[PDF DOWNLOAD] Fundamentals of Biochemistry: Life at the Molecular Level,Fundamentals of Biochemistry 5th edition
Fundamentals of Biochemistry PDF 5th Edition By Donald Voet, Judith G. Voet, Charlotte W. Pratt Fundamentals of Biochemistry By voet and voet PDF 5th Edition address the enormous advances in biochemistry, particularly in the areas of structural biology and Bioinformatics, by providing a solid biochemical foundation that is rooted in chemistry to prepare you for the scientific challenges of the future 21/10/ · Download BCH: Fundamentals of Biochemistry,5th edition PDF by Donald Voet, Judith Voet, Charlotte Pratt - You will find Fundamentals of Biochemistry,5th 30/08/ · Fundamentals of Biochemistry 5th Edition pdf Voet download August 30, Fundamentals of Biochemistry 5th edition is an authentic treatise on biochemistry and 20/08/ · Download Fundamentals of Biochemistry: Life at the Molecular Level 5th Edition PDF Free: You can easily download Fundamentals of Biochemistry: Life at the Molecular 22/01/ · DOWNLOAD NOW» Voet, Voet and Pratt’s Fundamentals of Biochemistry, 5th Edition addresses the enormous advances in biochemistry, particularly in the areas of ... read more
It is composed of agarose —neutral gelling fraction Agaropectin — sulfated non-gelling fraction It is the most effective gelling agents known andis soluble in hot water. It melts in the temperature range of 60 —90oC and sets between 32 and 39oC to form gel. Uses As Solidifying agent, emulsifier Pharmaceuticals, cosmetics and food Laxative Sizing material in tentile industry Emulsifierin dairy products Microbial lab. Seaweed Polysaccharides Structurally they are highly branched and composed of many different monosaccharides. Fenugreek gum Trigonella foenum —grae cum Monnose and galactose Prosopsis juliflora gum man: Gal 2. Figure 2. Strong alkali Under strong alkaline conditions sugar undergo caramelization reactions. The reducing property is mainly due to the ability of these sugars to reduce metal ions such as copper or silver to form insoluble cuprous oxide, under alkaline condition.
The aldehyde group of aldoses is oxidized to carboxylic acid. This reducing property is the basis for qualitative Fehling's, Benedict's, Barfoed's and Nylander's tests and quantitative reactions. All monosaccharides are reducing. In the case of oligosaccharides, if the molecule possesses a free aldehyde or ketone group it belongs to reducing sugar maltose and lactose. If the reducing groups are involved in the formation of glycosodic linkage. The precipitated compound is called as osazone. One molecule of reducing sugar reacts with three molecules of phenylhydrazine. The reaction of phenylhydrazine with glucose is shown in figure 2. D-mannose and D-fructose form same type of osazone as that of D-glucose since the configuration of C-3, C-4, C-5 and C-6 is same for all the three sugars.
The osazone of D- galactose is different. Different sugars form osazone at different rates. For example, D-fructose forms osazone more readily than D-glucose. The osazones are crystalline solids with characteristic shapes, decomposition points and specific optical rotations. The time of formation and crystalline shape of osazone is utilized for identification of sugars. This reaction serves to distinguish between aldose and ketose sugars. The derivaties of each sugar are named according to the name of the sugar, that is, the derivaties of glucose as glucosides, of galactose as galactosides and of arabinose as arabinosides etc. Glycosides are acid-labile but are relatively stable at alkaline pH. Since the formation of glycosides convert the aldehydic group to an acetal group, the glucosides are not a reducing sugars. Glycosides are also formed with a non-sugar component, the aglycone. The sugars which are connected to the non-sugar moiety are pentoses, hexoses, branched sugars or deoxy or dideoxy sugars.
The chain length varies from one to five monosaccharide sugar residues per glycosides. Apart from O-glycosides, three other classes of glycosides are found in higher plants namely S-glycosides, N-glycosides and C-glycosides. Heating a solution of hexoses in a strong non-oxidising acidic conditions, hydroxy methyl furfural is formed. The hydroxymethyl furfural from hexose is usually oxidized further to other products When phenolic compounds such as resorcinol, α-naphthol or anthrone are added, mixture of coloured compounds are formed Figure 2.
