Bchem 154 Biochemistry Course Outline PDF

GENERAL BIOCHEMISTRY BCHEM 154 F.O. MENSAH 1 COURSE OUTLINE 1. AMINO ACIDS AND PROTEINS A. Chemistry of amino acids a) Structure b) Properties c) Types/classiﬁcation d) Reaction – Carboxyl group – Amino group B. Peptides 2 2 C. Polypeptides and proteins a) Types and classiﬁcation b) Function c) Structure and properties I. Denaturation II. Determination of amino acid sequence III. Reactions of side chain R group d) Isolation and puriﬁcation of proteins 3 2. ENZYMES a) Naming and classiﬁcation b) Nature of enzyme activity c) Factors affecting rate of enzyme action I. Substrate concentration II. Enzyme concentration III. Temperature IV. pH V. Time VI. Inhibitors VII. Cofactors d) Regulation of enzyme activity 4 3. COFACTORS 4. NUCLEOTIDES AND NUCLEIC ACIDS 5 AMINO ACIDS AND PROTEINS Proteins are complex organic nitrogenous substances in the cells of plants and animals. They basically contain C, H, N, O and S. The building block units of proteins are amino acids. There are about 20 different naturally occurring amino acids and these contribute to the structure, properties and functions of proteins. 6 Chemistry of amino acids Amino acids have a general common structure, but differences occur in their side chain which distinguishes them from one another. They contain an amino or basic group and a carboxylic or acidic group. Both the amino and carboxylic groups are bound to the same carbon atom, α-carbon which is adjacent to the carboxylic group. The α-carbon is also bonded to a hydrogen atom and to a side chain R group. 7 The identity of a particular amino acid therefore depends on the nature of the R group. General structure + NH3 NH2  - or  H C C OO H C C OO H R R 8 Amino acids are often abbreviated by 3 letter symbols or one letter symbol. The carbon atoms in amino acids are designated α, β, γ, δ, ε, etc. in the order in which they are attached, starting with the C (carbon) adjacent to the carboxyl group. Almost all naturally occurring amino acids are optically active with the exception of glycine, and have L-conﬁguration at the α-carbon. Some D- amino acids have been found in bacterial cell walls. 9 Functions of the R-group The R-group may be acidic, basic or neutral, etc and may serve the following functions: 1. They contribute to the interaction between different parts of the protein molecule or different proteins, e.g. hydrophobic interactions, disulphide linkages, covalent modiﬁcation, etc. 2. They affect stability of proteins. 10 3. They affect reactivity as they serve as points of attachment of groups other than the amino acids, e.g. sugar, sulphates, phosphates group, etc. 4. They serve as sources of identiﬁcation by hydrolytic enzymes. 5. R-groups dictate the shape of the proteins due to their interactions, thus determining the ultimate functions of the protein i.e. they dictate folding into precise 3-dimensional conﬁguration. 11 Properties of amino acids 1. With the exception of glycine, amino acids have asymmetric centres and are therefore optically active. 2. They possess charges. Neutral amino acids are amphoteric in nature (have both acidic and basic properties). Amino acids with acidic R groups (COO- or COOH) are acidic while amino acids with basic R groups (NH3+ or NH2) are basic. 3. Due to the presence of both positive and negative charges, amino acids behave like salts and have high melting points normally above 200oC. 12 4. They are generally colourless, crystalline solids whose solubility in water varies with the nature of the constituent R-group. They are quite insoluble in non-polar solvents like ether and chloroform. 5. Some are sweet, e.g. valine, alanine, proline. Others are bitter, e.g. arginine, isoleucine or tasteless, e.g. leucine. 13 Classiﬁcation of amino acids Classiﬁcation may depend on: 1. their reactions, i.e. neutral, acidic or basic. 2. chemical structure i.e. presence of polar and non-polar side groups, aromatic groups, long hydrocarbon side chain, etc. A general classiﬁcation is based on the polar or non-polar nature of the R-group and the presence of acidic or basic groups in the side chain. There are four main groups; 14 methionine (M) isoleucine (I) valine (V) leucine (L) aspartic glutamic acid (D) acid (E) phenylalanine (F) tyrosine (Y) tryptophan (W) aspargine (N) glutamine (Q) serine (S) threonine (T) lysine (K) arginine (R) histidine (H) glycine (G) alanine (A) cysteine proline + 2 + 3 15 1. Amino acids with neutral hydrophobic (non-polar / apolar) side chain. e.g. glycine, valine, alanine, leucine and isoleucine. These have aliphatic side chains. Proline has an aliphatic cyclic structure and is actually an imino acid since the N is bonded to 2 carbon atoms. Phenylalanine and tryptophan have aromatic R- groups. (Aromatic ring of tryptophan is called indole ring). Methionine contains Sulphur in its aliphatic R group. 16 Non polar amino acids in a protein tend to produce a hydrophobic environment in the protein of which they are components. The amino acids in this group are generally found buried in the interior of the proteins where they can associate with one another and remain isolated from water. Amino acids in this group play an important role in maintaining the three dimensional structure of proteins. 17 2. Amino acids with neutral and hydrophilic (polar uncharged) R group in their side chain. These amino acids have polar side chains that are neutral at neutral pH (pH 7). They are serine and threonine (polar OH groups attached to aliphatic side chain). Tyrosine has OH group attached to aromatic chain. Cysteine has -SH polar side chain which can react with other cysteine –SH groups to form disulphide (-S-S-) bridges in proteins. Sometimes, glycine is put in this group because of the H (formation of H bonds in water). 18 The polar OH group of Ser, Thr and Tyr enables them to participate in H bonding, an important factor in protein structure. OH groups also serve as points for esteriﬁcation with phosphate and attachment of sugars or carbohydrates. Asparagine and glutamine bear highly polar amide side chains of different sizes. The amino acids within this group can associate with one another by hydrogen bonding which helps to maintain proper three dimensional structures of proteins. 19 3. Amino acids with acidic and hydrophilic (polar) side chains This group has an additional COOH group in the side chain. The carboxyl group can lose a proton to form the carboxylate ion (proton donor). They are negatively charged at neutral pH, e.g. glutamic acid (glutamate) and aspartic acid (aspartate). The side chain carboxyl groups frequently bond to NH2 to form the side chain amide groups yielding the analogous amino acids asparagine and glutamine. 20 4. Amino acids with basic and hydrophilic (polar) side chains These bear positive charges at neutral pH and have additional amino groups in the side chain, e. g. histidine (side chain of histidine referred to as imidazole). Lysine (side chain amino group attached to aliphatic hydrocarbon chain), and arginine (side chain guanidine group attached to aliphatic hydrocarbon chain). 21 The hydrophilic or polar amino acids are often found on the surface of protein in association with water. The negatively and positively charged amino acids within a protein can interact with one another to form ionic bridges, another strong force that helps keep the protein chain folded in a particular manner. 22 23 Non-protein amino acids These are amino acids that are found in free or ‘uncombined’ forms and are not constituents of proteins. Some play important roles in metabolism and may be intermediates in the biosynthesis of proteogenic amino acids, e.g. L-ornithine and L- citrulline are metabolic intermediates of the urea cycle and hence participate in the biosynthesis of the amino acid arginine. 24 O O + - + - H C H C O H N C H C O 3N 3 H 2 C H 2 C C H 2 C H 2 C H 2 C H 2 + H 3 N HN C=O NH L--Ornithine 2 L--Citrulline 25 β-alanine occurs free in nature and is a component of the water soluble vitamin pantothenic acid. Other non protein amino acids are homocysteine, homoserine, γ-amino butyric acid (GABA). Homoserine and homocysteine are intermediates in amino acid metabolism. GABA is involved in the transmission of nerve impulses. 26 H 2 N C H 2C H 2C O O H H 2 N C H 2C H 2C H 2C O O H a la n in e G A B A NH2 NH2 H S C H 2C H 2C C O O H H O C H 2C H 2C C O O H H H H o m o c y s te in e H o m o s e r in e 27 Other examples are serotonin, thyroxine and indoleacetic acid (IAA). I I - CH2 CO O - HO O C H 2C H C O O N + H NH3 I I T h y r o x in e I n d o le a c e tic a c id ( I A A ) HO + C H2C H2NH3 N H Serotonin 28 Rare amino acids These rarely occur but have been isolated from hydrolysate (hydrolytic products) of some speciﬁc proteins. They are all derivatives of some standard amino acids, eg. 4-hydroxyproline and 5-hydroxylysine are derivatives of proline and lysine respectively. They are abundant in ﬁbrous proteins and collagen. Hydroxyproline is an important component of animal supportive and connective tissues. 