate the isoelectric point, pI ; i.e., the ave. of the two pKa‟s bracketing the isoelectric structure. BONDING and PROTEIN STRUCTURE - the important properties and functions of proteins can be more easily understood if we know something about their structures and the bonding accounting for their structures - the structure of proteins is subdivided into 1o ; 2o ; 3o ; 4o according to the type of interactions of the amino acid 6 A. PRIMARY STRUCTURE - The 1o structure of a protein is the sequence of amino acid residues in a protein chain – i.e., the order in which the amino acids are connected to each other - frequently referred to as the backbone of the protein molecule - the 1o structure of proteins are translations of information contained in genes The Peptide Bond - holds amino acids together in chains; allows us to answer a very important question about proteins: “How can we account for the existence of over 100,000 different proteins when they are all constructed from the same 20 amino acids?” - peptides are formed through the union of 2 or more -amino acids via the peptide bond formation - peptide bonds are amide bonds that occur between the carboxyl group of one amino acid and the amino group of the next with the loss of water. A peptide bond joining 2 amino acids produces a dipeptide - may be termed di- ; tri- ; tetra- and so on based on the number of amino acids they contain - consider the combination of 2 amino acids, Gly and Ala, to form a dipeptide: the carboxyl group of gly reacts with the amino group of ala to form glycylalanine (Gly-Ala) O H O H2N – CH2 – C + N – CH – COOH  H2N – CH2 – C – NH – CH – COOH + H2O OH H CH3 CH3 Glycine Alanine Glycylalanine (Gly-Ala) - the carboxyl group of alanine may also react with the amino group of glycine to form another dipeptide, alanylglycine (Ala-Gly) - if we have 3 amino acids Gly, Cys, Ala, we will have 6 possible combinations of amino acids if each aa is used only once. Gly-Cys-Ala Gly-Ala-Cys Cys-Gly-Ala Cys-Ala-Gly Ala-Cys-Gly Ala-Gly-Cys - by convention, the structure of peptides is represented beginning with the amino acid whose amino group is free (N-terminal end). The other end contains a free carboxyl group and is the C-terminal end - amino acids are added to a peptide by forming peptide bonds with the C-terminal amino acid - peptide is named in accordance to the amino acid they contain; each amino acid in the sequence except the C- terminal amino acid is named as an acyl group (-ine  -yl). The name begins with the N-terminal amino acid & adds the suffix –yl to it. One proceeds in this manner until the C-terminal amino acid is reached, for which the original name is retained. E.g. ala-pro-glu-gly is named alanylprolylglutamylglysine 7 - the C – N bond in the peptide linkage has partial double bond character that makes it rigid and prevents the adjacent groups from rotating freely; the peptide bond is planar, and the two adjacent -carbons lie trans to it. The H of the amide nitrogen is also trans to the O of the carbonyl group. Almost all of the peptide bonds in proteins are planar and have a trans configuration. This is quite important physiologically because it makes protein structure relatively rigid. If they could not “hold their shapes”, they could not function; the peptide bonds are covalent and quite stable – can be disrupted by chemical or enzymatic hydrolysis but are not directly influenced by salt concentration, change in pH, or solvent Some examples of biochemically important small peptides: 1. Aspartame (Asp-Phe) Sold under the trade names Nutrasweet and Equal, aspartame is the artificial sweetener used in almost every diet food on the market today. Its caloric content is the same as sucrose but is ~ 180 times as sweet. Both aa‟s present in the dipeptide must be in the L- form for the sweet taste to occur; the L-D, D-L, and D-D forms have a bitter taste. 2. Glutathione, GSH (Glu-Cys-Gly) The tripeptide, produced by the body itself, is present in significant concentrations in most cells and is of considerable physiological importance as a regulator of oxidation-reduction reactions. It functions as an antioxidant, protecting cellular contents from oxidizing agents such as peroxides and superoxides, which are highly reactive forms of oxygen often generated within a cell. In GSH, Glu is bonded to Cys through the side- chain carboxyl group rather than through the α-carbon carboxyl group. 