When conc. H2SO4 is added slowly to a carbohydrate solution containing α-naphthol, a pink color is produced at the juncture. Summary Carbohydrates are chemically defined as polyhydroxy aldehydes or ketones and their polymers. They are classified into monosaccharides, oligosaccharides and polysaccharides. Monosaccharides, the simplest form of carbohydrates, are classified based on the functional groups as aldoses and ketoses or based on the carbon atoms they possess as triose, tetrose, pentose and hexose. Oligosaccarides contain two to ten monosaccharide units joined by glycosidic linkages. Monosaccharides and oligosaccharides are crystalline compounds and soluble in water having sweet taste. Most naturally occurring monosaccharides belong to D-family which is determined by the hydroxyl group attached on th right-hand side of the penultimate carbon.
Optical isomers differ in the configuration around the asymmetric carbon atoms. Mutarotation refers to the change in optical rotation when an aqueous sugar solution is allowed to stand. Monosaccharides exist either in cyclic or acyclic forms. The intramolecular hemiacetal or hemiketal formation is the cyclic structure of the monosaccharides. Cyclic form may possess a pyranose or a furanose ring structure. Sugars differing in the configuration of the hydroxyl and hydrogen attached to the carbonyl carbon are called anomers. Epimers differ in the configuration around a carbon atom other than the carbonyl carbon. Glucose and galactose are epimers. Derived monosaccharides are formed when the hydroxyl group forms esters or is replaced by hydrogen deoxysugars or amino group amino sugars.
The carbonyl group undergoes reduction to form polyols or oxidation to yield aldonic, uronic or aldaric acids. Non-reducing sugars are formed when both anomeric hydroxyl groups are involved in glycosididc bond formation. Thus maltose and lactose are linked reducing disaccharides. Sucrose and trehalose are non-reducing disaccharides. Sucrose is called as invert sugar because, the dextrorotatory sucrose is converted into a levorotatory sugar solution after hydrolysis. Starch and cellulose are important polysaccharides. Review Questions A. Multiple choice questions 1. Identify the non reducing sugar from the following a. Maltose b. Lactose c. Sucrose d. Glucose 2. Which one of the following is levorotatory a.
Glucose b. Mannose c. Fructose 3. The epimer of glucose is a. Galactose b. Fructose c. Arabinose d Ribose 4. Identify the aldopentose from the following a. Xylulose b. Ribulose c Ribose d. Erythrose 5. An example of heteropolysaccharide is a. Amylose b. Hemicellulose c. Cellulose d. Amylopectin B. Fill up the blanks 6. Number asymmetric carbons present in α-D-glucopyranose is Dihydroxyacetone is optically Starch and glycogen are polymers of The repeating unit in chitin is Acid catalysed dehydration of pentose yields Write short answers for the following Define the following a. Anomer b. Epimer c. Enantiomer d Diastereomer e. Reducing sugar f. Non-reducing sugar g. Mutarotation h. Optical activity Explain mutarotation with an example. What are glycosides? What are the evidences for the ring structure of glucose?
What is inversion sucrose? Which is the invert sugar? Write in detail on the following. How are carbohydrates classified? Give example for each group. Compare the structural differences between amylose, amylopectin and cellulose. Explain the reactions of carbohydrates with conc H2SO4, HNO3 and dilute alkali. Describe chemistry and functions of starch. Give the structure and functions of cellulose. Solve the following problems. Draw the structure of β-D-glucopyranose and answer the following a. Draw the anomeric form of this sigar. Name the enantiomer c. How many asymmetric carbon atoms it possess? How many steroisomers of this sugar are possible? Can you draw three epimeric structures and name them? Idetify X and draw its structure. Why there is a change in optical rotation upon hydrolysis? If a solution of D-mannose rotates the incident light by 1. One molar solution of sucrose is hydrolysed using hydrochloric acid. Calculate the molarity of glucose and fructose.
Can sucrose mutarotate? Justify your answer. Gain additional knowledge by further reading Industrial uses of starch Cell wall structure Pharmaceutical uses of sorbitol Flatulence Table 2. Low monosaccharides joined by hydrolysed further molecular weight carbohydrates glycosidic bonds. They can which can be hydrolysed by be hydrolysed by enzymes or enzymes or acids to yield acids. monosaccharides Crystalline, soluble in water, Powdery or crystalline, soluble Insoluble in water, tasteless, and sweet in taste. in water and sweet in taste linear or branched Classified into triose, tetrose, Classified into disaccharide, Classified into homoglycans pentose, hexose and heptose trisaccharide, tetrasaccharide and and heteroglycans depending depending upon the number of pentasaccharide depending upon upon the kind of carbon atoms.