29 - C OO + H 3N CH HO CH2 CH CH2 H 2C H 2C C C OO H + H C OH N H H H CH2 + H 3N 4 - h y d r o x y p r o lin e 5 - h y d r o x y ly s in e 30 Essential amino acids These can not be synthesized in the body of higher animals and have to be provided in the diet. The inability to synthesize them may be due to the absence of 1. The corresponding α-keto acid of the amino acid. 2. The enzyme involved in transamination i.e. a speciﬁc transaminase. 31 + + NH3 NH3 O tr a n s a m in a s e O 1 + R 2 C COO H R 1 C COO H + 2 R C COO H R C COO H H H A c c e p to r k e to D o n o r a m in o N e w a m in o N e w k e to a c id a c id a c id a c id The process is called transamination. Example; H O CH3 H C O + H 2N C C OO H H 2N C C OO H + H C C OO H C OO H H H 3C P y r u v a te G ly c in e A la n in e 32 Transamination Reactions 33 Transamination Reactions a la n in e L - a la n in e + - K G tr a n s a m in a s e P y r u v a te + L - g lu ta m a te o r g lu ta m a te p y r u v a te tr a n s a m in a s e a s p a r ta te L - a s p a r ta te + - K G tr a n s a m in a s e O A A + L - g lu ta m a te g lu ta m a te O A A tr a n s a m in a s e le u c in e L - le u c in e + - K G tr a n s a m in a s e - k e to is o c a p r o a te + L - g lu ta m a te L - ty r o s i n e + - K G p - h y d r o x y p h e n y l p y r u v a te + L - g l u ta m a te 34 Since most proteins in the body contain the full complement of amino acids, young animals fail to grow on a diet deﬁcient in even one essential amino acid. Without it they are unable to synthesize adequate proteins. Sufﬁcient quantities are needed to maintain the proper nitrogen balance in the body. 35 Prolonged deﬁciency leads to the disease ‘kwashiorkor’ in children. Other deﬁciencies are; 1. Fall in plasma protein level, and 2. Low haemoglobin levels in adults. 36 Essential amino acids Non-essential amino acids Histidine Alanine Leucine Glycine Isoleucine Asparagine Methionine Proline Phenylalanine Serine Threonine Tyrosine Tryptophan Cysteine Valine Glutamine Arginine Glutamic acid Lysine Aspartic acid Ideally, lysine and arginine are semi essential amino acids because they can be synthesized by the body but not in adequate quantities. Proteins from cereals are poor in lysine and those from legumes low in methionine 37 Acid base properties of amino acids Amino acids contain both acidic and basic groups and react with both alkali and acids to form salt and are thus amphoteric in nature. The acid group is a proton donor and the basic group is a proton acceptor. In the crystalline or solid state, amino acids exist as dipolar ions. In this case, the COOH group exists as the carboxylate ion (COO-) bearing a negative charge and the amino group (NH2) exists as ammonium ion (NH3+), bearing a positive charge. The dipolar ion is called a zwitterion. 38 In aqueous solution, equilibrium exists between the dipolar ion and other anionic and cationic forms of the amino acid. The position of the equilibrium depends on the pH of the solution and the nature of the amino acid especially contributed by the R-group. H H H + + - H + - H + H 2N C C OO - H 3N C C OO - H 3N C C OO H O H O H R R R B a s ic s o lu tio n Z w itte r io n C a tio n ic a n io n ic 39 In strongly acidic solution, all amino acids exist primarily as cations and in strongly basic solutions, they exist as anions. At some intermediate pH called the isoelectric point the concentration of the zwitterion is at its maximum and the concentration of the cation and anion are equal. At this pH (isoelectric point), there is no net migration of the amino acid when placed in an electric ﬁeld as the net charge is zero (0). The dissociation of an amino acid is therefore strongly dependent on the pH value of the solution. 40 Isoelectric pH It is the pH at which there is no net charge on the amino acid. It is denoted pI. Each amino acid has a speciﬁc pI and this has been the basis for precipitation of amino acids. At certain pH values, the amino acid may move either to the anode or cathode in an electric ﬁeld depending on the charge on it and the magnitude of that charge at that pH. 41 Amphoteric nature of amino acids The amphoteric nature of amino acids is responsible for the buffering action of proteins in the blood because as an acid it can donate proton and as a base, it can accept proton and therefore can resist small changes in pH. An amino acid with a neutral R group is somewhat more acidic than it is basic. As a result, its isoelectric point occurs at a pH slightly lower than a neutral solution of pH7. 42 Amphoteric nature of amino acids 43 Titration curves of amino acids When an amino acid is titrated, its titration curve indicates the reaction of each ionisable group that is capable of reacting with Hydrogen ion. The normal titration curve of an acid with a base is as follows; 44 45 Acid base reactions involve a conjugate acid base pair made up of proton donor and proton acceptor. (The biochemical behaviour of many important compounds depend on their acid base properties). The ability of acids or bases to readily lose or gain protons depends on the chemical nature of the compounds involved. E.g. the degree of dissociation of acids in water varies from complete dissociation for strong acids, to partial or no dissociation for weak acids. Intermediate values are possible. 46 Dissociation of weak acid in solution Typical example is dissociation of acetic acid; HA A- + H+ The strength of an acid, which is the amount of H+ released when a given amount of acid is dissolved in water, can be expressed numerically. The expression is called acid dissociation constant or Ka, and can be written for any acid HA according to the equation; 47 For each acid, Ka has a ﬁxed numerical value at a ﬁxed temperature. Ka = [H+] [A-] [HA] solving for [H+] [H+] = Ka[HA] [A-] According to the Henderson-Hasselbalch equation pH = pKa + log [A-] [HA] pH = pKa + log [proton acceptor/conjugate base] [proton donor/conjugate acid] 48 The numerical value of Ka is higher when the acid is more completely dissociated, i.e. the larger the Ka the stronger the acid or the smaller the pKa value the stronger the acid. At pH = pKa, half of the ionizable groups are dissociated. 49 Titration of acetic acid with NaOH During titration, a measured amount of base is added to a measured amount of acid thus changing the pH of the solution. The amount of base required for complete reaction with the acid is referred to as one equivalent. The point in titration at which the acid is exactly neutralized is called the equivalence point. In the course of titration of acetic acid, a point is reached when the pH = pKa of the acetic acid. 50 The point of the titration curve where pH=pKa is the inﬂection point. This corresponds to a solution with equal concentration of the weak acid and its conjugate base, in this case acetic acid and acetate respectively. The pH at the point of inﬂection is about 4.8 and is equal to the pKa of acetic acid. Inﬂection point is attained when 0.5 equivalent of base has been added. Near the inﬂection point the pH changes very slowly as more base is added. The equivalent point is reached when one equivalent of base has been added. At this point, practically all the acetic acid has been converted to acetate ion. 51 Titration of acetic acid with NaOH pH 9.2 - * CH3COO x 4.8 Inﬂection point * CH COOH 3 0 1.0 0.5 NaOH x CH3COOH = CH3COO- 52 Titration curves of some amino acids, e.g. alanine Alanine has 2 titrable groups; the carboxyl and amino groups. In it’s fully protonated form it can be considered as a dibasic acid and can therefore donate 2 protons during its titration with a base like NaOH. At very low pH, alanine has protonated carboxyl group (uncharged) and a positively charged amino group. 53 Under such conditions alanine has a net positive charge of one. As base is added, the carboxyl group loses its proton to become a negatively charged carboxylate ion as the pH of the solution increases. At this stage, alanine has no net charge. As more base is added with resulting increase in pH the protonated amino group (weak acid) loses its proton and the alanine has a negative charge of one. 54 Titration curve of Alanine 55 Ionization of alanine The pKa of the two stages of ionization of alanine is wide enough to yield two separate regions, each region showing a titratable group. The apparent pKa of the 2 dissociation curves can be determined from the midpoint of each stage. pKa1 is equal to 2.34 and pKa2 is 9.69. At pKa 2.34, NH3+CHRCOOH and NH3+CHRCOO- are present in equimolar concentrations and at pKa 9.69 H3N+CHRCOO- and H2NCHRCOO- are present in equimolar concentrations. 56 At pH 6.02 there is a point of inﬂection between the two separate phases of the titration curve of alanine. This is the isoelectric pH or pI and alanine bears no net charge and therefore there is no migration in an electric ﬁeld (at pH + - 6.02, the dominant specie is NH3 CHRCOO ). Mathematically, pI is expressed as the arithmetic mean of the 2 pKa values. pI = pKa1 + pKa2 2 pI alanine = 2.34 + 9.69 = 6.02 2 57 At pH above the pI (alkaline) amino acids exist as H2NCHRCOO- and migrate to the anode (positive pole). At pH below pI (acidic) amino acids exist as H3N+CHRCOOH and will migrate towards the cathode (negative pole). 