8 3. Enkephalins (Tyr-Gly-Gly-Phe-Leu & Tyr-Gly-Gly-Phe-Met) Enkephalins, of which there are two, are pentapeptides produced by the brain itself that bind at receptor sites in the brain to reduce pain. The two enkephalins differ only in the amino acid at the carboxyl end of the peptide chain. The order in which the amino acid residues of a peptide molecule are linked is the amino acid sequence of the molecule; differences in the chemical and physiologic properties of peptides result from differences in the amino acid sequence. e.g., a) Bradykinin vs Boguskinin - partly responsible for triggering pain, - completely inactive, hence, the name welt formation (as in scratches) , bogus or false movement of smooth muscle, and lowering of blood pressure Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg b) Normal Hb: …..Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Ser-Ala-….. Sickle-cell Hb: …..Val-His-Leu-Thr-Pro-Val-Glu-Lys-Ser-Ala-….. Georgetown anemia: …..Val-His-Leu-Thr-Pro-Glu-Lys-Lys-Ser-Ala-….. Drill: Write the structure of the tetrapeptide Lys-Phe-Asp-Ala that exists at isoelectric point. Calculate its pI. pKala SEQUENCING A PROTEIN (pp 598 – 600 , McKee) - the procedure in the determination of amino acid sequence basically involves the following steps: 1) hydrolysis - can be effected by acid, alkali, or enzyme 2) identification of the products of hydrolysis 3) fitting the pieces together as you would a jigsaw puzzle HYDROLYSIS A) Acid Hydrolysis - involves heating in the presence of 6N HCl in sealed tube at 110 oC for 10 – 100 hrs. depending on the nature of peptide or protein to be hydrolyzed - the protein is completely hydrolyzed, but trp, is destroyed completely and ser, thr, and tyr are partially destroyed B) Alkaline Hydrolysis - heating in the presence of 4N NaOH in sealed tube at 10 – 100 hrs as in acid hydrolysis - does not damage trp, but destroys arg, cys, cys-cys, thr, & ser; and some amino acids are partly deaminated - more disadvantageous but since it does not destroy trp, it is used in quantitative determination of this amino acid 9 C) Enzymatic Hydrolysis - by proteases/ peptidases 1) Exopeptidases - cleaves external peptide bonds a) Aminopeptidases - sequentially cleaves peptide bonds, beginning at the N-terminal end of the polypeptide - the liberated amino acids are identified one by one b) Carboxypeptidases - sequentially cleaves peptide bonds beginning at the C-terminal end of the polypeptide  The exopeptidases may be used in the determination of the amino acid sequence of peptides. By following the increase in amino acid liberated during hydrolysis by the two enzymes it is possible to determine the amino acid sequence in a peptide.  e.g., A time-course analysis of the free amino acid in the hydrolysate of the pentapeptide with aminopeptidase and carboxypeptidase gave the following results: a) with carboxypeptidase [Glu] > [Val] > [Ala] > [Cys] = [Arg] ; cyst and arg appear simultaneously on equal amounts b) with aminopeptidase [Arg] > [Cys] > [Ala] > [Val] = [Glu] ; val and glu appear simultaneously on equal amounts c) what is the primary structure of the pentapeptide? Other methods of determining the N-terminal end and the C-terminal end (chemical method) A. N-terminal end a) Sanger‟s method - 2,4-dinitrofluorobenzene (DNFB) binds to the N-terminal amino group of the polypeptide. The resultant dinitrophenyl-amino acid or DNP-amino acid can be separated from the other amino acids because it is more soluble in nonpolar solvents b) Edman‟s method - phenylisothiocyanate, PITC, (Ph – N = C = S) combines with the N-terminal amino acid to yield a phenylthiohydantoin- compound (PTH-compound), which may be identified by chromatography. PTh- compound can be extracted by organic solvent B. C-terminal end a) Hydrazine method - hydrazine reacts with all amino acids whose carboxyl group is bound in peptide linkage, creating amino acyl hydrazides. Only the C-terminal amino acid is spared 2) Endopeptidases - cleaves