They may be the number of monosaccharides monosaccharides present. either aldoses or ketoses they contain. Depending upon the depending upon whether they function, they are classified contain a potential free as storage and structural aldehyde or ketone group, polysaccharides. respectively All monosaccharides are Some of them are reducing and Non reducing in nature and reducing in nature some of them are non reducing give deep blue amylose or in nature red colour amylopectin with iodine. Table 2. and malt Fermentable by enzyme maltase present in yeast.
Hydrolysed to two molecules of D-glucose. Undergoes mutarotation. In It shows reactions of trace reducing sugars including amounts it mutarotation. Decomposed can be seen by alkali. Not in urine fermentable by yeast. Hydrolysed 2 sugar cane, by dilute acids or enzyme sorghum invertase sucrase to one and carrot molecule of glucose and roots one molecule of fructose. It is to glucose with difficulty. stored as a Not hydrolysed by reserve enzymes. food supply in insect's hemolymp h Table 2. Occurs in the storage organs such as Include both homo and seeds and tubers.
Proteins are macromolecular polymers composed of amino acids as the basic unit. These biopolymers contain carbon, hydrogen, oxygen, nitrogen and sulphur. Proteins are found in all living cells. They form essential constituent of protoplasm, cell membrane and nuclear material. They may be present as simple proteins or complexes with lipids or nucleic acids. Proteins from different tissues such as muscle, bone, brain, blood and other biological fluids differ in composition and properties. In cereal and leguminous plants, seeds contain comparatively higher amounts of protein than stem, leaves and flowers. Tuber crops usually contain less amounts of protein in all parts. Enzymes are specialized proteins with catalytic activities and are present in all living organisms.. Proteins serve as regulators of metabolic reactions, directly as components of enzymes and indirectly in the form of chemical messengers known as hormones as well as receptors for hormones. They regulate and integrate the numerous physiological and metabolic processes in the body.
Proteins are the center of action in many biological processes. The structure and properties of amino acids are described first since they are the fundamental structural units of all proteins. All the amino acids have trivial or common names based on the source from which they were first isolated or based on their properties. Asparagine was named so, as it was isolated from asparagus and glycine was so named because of its sweet taste Greek:'glykos' meaning sweet. They differ only in the side chains R groups. The 20 amino acids found in proteins are referred as the standard or normal or protein amino acids. There are many other amino acids found in nature but do not occur in proteins. They are referred as non-protein amino acids. More meaningful classification of amino acids is based on the polarity of the R groups. The polarity of the R groups varies widely from totally non-polar to highly polar.
The 20 amino acids are classified into four main classes whose structures, three-letter and one-letter symbols are shown in figure 3. Amino acids with non-polar or hydrophobic, aliphatic R groups This group of amino acids includes glycine, alanine, valine, leucine, isoleucine and proline Figure 3. The hydrocarbon R groups are non-polar and hydrophobic. The side chains of alanine, valine, leucine and isoleucine are important in promoting hydrophobic interactions within protein structures. The minimal steric hindrance of the glycine side chain hydrogen allows more flexibility than other amino acids. On the other hand, the imino group of proline is held in a rigid conformation and reduces the structural flexibility of the protein. b Amino acids with non-polar aromatic R groups This group includes phenylalanine, tyrosine and tryptophan Figure 3.
All these amino acids participate in hydrophobic interactions, which is stronger than aliphatic R groups because of stacking one another. Tyrosine and tryptophan are more polar than phenylalanine due to the presence of hydroxyl group in tyrosine and nitrogen in the indole ring of tryptophan. This property is exploited in the characterization and quantification of proteins. c Amino acids with polar, uncharged R groups This group of amino acids includes serine, threonine, cysteine, methionine, asparagine and glutamine Figure 3. The hydroxyl group of serine and threonine, the sulphur atom of cysteine and methionine and the amide group of asparagine and glutamine, contribute to the polarity. The R groups of these amino acids are more hydrophilic than the non-polar amino acids. d Amino acids with charged R groups i Acidic: The two amino acids with acidic R groups are aspartic and glutamic acids Figure 3.