58 Titration curve of diprotic amino acids A diprotic amino acid is an amino acid with an additional titratable group in the side chain e.g. Histidine Amino acids having additional NH2 or COOH group will have corresponding pKa2 values for them. Example, aspartate has a pKa of 2.1 for the α- carboxyl group and a pKa of 3.9 for the β- carboxyl group and pKa of 9.8 for the NH3+ 59 group. In histidine, the imidazole side chain also contains titratable group. At very low pH values, histidine has a net positive charge of 2 as both the imidazole and amino groups bear positive charges. As base is added, the pH increases and the carboxyl group loses a proton to become a carboxylate ion and histidine now has a net positive charge of +1. With further addition of more base, the imidazole charge group loses its proton. At this point histidine has no net charge (pI of histidine). 60 At still higher pH values the amino group loses its proton and histidine now has a net negative charge of -1. 61 Like acids, amino acids have characteristic values for Kas and pKas of their titratable groups. The pKas of the α-carboxyl groups are fairly low, approximately 2; that of the amino groups are between 9 and 10.5. The pKa of the side chain groups including the additional COOH and NH2 groups depend on the chemical nature of the group. The classiﬁcation of an amino acid as acidic or basic depends on the pKa of the side chain. These R groups can still be titrated within a protein but their pKas may not necessarily be the same as the value in the free amino acid. 62 63 64 Reactions of amino acids The characteristic reactions of amino acids are those of their functional groups, i.e. the COOH and the NH2 groups and the functional group present in the different side chains. These reactions are important in protein chemistry for; 1. Identiﬁcation and analysis of amino acids in protein hydrolysate (hydrolytic product). 65 2. Identiﬁcation of amino acid sequence in a protein. 3. Identiﬁcation of the speciﬁc amino acid residues of native protein that are required for their biological activity. 4. Chemical modiﬁcations of amino acid residues in protein molecule to produce changes in their biological activities and other properties. 5. Chemical synthesis of polypeptides. 66 Reactions of carboxyl group The carboxyl groups of amino acids can react to form salts, esters, acid chlorides and amides 1. Esteriﬁcation with alcohol and the formation of peptide bonds In the presence of HCl amino acids react with alcohol or ethanol to form esters. 67 When the amide bond involves an α-amino group of another amino acid instead of NH+3, a peptide bond is formed. This is the basis for the formation of peptides and proteins. Peptide bonds are links between amino acids in a protein (like glycosidic bonds). 68 69 70 2. Decarboxylation When α-amino acids are heated in the presence of Ba(OH)2, carbon dioxide is released and an amine is formed. Decarboxylation can also be achieved by the enzyme, decarboxylase. Decarboxylaton of amino acids is important in the body as it yields biologically important active amines, e.g. Tyrosine yields adrenaline, histidine yields histamine, and tryptophan yields serotonin. 71 72 Reactions of the Carboxyl group 73 Reactions of the amino group 1. Acylation Amino groups of amino acids may be acylated by treatment with acid anhydride or acid chlorides in cold alkaline medium. 74 This method is used to protect the α-amino group in the chemical synthesis of a peptide. Glycine readily reacts with benzoic acid to detoxify the benzoic acid in the body. 75 2. Reaction with mild oxidizing agent e.g. ninhydrin This reaction is to detect and estimate amino acids quantitatively in small amounts. The reaction involves oxidative deamination of the amino acid to form ammonia, carbon dioxide and an aldehyde obtained by loss of one carbon from the original amino acid. The reduced ninhydrin can react with another mole of ninhydrin and the ammonia to produce a complex with an intense violet blue colour known as Ruhemann’s purple. 76 Ruhemann's purple 77 78 All amino acids give intense purple blue colour whereas imino acids like proline and hydroxyproline give yellow colour. Arginine reacts to give a brown colour. Asparagine, because it has a free amide group also produces a characteristic brown colour with ninhydrin because of the amide group. The coloured complex produced forms the basis for quantitative determination of amino acids. The absorbance of the solution after heating with ninhydrin is proportional to the concentration of amino acid. 79 3. Reaction with strong oxidizing agent e.g. nitrous acid Nitrous acid (HNO2)/nitric acid (HNO3) reacts with amino group to form the corresponding hydroxyacids with the liberation of nitrogen. This reaction is important in estimation of α-amino groups in amino acids, peptides, polypeptides and proteins. Proline and hydroxyproline do not react. The ε-amino group of lysine reacts slowly. 80 Reaction with aldehydes The α-amino group of amino acids reacts reversibly with aldehydes to form a Schiff’s base. These appear to be intermediates in a number of enzymatic reactions involving interaction of the enzyme with the amino or carboxyl group of the substrate. 81 Reaction with cyanate Amino groups of amino acids react with cyanate to yield carbamoyl derivatives and this reaction has been used to modify the properties of sickle cell haemoglobin to make it more like adult haemoglobin. 82 Some very important reactions of amino acids involving amino group have become very useful in determining amino acid sequence of proteins. Such reactions include the following; 1. Reaction with Sanger’s reagent or 2,4- dinitroﬂuorobenzene. The reagent is used in determining the amino acid sequence in a protein or peptide since it reacts with the free N-terminal end to form an intense yellow dinitrophenyl (DNP) derivative. 83 Reaction occurs in cold, alkaline medium (normally HCO3- medium is used) and releases hydrogen ﬂuoride. The DNP derivative formed can be hydrolysed to yield individual amino acids and the DNP amino acid which is resistant to hydrolysis. The DNP amino acid can be distinguished by paper chromatography, thus identifying the amino terminal of the polypeptide chain. Disadvantage – The method is not reliable because amino acid sequence cannot be determined since free amino acids are released. 84 85 Reaction with Dansyl chloride (N-dimethyl aminonaphthalene-5-sulphonyl chloride) Dansyl chloride is a more sensitive agent for the detection and measurement of N- terminal amino acid residues. It reacts with N-terminal amino acid to yield a dansyl amino derivative which is stable. The dansyl group is highly ﬂuorescent and the intensity can be measured in a ﬂuorimeter. 86 A particular intensity will indicate the speciﬁc amino acid present. The reaction can be used to determine minute amounts of amino acid. Disadvantages are the same as that of Sanger’s reagent. 87 Dansyl’s method has an advantage over Sanger’s since smaller amounts of amino acids can be used. 88 Reaction with phenylisothiocyanate (Edman’s degradation) The reagent reacts with the N-terminal amino acid of peptide or protein under mild alkaline conditions to form the corresponding phenyl thiocarbamoyl peptide or derivative. On treatment with acid, the N-terminal residue is split off as a phenylthiohydantoin derivative and this can be identiﬁed by paper chromatography. 89 The Edman reaction has been used to determine the sequence of amino acids in peptides and proteins. Advantage of the method is that the derivative is very stable in acid and therefore step-wise degradation can occur, thus rest of the chain is left intact so further cycles of the procedure can occur. Disadvantage - accumulation of by-products can occur, interfering with the procedure. Can be limited to 25 cycles (automated). 90 91 Metal complexes The α-amino acids form stable complexes with metals such as Cu, Co, and Mn. When there are 2 or more peptide bonds, there is reaction with Cu2+ in alkaline solution to form a violet blue complex. This is the basis of the Biuret’s test. It is a quantitative test for proteins as intensity of colour determines concentration of proteins. 92 Identiﬁcation of C-terminal amino acid 1. Use of lithium borohydride (LiBH4) LiBH4 reduces C-terminal amino acid to form the α-amino alcohol. If the peptide chain is hydrolyzed, the hydrolysate will contain an α- amino alcohol which corresponds to the original C-terminal amino acid. This can be identiﬁed by chromatographic methods. 93 2. Use of hydrazine (NH2NH2) also known as hydrazinolysis or Akabori procedure Hydrazine is used to cleave all peptide bonds by converting all except the C- terminal amino acid into hydrazides. The C-terminal amino acid appears as a free amino acid which can be readily identiﬁed by chromatography. Usually, determination of N-terminal is more sensitive and common than C- terminal. 94 Hydrazinolysis 95 PEPTIDES They are intermediate compounds between amino acids and proteins. Peptides are named according to amino acid content. Peptides with 2 amino acids - dipeptide 3 amino acids - tripeptide 4 amino acids - tetrapeptide less than 10 - oligopeptide greater than 10 - polypeptide. 