internal peptide bonds a) Trypsin - cleaves peptide bonds at the carboxyl end of the two strongly basic amino acids: arginine and lysine b) Chymotrypsin - cleaves peptide bonds at the carboxyl end of the three aromatic amino acids: phenylalanine, tyrosine, & trptophan; and Leu c) Pepsin - cleaves peptide bonds at the amino end of the three aromatic amino acids: phenylalanine, tyrosine, tryptophan; acidic amino acids, Asp and Glu; and Ileu d) Thermolysin - cleaves peptide bonds at the amino end of the three aromatic amino acids, Phe, Tyr, Trp; and amino acids with bulky nonpolar R groups, Leu, Ileu, and Val 10  e) Elastase - cleaves on the carboxyl side of Gly and Ala Other methods (chemical method) a) Cyanogen Bromide (CNBr) – cleaves peptide bonds at the C-end of methionine Exercises: 1. Write the peptides generated from chymotrypsin, trypsin, and pepsin digestion of Ala-His-Tyr-Pro-Trp-Arg-Ileu-Phe-Glu-Lys-Cys 2. Total acid hydrolysis of a pentapeptide complemented by total alkaline hydrolysis yields an equimolqr mixture of five amino acids: ala, cys, lys, phe, and ser. N-terminal analysis with PITC generates PTH-serine. Trypsin digestion produces a tripeptide whose N-terminal residue is cys and a dipeptide with ser at its N-terminal. Chymotrypsin digestion of the above tripeptide yields ala plus another dipeptide. (a) What is the amino acid sequence of the tripeptide? (b) What is the amino acid composition of the dipeptide derived from trypsin digestion? (c) What is the primary structure of the original pentapeptide? 3. Consider a tripeptide that stimulates the thyroid to release thyroxine, a thyroid hormone. By using acid hydrolysis, all the peptide bonds in the protein can be broken and the resulting mixture analyzed to determine the number and kinds of amino acids. Upon hydrolysis the tripeptide contains His, Glu, and Pro. However, this does not tell us their sequence in the tripeptide. Six tripeptide sequences are possible from these three amino acids. Methods: N- terminal end determination complemented by C-terminal end determination. Another method is partial hydrolysis of the peptide to give smaller peptide fragments. B. SECONDARY STRUCTURE - when the primary sequence of the polypeptide folds into regularly repeating structures, secondary structure is formed; tells something about shape/conformation of protein molecule - the only bond responsible for the 2o structure of proteins is H-bonding between peptide bonds, the – C = O of one peptide group and the – N – H of another peptide linkage farther along the backbone. TYPES OF 2o STRUCTURE: 1. Helix structure - governed by intramolecular H-bonding ; the -helix is the most stable helical arrangement - in an α helix, all of the amino acid side chains (R groups) lie outside the helix; there is not enough room for them in the interior - the helix is so tightly wound that the space in the center is too small for solvent molecules to enter - every amide hydrogen and carbonyl oxygen is involved in a H-bond when the chain coils into an -helix. every carbonyl oxygen is H-bonded to an amide hydrogen four residues away in the chain - the H-bonds of the -helix are parallel to the long axis of the helix; these bonds lock the -helix into place - the polypeptide chain in an -helix is right-handed (left-handed helix for D-amino acids), it is oriented like a normal screw. If you turn the screw clockwise it goes into the wall; turned counterclockwise, it comes out of the wall - the repeat distance of the helix, or its pitch, is 5.4 Ao, and there are 3.6 aa residues per turn of the helix - certain amino acids do not foster α-helix formation (p 97 McKee) ; incompatible with α-helix structure eg. glycine - has R group (R = H) so small that the polypeptide chain may be too flexible proline - contains rigid ring and has no N – H group available for intramolecular H-bonding large # of charged aa‟s and bulky R groups (eg. Trp) in amino acid sequences are also incompatible 11  2. Triple helix or collagen 3. Random coil 4. Pleated-sheet structure or -conformation - governed by intermolecular H-bonds between protein chains to form a zigzag, sheetlike arrangement - all of the carbonyl oxygens and amide hydrogens