These amino acids have a net negative charge at pH 7. ii Basic: This group includes lysine, arginine and histidine Figure 3. The R groups have a net positive charge at pH 7. The lysine has a second ε-amino group; arginine has a positively charged guanidino group; and histidine has an imidazole group. Most of them are soluble in water and insoluble in non-polar organic solvents e. Aliphatic and aromatic amino acids particularly those having several carbon atoms have limited solubility in water but readily soluble in polar organic solvents. They have high melting points varying from oC or even more. They are tasteless, sweet or bitter.
Some are having good flavour. Sodium glutamate is a valuable flavouring agent and is used in the preparation of certain dishes and sauces. They can react with both alkalies and acids to form salts. In acid solution amino acids carry positive charges and hence they move towards cathode in an electric field. In alkaline solution, on the other hand, the amino acids carry negative charges and therefore move towards anode. But when an amino acid is dissolved in water, it exists as inner salt carrying both positive and negative charges. The amino acids possessing both positive and negative charges are called zwitterions. Figure 3. These reactions are reversible. The pH at which the amino acid has no tendency to move either towards positive or negative electrode is called isoelectric pH or isoelectric point. At this pH, the amino acid molecule bears a net charge of zero.
Isomerism All amino acids except proline, found in protein are α-amino acids because NH2 group is attached to the α-carbon atom, which is next to the COOH group. Examination of the structure of amino acids reveals that except glycine, all other amino acids possess asymmetric carbon atom at the alpha position. Because of the presence of asymmetric carbon atom, amino acids exist in optically active forms. For example, in the steric configuration for serine, the carboxyl group is written on the top, while the amino group is written to the left in the case of L-serine and to the right in the case of D-serine Figure 3. This distinction will hold good for all the amino acids having asymmetric carbon atoms. L-forms are more common than D-forms and most of the naturally occurring amino acids are L-amino acids.
If an amino acid solution is treated with excess of neutralized formaldehyde solution, the amino group combines with formaldehyde forming dimethylol amino acid which is an amino acid formaldehyde complex Figure 3. Hence the amino group is protected and the proton released is titrated against alkali. This method is used to find out the amount of total free amino acids in plant samples. Reaction with nitrous acid Nitrous acid reacts with the amino group of amino acids to form the corresponding hydroxyacids and liberate nitrogen gas Figure 3. Reaction with ninhydrin Ninhydrin is a strong oxidizing agent. When a solution of amino acid is boiled with ninhydrin, the amino acid is oxidatively deaminated to produce ammonia and a ketoacid.
The keto acid is decarboxylated to produce an aldehyde with one carbon atom less than decarboxylates α-amino acids to CO2, NH3 and an aldehyde. The reduced ninhydrin then the parent amino acid. The net reaction is that ninhydrin oxidatively deaminates and reacts with the liberated ammonia and another molecule of intact ninhydrin to produce a purple coloured compound known as Ruhemann's purple Figure 3. This ninhydrin reaction is employed in the quantitative determination of amino acids. Proteins and peptides that have free amino group s in the side chain will also react and give colour with ninhydrin. Thus, the vasoconstrictor agent, histamine is produced from histidine Figure 3. Histamine stimulates the flow of gastric juice into the stomach and the dilation and constriction of specific blood vessels.
Excess reaction to histamine causes the symptoms of asthma and various allergic reactions. But higher animals including man possess this ability only for certain amino acids. The other amino acids, which are needed for normal functioning of the body but cannot be synthesized from metabolic intermediates, are called essential amino acids. These must be obtained from the diet and a deficiency in any one of the amino acids prevents growth and may even cause death. The covalent bond is formed between the α-carboxyl group of one amino acid and the α-amino group of the next amino acid.
The bond so formed between the carboxyl and the amino groups, after elimination of a water molecule is called as a peptide bond and the compound formed is a peptide Figure 3. The peptide formed between two amino acids is a dipeptide; three amino acids is a tripeptide; few amino acids are an oligopeptide and many amino acids is a polypeptide. In writing the peptide structure, the amino terminal N-terminal amino acid is written first and carboxyl terminal C-terminal amino acid written last. It has a role in detoxification of toxic compounds in physiological system. The nanapeptides nine amino acids , oxytocin and vasopressin are important animal peptide hormones. Oxytocin induces labor in pregnant women and controls contraction of uterine muscle. Vasopressin plays a role in control of blood pressure by regulating the contraction of smooth muscles. A dipeptide L-aspartyl-L-phenylalanine, is of commercial importance. This dipeptide is about times sweeter than cane sugar.