96 96 By convention, H2N is to the left and COOH to the right or H2N is up and COOH down (vertically). Speciﬁc name for peptides is derived by attaching the ending –yl to the amino acid whose carbonyl group is involved in the peptide link. 97 Peptides have amino group (NH2) at one end (N-terminal) and a COOH at the other end (C-terminal). Peptides also have characteristic chemical reactions based on the NH2, COOH and the R-groups. Examples of naturally occurring peptides are vasopressin, oxytocin, glutathione, opioid peptides and enkephalins. 98 Hydrolysis of peptides This can be achieved by boiling with either strong acid or base to yield the constituent amino acids. The peptide bond has a partial double bond character and is therefore rigid. This prevents free rotation. The peptide bond is generally a trans bond. Hydrolysis of peptide bonds can be achieved by the use of enzymes. 99 Hydrolysis of peptides 100 POLYPEPTIDES AND PROTEINS Proteins are large molecular weight compounds which may contain a single polypeptide chain like myoglobin or 2 or more polypeptide chains like haemoglobin (4 peptide chains). The various polypeptide chains in a protein are held together by hydrophobic interactions, covalent linkages and disulphide bonds, etc. 101 Classiﬁcation of proteins This may be based on their composition. There are 2 main types of this classiﬁcation: 1.Simple proteins 2.Conjugated proteins Simple proteins These are made up of amino acids only, e. g. albumin, globulins, glutelins, collagen, keratin, myosin. 102 Conjugated proteins These contain non protein portions and on hydrolysis yield amino acids and other organic or inorganic components. The non protein portions are called the prosthetic group and they are classiﬁed according to the nature of the non-protein portion. 103 Examples of conjugated proteins Nucleoproteins - nucleic acids + protein portion Glycoproteins -carbohydrates + protein portion Lipoproteins - lipids + protein portion Haemoglobin - haem + protein portion Another mode of classiﬁcation is with regard to the shape or structure of the protein. On this basis, proteins are divided into two main groups; 104 1. Fibrous proteins They are water insoluble long thread-like molecules, highly resistant to digestion by proteolytic enzymes. They consist of several coiled peptide chains which are highly linked. They are physically tough and have structural and protective or supportive functions. Secondary structure (α-helix or β-pleated sheet) forms dominant structure. 105 They often have regular repeating structures. Involved in movement (as in muscle and ciliary proteins). E.g. collagen of muscles, tendons, keratin of hair, proteins of silk (silk ﬁbroin), nails, connective tissues (collagen) and bone elastin. 106 2. Globular proteins These are highly diverse and form compact spherical molecules in water. They are soluble in aqueous system. All have tertiary structure and some quaternary structure in addition to secondary structure. E.g. enzymes, food proteins like albumin and casein of milk, transport proteins (haemoglobin). 107 Fibrous proteins Globular proteins These contain higher amounts of Have variable molecular weight and regular secondary structure more of tertiary structure and some quaternary structure. Long, cylindrical, rod-like shapes Spherical in shape Have no water solubility Have high water solubility They play structural rather than Play functional role like catalyst, dynamic role transporters, controller protein, regulation of metabolic pathways and gene expression 108 Properties of proteins 1. They contain L-amino acids in peptide linkages. 2. They form colloids in solution 3. They are generally tasteless, but their hydrolysate may be bitter or sweet. 4. They are colourless, but heating turns them brown and continuous heating results in charring, giving off the odour of burning hair. 5. Each type of protein is characterized by; a. speciﬁc chemical composition b. speciﬁc amino acid sequence c. speciﬁc molecular weight 109 Functions of proteins Proteins play important role in all biological processes and their functions are exempliﬁed in; 1. Enzymatic catalysis – enzymes catalyze chemical reactions in biological systems, thus proteins play a unique role in determining the pattern of chemical transformations in biological systems. 2. Transport – speciﬁc proteins transport small molecules and ions, e.g. haemoglobin carries oxygen in erythrocytes or red blood cells, and myoglobin carries oxygen in the muscles. 110 3. Coordinated motion (movement) – proteins are major components of muscle and muscle contraction is accomplished by the steady motion of two kinds of protein ﬁlaments, actin and myosin. 4. Mechanical support – collagen, a ﬁbrous protein is found in bone and skin and this gives the high tensile strength of the skin. Others are ﬁbrous (silk protein) for mechanical strength in silk, elastin/rubber-like protein found in elastin ﬁbres present in several tissues in the body eg blood vessels and skin, α-keratin – major protein of hair and nails. 111 5. Immune protection or defence (antibodies) – these are highly speciﬁc proteins that recognise and attack foreign substances or organisms such as viruses and bacteria and nullify their effects in the body. 6. Generation and transmission of nerve impulses (hormones) – certain proteins help in the transmission of nerve impulses. The response of nerve cells to speciﬁc stimuli is mediated by speciﬁc proteins. 112 7. Growth control and differentiation (genetical) – controlled sequential expression of genetic information is essential for the orderly growth and differentiation of cells. These processes are mediated by proteins. 113 Organisation of amino acids in protein (protein structure) Amino acids present in proteins have several functional groups and therefore contribute to structure, properties and functions of a particular protein. Protein structure may be considered under four levels of organisation. These are the primary, secondary, tertiary and quaternary structures. Differences arise due to the types of bond in these structures. 114 The primary structure This refers to the order in which the amino acids are covalently linked together in a protein, i.e. the amino acid sequence. It indicates the number and types of amino acids and in which fashion or manner they are linked. E.g. H2N-ala-cys-pro-met-leu-ala-ala-glu-gly-COO- 115 The secondary structure This determines the coiling of the polypeptide chain into a helical structure. This structure arises from the folding and twisting of the polypeptide chain into a coil or spiral form as a result of H bonding. In many proteins, the H bonding produces a regular coiled arrangement called α-helix. H bonding in secondary structure usually involves amino acids that are quite close in the polypeptide chain. 116 Although H bonding is weak, they are so numerous that they are able to stabilize the molecule. Another type of secondary structure is the β- pleated sheet which may be parallel or anti- parallel. α-helix is predominant in ﬁbrous proteins like myosin and α-keratin of hair, wool and nails. β- pleated sheet is common in silk protein. 117 118 119 Factors that affect α-helix 1. Proline creates a bend in the backbone of its cyclic structure, it cannot ﬁt into the α-helix. 2. Localized factors involving the side chains include strong electrostatic repulsion due to the proximity of several charged groups of the same sign, eg positively charged group of lysine or arginine or negatively charged groups of glutamate or aspartate. 3. Steric repulsion or crowding due to the proximity of several bulky side chains. 120 In the α-helix conformation, all side chains lie outside the helix as there is not enough space for them in the interior. α-helix is stabilized by intramolecular H bonding (between –C=O-and --H-N group ie between carbonyl O and amide H of the same molecule). There are about 3.6 amino acids per turn of the helix. 121 The H-bonds in β-sheet are perpendicular to the polypeptide backbone. β-pleated sheet is stabilized by intermolecular forces ie C=O from one molecule and the NH of another molecule. Unlike the α-helix, β-sheets have two or more peptide chains or β-strands. 122 The tertiary structure This involves coiling or folding of the helical structure into a three dimensional structure of the biologically active native conformation. There are other associated forces between the amino acid residues relatively far apart in the chain. These include H bonding, disulphide linkages, ionic bonding, ester bonding and hydrophobic interactions or Van der Waals forces. 123 Reactivity between the R-groups of amino acids contributes to stability of the proteins. The folding occurs such that maximum numbers of polar (hydrophilic) groups are on the exterior of the molecule exposed to the aqueous environment and the maximum number of non- polar (hydrophobic) groups are within the interior. 124 125 The tertiary structure determines the structure of the protein and dictates the catalytic properties of biologically active protein. Types of interactions include the following: 1.Hydrophobic interactions (Van der Waals forces) occur between R groups of non- polar amino acids. It is the most important non-covalent force that causes proteins to fold into their native structure. 126 2. Hydrogen bonds between the polar R groups of the polar amino acids. It can also occur between the OH groups of serine and threonine and tyrosine and amino groups and carbonyl oxygen of asparagine or glutamine and ring N of histidine. 