are involved in H-bonds, and the polypeptide chain is nearly completely extended; R groups are located above and below the plane of the sheet. - proteins with this structure are crystalline and quite insoluble in aqeous solvents a) parallel structure - chains are running in the same direction, the –COOH and -NH2 ends of the proteins lying all at the top or all at the bottom of the sheet b) antiparallel structure - protein chains alternate in such a way that the –COOH end of one chain is next to the –NH2 end of the other; chains are running in opposite directions ; more stable than parallel because of fully collinear H-bonds form 12 5. Supersecondary structures - combinations of α-helix and β-pleated sheets Very few proteins have 100% α helix or β pleated sheet structures. Most proteins have only certain portions of their amino acid residues in these conformations; the rest of the molecule assumes a “random structure”; it is also possible to have both α helix and β pleated sheet structures with the same protein. C. TERTIARY STRUCTURE - the overall three-dimensional shape that results from the interaction of groups in the side chain (-R) of the amino acids widely separated from each other within the chain; defines the biological function of proteins - proteins may have, in general, either of the two forms of tertiary structure: (a) fibrous and (b) globular - fibrous proteins (insoluble): mechanical strength, structural components, movement - globular proteins (soluble): transport, regulatory, enzymes INTERACTIONS/LINKAGES Responsible for 3o Structure - these linkages aid in holding the protein in a rather rigid structure 1. salt linkages / ionic interaction/ electrostatic attraction 2. H-bonds 3. disulfide bonds 4. hydrophobic bond 5. van der Waals forces 6. polar group interactions with water 1. Salt linkages - some amino acid side chains may contain positively charged groups, others negatively charged groups. If properly positioned these groups may give rise to bonding between different portions of given molecule, or between 2 or more protein chains 2. Disulfide bonds - the only covalent bond, the strongest of the 3o bonds; link chains together and cause chains to twist and bend - a covalent bond between 2 S atoms formed by the oxidation of the – SH groups on two cysteine amino acids - S – S linkage can occur within the same chain (intrachain), or between 2 or more chains (interchain), or both inter- and intrachain 13 3. Hydrophobic bonds - a number of amino acids have side chains which are of hydrocarbon nature. These are hydrophobic groups in that they do not form H-bonds with water molecules. On the other hand, water molecules have a strong tendency to form H-bonds among themselves. As a result of the H-bonding among water molecules, the hydrocarbons are forced out of any water phase in which they may be placed. Similarly, the HC side chains of the various amino acids tend to be forced together as a result of H-bonding among water molecules D. QUATERNARY STRUCTURE - the functional form of many proteins is not that of a single polypeptide chain, but actually an aggregate of several globular peptides - quaternary structure: the arrangement of subunits or peptides that form a larger protein - subunit: a polypeptide chain having primary, secondary, and tertiary structural features that is a part of a larger protein  myoglobin has no 4ostructure; it has only 1 chain  hemoglobin has 4ostructure; it consists of 4 separate chains called protein subunits: 2 and 2 chains – each chain is bound to a separate heme group  collagen has 4ostructure; composed of 3 strands of the same protein twisted together in a helical fashion like a piece of a rope 14  Types of Protein Structure and Their Interrelationships PROTEIN DENATURATION - refers to the breaking of any bond in protein except the 1o bond (peptide bond) - when protein is in the cell, it is in its natural conformation, in its native state. If this native state is changed in any way, the protein is said to be denatured. - denaturation often radically changes both the physical and chemical properties of the protein; - proteins lose their activity, although under certain conditions this loss may be reversible (renaturation) Some selected physical and chemical denaturing agents 1. heat – disrupts H-bonds and hydrophobic attractions by making molecules vibrate too violently; produces coagulation, as in the frying of an egg 2. microwave radiation – causes violent vibrations of molecules that disrupt H-bonds 3. ultraviolet radiation – operates very similarly to the action of heat (e.g., sunburning) 4. acids and bases – disrupts salt linkages 5. violent whipping or shaking – causes molecules in globular shapes to extend to longer lengths, which then entangle (e.g., beating egg white into meringue) 6. detergent – affects H-bonds and salt linkages 7. organic solvents (e.g., ethanol, 2-propanol, acetone) – interfere with H-bonds, because these solvents also can form H-bonds; quickly denatures protein in bacteria, killing them (the disinfectant action of 70% ethanol) 8. strong acids and bases – disrupt H-bonds and salt bridges; prolonged action leads to actual hydrolysis of peptide bonds 15 9. salts of heavy metals (e.g., salts of Hg2+, Ag+, Pb2+) – metal ions combine with – SH groups and form poisonous salts; precipitates proteins 10. reducing agents – oxidize disulfide linkages to produce – SH groups Some practical aspects of protein denaturation 1. Heat and UV - cooking denatures proteins ; e.g., egg white proteins have to be denatured by cooking for them to become utilizable by our system - any burn, including sunburn, causes denaturation of protein in the body - sterilization uses UV and heat in the form of steam to coagulate the proteins of bacteria 2. Salts of heavy metal ions esp. Hg2+, Pb2+, Ag+ - used as antiseptics in low concentrations, in higher concentrations they act as poisons. When ingested they precipitate the proteins in cells of body tissues. Effective treatment consists of feeding with egg white, followed by an emetic (the egg white forms complex with the poison and taken out of circulation by emetic) 3. Organic compounds such as soap, detergents, phenol, and aliphatic alcohol - the hydrophobic portions of these compounds interact with the hydrophobic core of the protein, while the hydrophilic portion is H-bonded with the aqueous environment. This causes swelling and concomitant unfolding of the protein molecules. 4. Permanent hair waving - keratin has a high proportion of S-containing amino acids; the –S – S – linkages give shape to the hair - process involves the breaking of these linkages by reducing agent  brings disorderliness of linkages  placed on curlers and set in the desired pattern  finally neutralized with an oxidizing agent to reform – S – S – linkages this time between different amino acids HORMONES Some Peptide Hormones and Their Functions Hormone Composition Target Organ Biological Effect From the pituitary gland Oxytocin 9 amino acids Uterus, mammary Stimulates contraction of uterus, glands and production of milk Vasopressin 9 amino acids Kidneys Increases reabsorption of water by kidneys; decreases urine formation Adrenocorticotropin 39 amino acids Adrenal cortex Stimulates production of corticosteroids Growth hormone 188 amino acids Body tissues Increases rate of protein synthesis (GH) promotes growth of cells Follicle-stimulating Glycoprotein Ovaries Stimulates follicle growth hormone (FSH) Luteinizing hormone Glycoprotein Testes, Overies Stimulates production of testosterone, (LH) estrogens, & progesterone ----------------------------------------------------------------------------------------------------------------------------------------- From the pancreas Insulin 51 amino acids Body tissues Stimulates glucose metabolism, lowers glucose, increase glycogen storage Glucagon 29 amino acids Liver Inc. blood glucose by converting glycogen to glucose, stimulates glucose synthesis From parathyroid from amino acids Parathyroid 84 amino acids Body tissues Regulates Ca & PO3 levels in body fluids From the thyroid Thyroxine I2-containing Body tissues Inc. metabolic rate, utilization of food, derivative of tyr protein synthesis, and growth ================================================================================= 16 Reading Assignment: Characteristics of the Immune System Types and functions of immunoglobulin 17 18