The methyl ester of this dipeptide is called as aspartame and marketed as an artificial sweetener for diabetics. i Simple proteins Simple proteins yield on hydrolysis, only amino acids. These proteins are further classified based on their solubility in different solvents as well as their heat coagulability. Seed proteins contain albumin in lesser quantities. Albumins may be precipitated out from solution using high salt concentration, a process called 'salting out'. They are deficient in glycine. Serum albumin and ovalbumin egg white are examples. Globulins Globulins are insoluble or sparingly soluble in water, but their solubility is greatly increased by the addition of neutral salts such as sodium chloride. These proteins are coagulated by heat. They are deficient in methionine. Serum globulin, fibrinogen, myosin of muscle and globulins of pulses are examples.
Upon hydrolysis they yield much proline and amide nitrogen, hence the name prolamin. They are deficient in lysine. Gliadin of wheat and zein of corn are examples of prolamins. Glutelins Glutelins are insoluble in water and absolute alcohol but soluble in dilute alkalies and acids. They are plant proteins e. Histones Histones are small and stable basic proteins and contain fairly large amounts of basic amino acid, histidine. They are soluble in water, but insoluble in ammonium hydroxide. They are not readily coagulated by heat. They occur in globin of hemoglobin and nucleoproteins. They are soluble in water and are not coagulated by heat. They are basic in nature due to the presence of large quantities of arginine. Protamines are found in association with nucleic acid in the sperm cells of certain fish. Tyrosine and tryptophan are usually absent in protamines.
Albuminoids These are characterized by great stability and insolubility in water and salt solutions. These are called albuminoids because they are essentially similar to albumin and globulins. They are highly resistant to proteolytic enzymes. They are fibrous in nature and form most of the supporting structures of animals. They occur as chief constituent of exoskeleton structure such as hair, horn and nails. Conjugated or compound proteins These are simple proteins combined with some non-protein substances known as prosthetic groups. The nature of the non-protein or prosthetic groups is the basis for the sub classification of conjugated proteins.
Nucleoproteins Nucleoproteins are simple basic proteins protamines or histones in salt combination with nucleic acids as the prosthetic group. They are the important constituents of nuclei and chromatin. Mucoproteins These proteins are composed of simple proteins in combination with carbohydrates like mucopolysaccharides, which include hyaluronic acid and chondroitin sulphates. Chromoproteins These are proteins containing coloured prosthetic groups e. Lipoproteins These are proteins conjugated with lipids such as neutral fat, phospholipids and cholesterol Refer chapter 4. Metalloproteins These are metal-binding proteins. A β-globulin, termed transferrin is capable of combining with iron, copper and zinc. Another example is ceruloplasmin, which contains copper. Phosphoproteins These are proteins containing phosphoric acid. Phosphoric acid is linked to the hydroxyl group of certain amino acids like serine in the protein e. Derived proteins These are proteins derived by partial to complete hydrolysis from the simple or conjugated proteins by the action of acids, alkalies or enzymes.
They include two types of derivatives, primary-derived proteins and secondary-derived proteins. There is little or no hydrolytic cleavage of peptide bonds. Proteans Proteans are insoluble products formed by the action of water, dilute acids and enzymes. These are particularly formed from globulins but are insoluble in dilute salt solutions e. Metaproteins These are formed by the action of acids and alkalies upon protein. They are insoluble in neutral solvents. Coagulated proteins Coagulated proteins are insoluble products formed by the action of heat or alcohol on natural proteins e. Secondary-derived proteins These proteins are formed in the progressive hydrolytic cleavage of the peptide bonds of protein molecule. They are roughly grouped into proteoses, peptones and peptides according to average molecular weight.