3. Ionic bonds (electrostatic interaction)-this occurs between the R-groups of positively charged and negatively charged amino acids, e.g. lysine and glutamate. 127 4. Covalent bonds – these occur between the sulphydryl containing amino acids (cysteine) i.e. the disulphide link. All these bonds are in addition to the H bonds and peptide bonds of the helical structure. 128 The quaternary structure This is found in proteins with more than one polypeptide chain. The individual chains are arranged in relation to each other in such a way as to produce a simple 3-dimensional structure of the overall protein molecule. Each polypeptide chain in such a protein is called sub-unit or monomer and the assembly is called oligomer. 129 The sub units are linked together primarily by non covalent forces. The primary structure is very vital in determining the 3-dimensional structure or shape, i.e. the primary structure speciﬁes or dictates the 3-dimensional shape of the protein. 130 131 Thus the critical determinant of the biological function of a protein is its conformation which is deﬁned as its 3- dimensional arrangement of the atoms within the molecule. Non-covalent forces cause a polypeptide to fold into a unique conformation and then stabilize the native structure against denaturation. 132 133 Proteins have different levels of structural organization; 134 135 Importance of the primary structure The amino acid sequence (primary structure) of a protein determines its three dimensional structure which in turn determines its properties and functions. E.g. in enzymes, the 3-dimensional structure serves to place the crucial amino acids that are directly involved in catalyzing reactions close to each other. 136 Alteration in the amino acid sequence can affect the function of the protein. In some cases, large changes may occur without affecting function of a particular protein. In other cases, a change in only a single amino acid residue can effect a profound alteration in the properties of the protein. 137 Primary structure and species variation Studies on cytochrome C from about 40 different organisms show that there isn’t much difference in the primary structure. The cytochrome C from these organisms may differ in the positions of one or a few amino acids in the chain. Though amino acids in certain positions may be different, the proteins perform the same function irrespective of the organism. Scientists concluded that differences in amino acid sequence have given rise to variation in the different species. Such a change is called conservative change, e.g. in man and monkey position 1 is different and in man and horse, position 12 is different. 138 Primary structure and genetic defect This is exempliﬁed in the haemoglobin associated with sickle cell anaemia. In this genetic disease, red blood cells are unable to bind oxygen efﬁciently. The red cells also assume a characteristic sickled shape hence the name of the disease. The sickle cells tend to become trapped in small blood vessels cutting off circulation and thus causing organ damage. A change in one amino acid residue in the sequence or the primary structure causes these drastic consequences. 139 Haemoglobin is made up of 4 subunits consisting of two α chains and two β chains bound together. In HbS, the α–chains are intact but the β–chains are affected. In one of these β–chains, glutamic acid (acidic) is replaced with valine (hydrophobic) in position 6. HbA H2N-Val-His-Leu-Thr-Pro-Glu-Glu-Lys— HbS H2N-Val-His-Leu-Thr-Pro-Val-Glu-Lys— 140 Protein Structure Importance of Primary structure Example :sickle cell disease In sickle cell disease there is a change in the primary protein structure of one of the polypeptide chains that form hemoglobin: The amino acid glutamic acid is substituted by the amino acid valine in the β chain. 37 Protein Structure Importance of Primary structure Example :sickle cell disease The primary “mistake”, which also creates a different (sickle) shape for red blood cells. They obstruct peripheral blood circulation, causing tissue hypoxia and the acute pain typical of sickle cell anemia. 38 The highly polar side chain of glutamate containing an ionisable carboxyl group is replaced by a non polar one, the isopropyl group of valine. In the 3-dimensional structure of haemoglobin, this residue is on the outside of the molecule. One molecule of HbS can become involved in hydrophobic interactions with other haemoglobin molecules because of the presence of non polar residue. 143 Such an interaction does not occur in HbA with a polar residue in the same position. As a result groups of molecules of HbS aggregate with each other. These aggregates distort the shape of the blood cells resulting in the disease. Such a change is non conservative because it alters the function and property of the molecule. 144 Primary structure and functional differentiation This is also a conservative replacement but does not result in species variation but variation in function. Replacement of one amino acid in the chain gives rise to differences in function. E.g. oxytocin and vasopressin. 145 Vasopressin 1 2 3 4 5 6 7 8 9 Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly S S Oxytocin 1 2 3 4 5 6 7 8 9 Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly S S 146 Protein denaturation In the native state, a globular protein is a highly ordered conformation in which the biological activity is manifested. The non covalent interactions responsible for maintaining the 3-dimensional structure are weak and can easily be disrupted leading to unfolding of the protein. The process of unfolding is called denaturation and this leads to loss of activity. Under proper conditions, the 3-dimensional structure can be restored or recovered in some cases 147 (renaturation). Denaturation arises from the following; 1. Changes in peptide structure due to unfolding. 2. Destruction of ionic and other bonds discussed in protein structure. Generally, denaturation results in changes in solubility at the isoelectric pH where normally, insolubility is high. The consequences of denaturation is coagulation, i. e. when protein is thrown out of solution. 148 Water is also very vital because in its presence proteins are easily denatured. Dry proteins are less susceptible to heat denaturation or coagulation than hydrated ones or those in solution. Denatured or coagulated proteins have little tendency to associate with water. AGENTS OF DENATURATION There are three main agents, these are; Physical, Chemical and Biological. 149 PHYSICAL E.g. heat, pressure, freezing, shaking and foaming, e.g. beating of eggs, autoclaving (sterilizing). In sterilizing surgical instruments, heat and pressure are employed in the use of autoclave. The heat and pressure denature the bacterial protein (cell wall) thus killing the 150 bacteria. CHEMICAL Extreme of pH leads to changes in the charge on the protein. Each protein has a characteristic charge because of the R groups of amino acids and speciﬁc amino acid composition. These positively and negatively charged R- groups on the surface interact with ions and water molecules keeping the proteins in solution within the cytoplasm. 151 When the charges on the protein are neutralised the net charge on the protein is zero and it becomes isoelectric. Once this happens the proteins no longer have means of interacting with the surrounding water molecules and cannot remain in solution. Under such conditions the protein molecules aggregate with each other and coagulation occurs. 152 COO- NH3+ NH3+ COO- NH2 NH3+ Protein + 2OH- Protein NH3+ COO- NH3+ COO- NH3 + NH2 Net Charge +2 Net charge 0 153 When a base is added to a protein with +2 overall charge, some of the protonated amino groups lose their protons and the protein becomes isoelectric. NH3+ NH3+ COO- COO- COO- COOH Protein Protein + 2H+ COO- COOH NH3+ NH2 COO- COOH Net Charge - 2 Net Charge 0 154 If a protein has 2 excess negative charges, on the addition of acid, some of the carboxyl groups become protonated and the proteins become isoelectric. E.g. When milk is stored in the refrigerator for a long time, the bacteria in the milk begin to grow. These use milk sugar lactose as a source of energy during fermentation and produce lactic acid as a by product. 155 As the bacteria population increase, lactic acid concentration correspondingly increases decreasing the pH of the milk. The additional acid results in the protonation of the exposed carboxylate groups on the surface of the dissolved milk proteins. They become isoelectric and coagulate into a solid curd. 156 Again if pH of the blood becomes too acidic or basic, blood proteins like albumin (carriers) ﬁbrinogen (involved in blood clotting) and immunoglobulin(protection from disease) will become isoelectric, denature, and can’t carry out required functions. This eventually results in death as enzymes become denatured and oxygen cannot be transported by haemoglobin. 157 Also at extreme pH values strong intramolecular electrostatic repulsion caused by high net charge results in swelling and unfolding of the protein. The degree of unfolding is greater at extreme alkaline pH than extreme acid pH values. Denaturation under extreme alkaline conditions is due to ionization of partially buried carboxyl, phenolic and sulfhydryl groups which causes unfolding of the polypeptide chain as they attempt to expose themselves to aqueous environment. 158 ORGANIC SOLVENT E.g. detergents like sodium dodecyl sulphate Organic solvents that are miscible with water e.g. alcohol and acetone denature proteins. Urea forms H-bonds with the proteins that are stronger than those within the protein. Urea and detergents disrupt hydrophobic interactions. Mercaptoethanol reduces disulﬁde bonds to sulfhydryl groups thus disrupting three dimensional structure of proteins. 159 160 Application of denaturation using organic solvents. Ethyl alcohol and isopropyl alcohol are good germicides because they denature bacteria proteins thus killing them. Treatment of Burns A complex organic compound, tannic acid is incorporated in burn ointment. On application to the skin the tannic acid causes a protective layer of denatured protein to form that prevents water loss from the burnt area. 161 CHARACTERISTICS OF DENATURED PROTEINS 1. Unfolding or uncoiling of peptide or polypeptide chains. 2. Increased viscosity of denatured proteins in urea solution. The increase is due to the formation of more elongated and ﬁbrous structures. 3. Chemical characteristics differ from those of the original proteins. 4. Isoelectric pH may be altered. 162 5. Denatured proteins have less capacity to interact with water than with native protein. 6. They are difﬁcult to crystallize. 7. Denaturation of enzyme proteins lead to their inactivation. 8. Digestibility of protein by proteolytic enzymes may be altered. E.g. native haemoglobin is not digested by trypsin whereas denatured ones are easily digested. 163 DETERMINATION OF PRIMARY STRUCTURE OF PROTEINS In determining the primary structure of proteins the following questions must be addressed. 1. What is the amino acid composition of the protein? 2. Which amino acids occur at the N- terminal and C- terminal ends? 3. What is the exact order or sequence of amino acids in the protein? 164 165 Determination of amino acid composition The protein is digested by reﬂuxing with 6N HCl for 12 to 36 hours (usually 24hrs) at 100 to 110oC. This hydrolyses the protein to yield a mixture of amino acids. Separation and identiﬁcation of the amino acids in the hydrolysate Ion exchange chromatography can be used. This technique has been automated to produce the amino acid analyser. 166 Principle behind amino acids separation The technique is based on separation by virtue of differences in sign (positive or negative or neutral) and magnitude (which is more positive or negative) of charge on the amino acids. The acidic solution of pH 3 is passed through a long column packed with cation resin (sodium sulphonate). 167 As the hydrolysate travels (percolates) through the resin the amino acids are exchanged for sodium ions. At this pH most of the amino acids are cations since their pI are usually above three but the magnitude of the charges differ. The positively charged amino acids are adsorbed by the resin because of the attractive forces between the negatively charged sulphonate groups and the positively charged amino acids. 168 Those with larger positive charges (i.e. most basic e. g. lysine and histidine) will have more afﬁnity for the negatively charged resin particles and will be held more strongly. Those with least positive charges at pH 3 e.g. acidic as aspartate and glutamate will be bound more loosely. All other amino acids will have intermediate afﬁnity. Amino acids will therefore move down the column at different rates and be separated if the column is eluted with buffered solution. 169 H HOOC C R + SO3-Na+ + H3N SO3- NH3 R C COOH Cation Resin Resin + exchange H SO3-Na+ + Cation exchanger Na+ 170 At a given pH the amino acids will ultimately separate and be collected into small vials treated with ninhydrin and the absorbance of the solution measured at wavelength 570 nm. The absorbance is recorded as a function of volume of efﬂuent (solution that comes out) and represented on a graph. The area under the curve or peaks corresponds to the relative amount of the amino acid. (Illustrate) 171 172 Absorbance pH 173 Identiﬁcation of C-terminal and the N- terminal ends of proteins This can be achieved in several ways by terminal residue analysis and partial hydrolysis. These involve both chemical and enzymatic methods for identifying the amino acid at the ends of the molecule. 174 Identiﬁcation of the N-terminal end 1. Use of Dansyl chloride 2. Use of phenylisothiocyanate 3. Use of Sanger’s reagent 4. Use of aminopeptidases (enzymatic) 175 Use of aminopeptidases (enzymatic) These are enzymes that cleave the N-terminal amino acid of proteins or peptides. (These together with carboxypeptidase, are called exopeptidases as they attack only peptide bonds at the end of the polypeptide chain.) The disadvantage is that they continue to cleave off other amino acids in the sequence. If conditions are not controlled, unreliable results are obtained. 176 Identiﬁcation of the C-terminal 1. Treatment with hydrazine (Akabori procedure). 2. Use of LiBH4. 3. Use of carboxypeptidases: these cleave polypeptides from the C-terminal end and present similar problems as aminopeptidases. 177 Separation of peptide chains Determination of N-terminal and C-terminal residues can also indicate whether a given protein has a single polypeptide or more. If the protein contains more than one polypeptide chain, the individual chains which make up the complete protein must be separated before the sequence is determined. 178 The disulphide bond of cysteine which joins 2 parts of the same chain or two different chains can be cleaved by either oxidation or reduction. If the disulphide is reduced, the resulting sulphydryls are alkylated to prevent spontaneous re-oxidation to the disulphide form. (Illustrate) 179 Oxidative Cleavage O S SO3H H C OOH SO3H S Performic acid Cysteic acid residues (stable) 180 181 Determination of amino acid sequence of the isolated polypeptide After identifying the N and C-terminal ends, the next step is to determine the amino acid sequence of the polypeptide. This is achieved by cleaving the parent polypeptide into a number of smaller fragments and characterizing each of them. Points of overlap are identiﬁed and the peptide pieced together to determine the amino acid sequence of the original polypeptide. Determination of points of overlap can be carried out by partial or selective hydrolysis of the polypeptide chain. Agents for such hydrolysis may be chemical or enzymatic. 182 Chemical 1. Use of dilute acids. These hydrolyse peptide bonds but bonds between certain pairs of amino acids are more susceptible to acid hydrolysis than others. 2. Cyanogen bromide (CNBr). This cleaves peptide bonds whose carbonyl functional group is donated by methionine. After hydrolysis, the methionine is converted to homoserine lactone. 183 Use of proteases Method Peptide bonds cleaved Trypsin (from digestive tract of Hydrolysis of peptide bonds whose carbonyl animals) C is provided by basic amino acids like arginine and lysine Chymotrypsin (from digestive tract of This hydrolyses peptide bonds whose animals) carbonyl group is donated by aromatic amino acids like phenylalanine, tyrosine, tryptophan or by amino acids with large apolar (non polar) side chain like leucine. These may yield fragments that overlap those produced by trypsin. Thermolysin (bacterial enzyme) Hydrolyses peptide bonds whose carbonyl group is donated by non polar amino acids e. g. leucine, isoleucine and valine in addition to those cleaved by chymotrypsin. Papain (pawpaw) Carbonyl group donated by lysine and leucine Pepsin Same as chymotrypsin 184 Ordering of peptide fragments A polypeptide can be broken into a number of small fragments that can be easily sequenced. However this information is not sufﬁcient to obtain the full structure of the original polypeptide or protein since it won’t give the order of the fragments in the original chain. This problem is overcome by obtaining a new set of fragments that overlap the sequences determined in the ﬁrst step. Combine information of overlapping peptides to get complete sequence. (Illustrate) 185 Peptides from method A Peptide I II III IV Ala-Glu-Lys Gly-Phe-Ala-Arg Ser-Asp-Tyr-Lys Leu-Met-Tyr Bonds cleaved by method A Ala-Glu-Lys-Gly-Phe-Ala-Arg-Ser-Asp-Tyr-Lys-Leu-Met-Tyr Original peptide Bonds cleaved by method B Ala-Glu-Lys-Gly-Phe Ala-Arg-Ser-Asp-Tyr Lys-Leu-Met-Tyr Peptide 1 2 3 186 Partial hydrolysis using chymotrypsin and CNBr + Chymotrypsin H3N-Leu-Asn-Asp-Phe + CNBr H3N-Leu-Asn-Asp-Phe-His-Met Chymotrypsin His-Met-Thr-Met-Ala-Trp CNBr Thr-Met CNBr Ala-Trp-Val-Lys-COO- Chymotrypsin Val-Lys-COO- Overall sequence + H3N-Leu-Asn-Asp-Phe-His-Met-Thr-Met-Ala-Trp-Val-Lys-COO- 187 Steps involved in the determination of amino acid sequence of a protein 1. If the protein contains more than one polypeptide chain, the individual chains are ﬁrst separated and puriﬁed. 2. All disulphide groups are reduced and the resulting sulphydryl groups are alkylated. 3. Subject each polypeptide chain to total hydrolysis and determine its amino acid composition. 