Proteoses are hydrolytic products of proteins, which are soluble in water and are not coagulated by heat. Peptones are hydrolytic products, which have simpler structure than proteoses. Peptides are composed of relatively few amino acids. They are water-soluble and not coagulated by heat. These biocatalysts are called as enzymes. Enzymes represent the largest class. Nearly different kinds of enzymes are known, each catalyzing a different kind of reaction. They enhance the reaction rates a million fold. Refer chapter 6 for more detail. Regulatory proteins - Hormones These are polypeptides and small proteins found in relatively lower concentrations in animal kingdom but play highly important regulatory role in maintaining order in complex metabolic reactions e.
Protective proteins - Antibodies Some proteins have protective defense function. These proteins combine with foreign protein and other substances and fight against certain diseases. These proteins are produced in the spleen and lymphatic cells in response to foreign substances called antigen. The newly formed protein is called antibody which specifically combines with the antigen which triggered its synthesis thereby prevents the development of diseases. Fibrin present in the blood is also a protective protein. Storage proteins A major class of proteins which has the function of storing amino acids as nutrients and as building blocks for the growing embryo.
Storage proteins are source of essential amino acids, which cannot be synthesized by human beings. The major storage protein in pulses is globulins and prolamins in cereals. But in rice the major storage protein is glutelins. Albumin of egg and casein of milk are also storage proteins. Haemoglobin is a conjugated protein composed of colourless basic protein, the globin and ferroprotoporphyrin or haem. It has the capacity to bind with oxygen and transport through blood to various tissues. Myoglobin, a related protein, transports oxygen in muscle. Lipids bind to serum proteins, principally, albumin and transported as lipoproteins in the blood. Toxic proteins Some of the proteins are toxic in nature. Ricin present in castor bean is extremely toxic to higher animals in very small amounts. Enzyme inhibitors such as trypsin inhibitor bind to digestive enzyme and prevent the availability of the protein. Lectin, a toxic protein present commonly in legumes, agglutinates red blood cells.
A bacterial toxin causes cholera, which is a protein. Snake venom is protein in nature. Structural proteins Some proteins serve as structural materials or as important components of extra cellular fluid. Examples of structural proteins are myosin of muscles, keratin of skin and hair and collagen of connective tissue. Carbohydrates, fats, minerals and other cellular components are organized around such structural proteins that form the molecular framework of living material. Contractile proteins Proteins like actin and myosin function as essential elements in contractile system of skeletal muscle. Secretary proteins Fibroin is a protein secreted by spiders and silkworms to form webs and cocoons. Exotic proteins Antarctic fishes live in These fishes are prevented from freezing by antifreeze glycoproteins present in their body.
Globular proteins are mostly water-soluble and fragile in nature e. Fibrous proteins are tough and water-insoluble. They are used to build a variety of materials that support and protect specific tissues, e. There are many different possible conformations for a molecule as large as a protein. A protein can perform its function only when it is in its native condition. Due to the complexity of three-dimensional structures, the structure of protein is discussed at different levels of its organization. Four levels of structural organization can be distinguished in proteins: 1.
Primary 2. Secondary 3. Tertiary 4. Quaternary 3. It also refers to the location of disulfide bridges, if there are any, in a polypeptide chain. The peptide bond is covalent in nature, quiet stable and referred as backbone of the protein. They can be disrupted by chemical or enzymatic hydrolysis but are not directly influenced by salt concentration, change in pH or solvent. The important steps involved in determining the primary structure of protein are a. Determination of number of chemically different polypeptide chains or subunits in the protein. Separation of polypeptide chains if more than one are present in a protein. Determination of the amino acid sequence of the subunits. Elucidation of the position of the disulfide bonds, if any, between and within the subunits. Determination of number of polypeptides or subunits Determination of the number of C-terminal or N-terminal amino acids will indicate the number of polypeptides in a protein.
This reagent was replaced by dansyl chloride and Edman's reagent phenyl isothiocyanate, PITC. Edman's reagent is also used to determine the amino acid sequence of a polypeptide chain from the N-terminal by subjecting the polypeptide to repeated cycles of Edman degradation. After every cycle, the newly liberated phenylthiohydantoin PTH amino acid was identified Figure 3. The sequence of peptides containing amino acids can be determined using a sequencer by adopting the Edman's degradation method. C-terminal identification C-terminal amino acid can be determined by methods similar to those used for the N-terminal acid. Hydrazine is used to find out the C-terminal amino acid. It reacts with the carbonyl group of each peptide bond except C-terminal amino acid.