4. Identify N and C-terminal residues of another sample of the polypeptide chain. 188 5. Cleave intact polypeptide chain into a series of smaller peptides by enzymatic or chemical hydrolysis. 6. Separate peptide fragments in 5 and determine amino acid composition and sequence of each. 7. Partially hydrolyse another sample of the original polypeptide by a second procedure to fragment the chain at points other than those cleaved by the ﬁrst partial hydrolysis. Separate the fragments and determine amino acid composition and sequence as in 5 and 6. 189 8. By comparing the amino acid sequence of the two sets of peptide fragments particularly where there is overlapping, the peptide fragment can be placed in the proper order to yield the complete amino acid sequence. 9. The position of disulphide bonds and amide groups in the original polypeptide chains are determined. 190 Importance of determination of the amino acid sequence i.e. primary structure 1. It helps in the elucidation of the molecular basis of the protein’s biological activity (i.e. whether it is an enzyme, hormone etc) 2. It helps in deducing the 3-dimensional structure of the protein 3. Alterations in the amino acid can produce abnormal function and disease, e.g. sickle cell anaemia. 4. Amino acid sequence of a protein reveals much about its evolutionary activity – proteins resemble one another in their amino acid sequence only if they have common ancestor. Consequently molecular events in evolution can be traced from amino acid sequence. 191 Roles of speciﬁc amino acid in proteins The uncharged polar amino acid residues in a protein are the sites for H-bonding leading to potential cross linking of chains. Charged polar groups are susceptible to pH changes and may markedly affect the activity of functional proteins. 192 These amino acids include: Cysteine This forms cross linkages with other cysteine sulphydryl groups in the same or different polypeptide chain by oxidation to form a covalent disulphide bond. The reduced cysteine serves as a site of attachment for substrate in a number of enzymes. 193 Histidine This contains a lone pair of electrons in the ring N and may serve as a potential metal ligand site in the iron (Fe) containing proteins, e.g. haemoglobin and cytochrome C. Lysine The ε amino group of lysine forms a Schiff’s base with substrate at active sites of enzymes. It is intimately involved in binding with pyridoxal phosphate, lipoic acid and biotin. 194 Serine It has a primary alcoholic group and may serve as a nucleophile in a number of proteolytic enzymes. Together with histidine it serves as component of active site of chymotrypsin. Proline Due to its relatively rigid ring it forces a bend in a polypeptide chain and disrupts α-helicity. 195 Reactions of side chain R-groups (colour reactions of proteins) Amino acids show qualitative colour reactions typical of certain functional groups present in the side chain, e.g. thiol group, phenol group, indole group, etc. 1. Reaction due to the presence of thiol group (SH) Proteins containing cysteine or cystine are heated with strong alkali. H2S is formed and this can be detected by forming insoluble brown to black lead sulphide (PbS) on addition of lead acetate. 196 2. Presence of indole ring Indole ring can be detected by the Hopkins- Cole reaction. In the presence of concentrated H2SO4, the indole ring of tryptophan forms coloured (violet) condensation products with glyoxylic acid. Proteins that do not contain tryptophan do not give a positive test. 3. Folin’s reaction uses Folin’s reagent (sodium 1, 2-naphthoquinone-4-sulphonate) The reagent gives a deep red colour with amino acids in the presence of alkaline. 197 4. Detection of aromatic group – phenylalanine, tyrosine, tryptophan The xanthoproteic test is used in this. The phenyl group is nitrated by concentrated HNO3 to form white, then yellow nitro substitution product or precipitate on heating. On the addition of NaOH, the sodium salts of the nitro derivatives turn orange. 5. Presence of arginine or guanidine group Arginine, the only amino acid containing a guanidine group reacts with α-naphthol and an oxidizing agent such as bromine water or sodium hypochlorite to give a red colour. The reaction is called Sakaguchi reaction. 198 6. Test for phenolic group (speciﬁc for tyrosine) Millon’s reagent is used. This reacts with phenolic groups to give a faint pink colour which changes to red on heating. Millon’s reagent is a solution of mercurous and mercuric nitrate containing HNO3. When added to a protein solution, a white/ yellow precipitate is ﬁrst formed which turns red on heating. Reaction is dependent on the formation of a coloured Hg compound with the OH of the 199 phenol group. 7. Biuret’s test This is a popular quantitative test for proteins just like ninhydrin. This involves peptides with 2 or more peptide bonds. Protein solutions are made strongly alkaline with Na or K hydroxides and very dilute CuSO4 is added. A pinkish to purple colour develops. 200 The colour depends on the complexity of the protein. Proteins give purple colour; peptones give a pink colour and peptides very light pink colour. Gelatin gives an almost blue to violet colour. In the Biuret’s test, there is coordination of cupric ions with the unshared electron pairs of peptide N and the O of H2O to form a coloured coordination complex. MgSO4 interferes with the reaction as precipitate of Mg(OH)2 is formed. 201 Evidence supporting peptide bonds in proteins These are obtained from both chemical and enzymatic degradation as well as physical measurement. 1.Intact proteins show little free amino N (hydrolysis produces a large amount of N2). 2. Proteins have absorption band in the far UV and IR regions that are similar to genuine peptides. 3.X-ray diffraction analysis conﬁrms the presence of peptide bonds. 202 4. Enzymes that can hydrolyse proteins also hydrolyse synthetic peptides. 5. Most proteins and peptides give the same colour reaction with Biuret’s test. 6. Several polypeptide hormones have been isolated in pure forms and have been synthesized from the constituent amino acids. 203 Determination of protein size Lots of methods have been employed to determine size of proteins. They include; 1. Determination of mini molecular weight which can be computed from the quantity of constituents. 2. Use of osmotic pressure – there are lots of difﬁculties in using osmotic pressure in calculation. 204 Difﬁculties in using osmotic pressure These include: i. Equilibrium is attained slowly and hence reading must be taken within a long range of time. Due to this there may be bacteria contamination and decomposition depending on environmental conditions. ii. The gas law equation is valid only at low solvent concentration and osmotic pressure determination makes use of the gas law equation. Therefore unless solution is diluted to inﬁnite dilution values may be wrong. 205 3. Sedimentation This is the most important method for determining the shape, size and molecular weight of proteins and in this determination, ultra centrifugation is made use of. The principle underlying this is the rate at which a particle is driven down a centrifuge tube under the action of centrifugal force which depends on; 206 i. initial force applied when centrifuging, ii. size, shape and density of the particle being measured, iii. density and viscosity of solvent system in which the molecular weight is being determined. 207 Isolation and puriﬁcation of a protein (same as isolation and puriﬁcation of amino acid) Amino acids and proteins can be separated from each other and from other kinds of molecules on the basis of such characteristics as size, solubility, charge and speciﬁc binding afﬁnity. In purifying proteins, various separation methods are employed and their efﬁciency is evaluated by assaying for distinctive properties of the protein of interest. 208 Separation Based on Size 1. Dialysis and ultracentrifugation Proteins can be separated from smaller molecules by dialysis through a semi permeable membrane. The membrane retains protein molecules and allows small solute molecules and water to pass through. In ultra centrifugation, pressure or centrifugal force is used to ﬁlter the aqueous medium and small solute molecules through a semi permeable membrane. 209 2. Gel ﬁltration chromatography (molecular exclusion/sieve chromatography) This is also a separation procedure based on size. It is a form of column chromatography in which the stationary phase consists of cross linked gel particles which are hydrated. The gel particles are usually in bead form and there are two types of polymers. One includes carbohydrate polymer such as dextran and agarose and the other type is polyacrylamide. 210 The cross linking produces pores in the material. The sample is applied to the top of the column. Smaller molecules enter the pores and appear in solution within the beads and in between them, but larger ones cannot. As a result, smaller molecules are delayed in their progress down the column. 211 As the sample is eluted by the mobile phase, the larger molecules are eluted ﬁrst followed by the smaller ones. Advantages are; 1. It is a convenient way to separate molecules based on their size. 2. It can be used to estimate molecular weight by comparison with standard samples. 212 Separation based on charge Ion exchange chromatography is used and the procedure relies on the attraction between oppositely charged particles. There are anion exchangers and cation exchangers. Cation exchangers possess negatively charged groups and will attract positively charged cations. Anion exchangers have positively charged groups that will attract negatively charged anions. 213 If the pH is below the pI, cation exchangers are used. For pH above the pI, anion exchangers are used. The method is based on the net charge of the protein (or amino acid) which is largely determined by the number and charge of the ionisable R groups in the polypeptide chain (or amino acid). 214 Synthetic resins which are cross linked long chain polymers available commercially in bead form are used as ion exchangers. Each bead can contains several thousand charged groups of the same sign, all positive or all negative. Resins usually contain negatively charged sulfonate groups (-SO3-). 215 The beads are soaked in a solution containing the counter ion desired for the start of the experiment. eg beads can be treated with NaCl before packing the column if Na+ is the desired counter ion. Other positively charged ions can replace the Na+ by an exchange process. The separation of proteins (or amino acids) occur on the basis of the extent to which each protein is positively charged and the extent to which it can replace the Na+ as the counter ion that balances the negative charge of each sulfonate group of the cation exchange resin. 216 Example using a mixture of 3 amino acids, aspartate, serine and histidine in a buffer of pH 3.25. At this pH, all the amino acids are protonated including the side chain imidazole group of histidine. Histidine has a positive charge of 1, serine is electrically neutral and aspartate has a net charge of -1. The histidine is bound strongly to the cation exchanger, the serine next and the aspartate least bound. 217 Aspartate is eluted ﬁrst, followed by the serine and histidine remains bound to the resin. The pH of eluting buffer is raised in stages to facilitate release of amino acid with positively charged R groups. Collect eluent, react with ninhydrin and determine absorbance and compare with standards. 218 Anion Exchanger: (R)4N+…Cl- + -OOCR’ ↔ (R)4N+…OOCR’ + Cl- Cation Exchanger: RSO3-.. Na+ + +NH3R’ ↔ RSO3-..+NH3R’ + Na+ Counter Charged Exchanged Exchanger Bound ion Molecule Ion Molecular To be ion exchanged 219 Electrophoresis The underlying principle in this procedure is electrostatic attraction. In addition, size and shape of the molecule can also inﬂuence separation. Electrophoresis depends on different rates of migration of particles of different charges in an electric ﬁeld. 220 The charged molecule moves through a liquid that conducts an electric current. Inert substances like paper and gel (SDS polyacrylamide gel electrophoresis : SDS-PAGE) are used as support for the conducting liquid. The sample to be separated is applied to a strip of paper moistened with the conducting solution, usually a buffer. The ends of the paper strip are placed in reservoirs of buffer solution. 221 A positive electrode is placed at one end of the reservoir and a negative electrode at the other end. A high voltage is then applied. Proteins with a net positive charge will migrate towards the negative electrode; those with high positive charge will move faster than those with lower positive charges. 222 223 Proteins with net negative charge will move to the positive electrode; those with higher negative charge will move faster than those with lower charge. A protein with no net charge will not migrate in an electric ﬁeld. The net charge of each protein depends on pH. The net charge on a protein or amino acid inﬂuences the rate of migration in an electric ﬁeld. 224 The principle is that, the velocity of migration (v) of the protein in an electric ﬁeld depends on; 1. the strength of the electric ﬁeld (E), 2. the net electric charge on the protein (z) and 3. the frictional resistance (f) which is a function of size and shape of the protein (retards movement of charged molecule). 225 Hence; V =Ez f At the isoelectric pH, there is no net charge on the protein. Therefore electrophoretic mobility (v) is zero. Molecules of the same charge but different molecular mass move at different rates in an electric ﬁeld. Bulky ones will move at a slower rate than non bulky ones. 226 Separation based on polarity Example: paper chromatography This technique is based on the principle that polar organic molecules will dissolve more easily in water than in a non polar organic solvent. The mobile phase which carries the sample to be separated along with it is less polar than water and ﬂows over the stationary phase which is polar. 227 In paper chromatography, the stationary phase water, is adsorbed on the cellulose ﬁbre of the paper which serves as the inert support. The various components in the sample interact with the stationary phase to different extents based on their polarity. The more polar components of the sample are carried along more slowly by the mobile phase than the less polar ones which interact less strongly with the stationary phase. 228 Mobile phase is frequently a mixture of solvents like N-butyl alcohol and water or N- butyl alcohol, butyric acid and water. The various components of the sample can be characterized by the -distance travelled from the origin (site of application of the sample) compared with -distance travelled by the solvent front. 229 The ratio of these 2 distances is called the Rf and its numerical value can be used in identifying proteins or amino acids by comparison with standards. Rf =Distance travelled by substance Distance travelled by solvent front Rf is never 1 or more than 1. 230 Paper Chromatography a – aspartate b – alanine, c – methionine S – solvent front Paper c S b a o Spot of sample Non-polar solvent (mobile phase) 231 232 Afﬁnity chromatography This makes use of the binding properties of many proteins. The column is made up of a polymer which is covalently linked to a substrate which binds speciﬁcally to the desired protein. The other proteins in the sample don’t bind to the column and can easily be eluted with buffer while the bound protein remains in the column. 233 The bound protein is then eluted out by adding high concentration of the substrate in soluble form. The protein therefore binds to the substrate in the mobile phase and is eluted or recovered from the column. Method has advantage of producing pure proteins. 234 235 Separation based on solubility differences The solubility of proteins can be affected by the pH of the system. A protein is least soluble at its pI and since different proteins have different pIs, they can often be separated from each other by isoelectric precipitation. 236 Solubility and salting-out of proteins Every protein has its own characteristic solubility curve at ﬁxed -pH, -temperature and -concentration of solutes. If conditions are controlled, the amount of protein that dissolves to form a saturated solution is not really dependent on the other solute particles present. 237 Its solubility depends on the polar hydrophilic groups and the non polar hydrophobic groups. Solubility is highly inﬂuenced by pH due to the amphoteric nature of proteins. Solubility increases with rise or fall in pH, e. g. isoelectric precipitation (i.e. a protein is only insoluble at its pI). 238 The effect of pH and salt concentration on the solubility of β-lactoglobulin at 25oC β-lactoglobulin is a milk protein with pI about 5.3. Above or below this pH all the molecules have either negative or positive charges and repel one another so the protein is very soluble at either acidic or alkaline pH. 239 At the pI, there is no net charge though molecules still bear positive and negative charges. However ionic interactions, Van der Waals forces etc make the molecules clump together and precipitate. Therefore solubility is minimal at pI. (Illustrate) 240 Figs. Show concentration of NaCl 4 3 Solubility mg/ml 20mM 2 10mM 1 5mM 0 1mM 4.8 5.0 5.2 pI 5.4 5.6 5.8 pH 241 When ionic strength is increased solubility increases even at pI. This effect of putting proteins in solution by increasing salt concentration is called ‘salting-in’. At very high salt concentration, much of the water that will solvate proteins is used for the hydration shells of the numerous salt ions. 242 At such high salt concentration then, solubility of proteins again decreases and the effect is called ‘salting-out’. Divalent and trivalent ions are much more effective than univalent ions for salting out. Commonly used salts are ammonium sulphate, magnesium salts or phosphates and sodium sulphates. Principle behind salting out is that high concentration of salts may remove water of hydration from the protein molecules thus reducing their solubilities. 243 Thus anything that reduces the activity of water reduces the solubility of the proteins. In summary, low concentration of neutral salts increases the solubility of proteins in water (salting in) by stabilizing the surface charged groups. At high concentration, salt ions compete with proteins for water molecules

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