Since the carboxyl group of C-terminal amino acid is not involved in a peptide bond, it remains in the mixture as the only unmodified amino acid Figure 3. After chromatographic separation and comparison with the standards, the C-terminal amino acid can be identified. Carboxypeptidases are used for enzymic determination of the C-terminal amio acid. If the protein contains more than one polypeptide chain, separation of polypeptide chain is essential. If the polypeptide chains are connected by disulfide bond, they are cleaved to separate the individual peptide chains. The polypeptide is treated with 2-mercaptoethanol HS-CH2-CH2OH so that reductive cleavage occurs and the polypeptide chains are separated. The resulting free-SH groups are usually alkylated by treatment with iodoacetic acid Figure 3. After cleaving the disulfide links using mercaptoethanol, subunits are dissociated using denaturing agents such as urea or guinidinum ion or detergents such as sodium dodecyl sulphate SDS.
The dissociated subunits are then separated using ion exchange or gel filtration chromatographic method. Amino acid sequencing of polypeptides The amino acid sequence in polypeptides with amino acids can be determined by Edman reaction. For polypeptides containing more than 40 amino acids, both enzymatic and chemical methods are employed to break polypeptide chains into smaller peptides. The enzyme, trypsin hydrolyses the peptide bond on the carboxyl side of the basic amino acid residues of lysine or arginine. The chemical reagent, cyanogen bromide cleaves peptide bond on the carboxyl side of methionine residues. The hydrolyzed peptides are separated and the amino acid sequence is determined by Edman reaction. The hydrolysis of the original polypeptide by two different methods separately gives overlapping regions, from which the sequence is derived Figure 3.
The folding of a linear polypeptide chain occurs to form a specific coiled structure. Such coiling or folding is maintained by hydrogen bonds and hydrogen bond is the only bond responsible for secondary structure. Peptide groups mostly assume the trans- conformation in which successive C2 atoms are on opposite sides of peptide bond joining them. The cis configuration creates steric interference. If a polypeptide chain is twisted by the same amount each of its C atoms, it assumes a helical conformation Figure 3. Helix structure The α-helix is the most stable arrangement of polypeptides Figure 3. The helix structure of proteins is stabilized by intramolecular hydrogen bonding. The polypeptide chain constituted by L-amino acids form a right-handed helix, whereas the polypeptide chains made up of D-amino acids form a left-handed helix.
In the α-helical conformation, all the side chains lie outside the helix whereas C, N, O and H of the peptide bond lie in the same plane. Certain amino acids tend to disrupt the α-helix. Among these are proline the N- atoms is part of the rigid ring and no rotation of the N-C bond can occur and amino acid with charged or bulk R groups that either electrostatically or physically interferes with helix formation. The β-pleated sheet structure Pauling and Corey also proposed a second ordered structure, the β-pleated sheet for polypeptide. This structure is a result of intermolecular hydrogen bonding between the polypeptide chains to form a sheet like arrangement Figure 3. There are two ways in which proteins chains can form the pleated sheet structure.
One is with the chains running in the same direction i. the -COOH or NH2 ends of the polypeptide chains lying all at the top or all at the bottom of the sheet. In another type, known as antiparallel β-pleated sheet structure, the polypeptide chains alternate in such a way that the -COOH end of the one polypeptide is next to the -NH2 end of the other i. polypeptide chains run in opposite directions. The random coil Regions of proteins that are not identifiably organized as helices or pleated sheets are said to be present in random coil conformation. Considerable portion of the protein may be present in this conformation. The term 'random' is unfortunate which imply less biological significance than more highly repeating regions. But in terms of biological function, the regions of random coil are of equal importance to those of helix and pleated sheet. This leads to the twisting of polypeptide chains into specific loops and bends which are maintained chiefly by five kinds of bonds.
Hydrogen bonds Hydrogen bonds are formed between the side chain R group of amino acids having a hydrogen donor group and an acceptor group Figure 3. Salt-linkages electrostatic forces; ionic bonds Salt linkages are due to the interaction between amino groups of basic amino acids and the carboxyl group of acidic amino acids present in the R group Figure 3. Disulfide bonds S-S linkages The S-S linkages are formed by the oxidation of sulfhydryl -SH group of two cysteine side chains Figure 3. Dipole-dipole interaction This interaction occurs between polar unionized side chains Figure 3. The folding of a polypeptide chain due to different covalent and non-covalent interactions is shown in figure 3. Out of the above bonds, the disulfide bond covalent bond is the strongest and cannot be affected by solvent, pH, temperature and salts whereas the above conditions. The tertiary structure gains special importance in the case of enzymes. Domain Domains are structurally independent units that have the characteristics of a small globular protein.
Domains often have a specific function such as the binding of a small molecule. A long peptide strand of a protein will often fold into multiple, compact semi- independent folded regions or domains. Each domain having a characteristic spherical geometry with a hydrophobic core and polar surface very much like the tertiary structure of a whole globular protein. The domains of a multidomain protein are often interconnected by a segment of polypeptide chain lacking regular secondary structure. In enzymes with more than one substrate or allosteric effector sites the different binding sites are often located in different domains. In multifunctional proteins, the different domains perform different tasks.
These subunits are held together by noncovalent surface interaction between the polar side chains. Proteins formed like above are termed oligomers and the individual polypeptide chains are variously termed protomers, monomers or subunits. The most common oligomeric proteins contain two or four protomers and are termed dimers or tetramers, respectively. Myoglobin has no quaternary structure since, it is composed of a single polypeptide chain. Hemoglobin molecule, which consists of four separate polypeptide chains, exhibits quaternary structure. Quaternary structure may influence the activity of enzymes. Some enzymes are active only in their quaternary state and become inactive when split into smaller units. Other enzymes are inactive in the quaternary state and are activated only when they are dissociated to form monomeric state.
Pure proteins are odourless. Because of the large size of the molecules, proteins exhibit many properties that are colloidal in nature. Proteins, like amino acids, are amphoteric and contain both acidic and basic groups. They possess electrically charged groups and hence migrate in an electric field. Many proteins are labile and readily modified by alterations in pH, UV radiation, heat and by many organic solvents. The absorption spectrum of protein is maximum at nm due to the presence of tyrosine and tryptophan, which are the strongest chromophores in that region. Hence the absorbance of the protein at this wavelength is adapted for its determination. This disruption of native structure is termed denaturation.
Physically, denaturation is viewed as randomizing the conformation of a polypeptide chain without affecting its primary structure Figure 3. Physical and chemical factors are involved in the denaturation of protein a Heat and UV radiation supply kinetic energy to protein molecules causing their atoms to vibrate rapidly, thus disrupting the relatively weak hydrogen bonds and salt linkages. This results in denaturation of protein leading to coagulation. Enzymes easily digest denatured or coagulated proteins. b Organic solvents such as ethyl alcohol and acetone are capable of forming intermolecular hydrogen bonds with protein disrupting the intramolecular hydrogen bonding.
This causes precipitation of protein. c Acidic and basic reagents cause changes in pH, which alter the charges present on the side chain of protein disrupting the salt linkages. This property makes some of the heavy metal salts suitable for use as antiseptics. Renaturation Renaturation refers to the attainment of an original, regular three-dimensional functional protein after its denaturation. When active pancreatic ribonuclease A is treated with 8M urea or β- mercaptoethanol, it is converted to an inactive, denatured molecule. When urea or mercaptoethanol is removed, it attains its native active conformation.
Biuret test is extensively used as a test to detect proteins in biological materials. Biuret reaction A compound, which is having more than one peptide bond when treated with Biuret reagent, produces a violet colour. This is due to the formation of coordination complex between four nitrogen atoms of two polypeptide chains and one copper atom Figure 3. Xanthoproteic reaction Addition of concentrated nitric acid to protein produces yellow colour on heating, the colour changes to orange when the solution is made alkaline. The yellow stains upon the skin caused by nitric acid are the result of this xanthoproteic reaction. This is due to the nitration of the phenyl rings of aromatic amino acids. Hopkins-Cole reaction Indole ring of tryptophan reacts with glacial acetic acid in the presence of concentrated sulphuric acid and forms a purple coloured product. Glacial acetic acid reacts with concentrated sulphuric acid and forms glyoxalic acid, which in turn reacts with indole ring of tryptophan in the presence of sulphuric acid forming a purple coloured product.
Nutroitional quality of proteins In judging the adequacy of dietary proteins to meet the human needs, not only the quantity, but the nutritional quality of the dietary proteins also matters. Proteins present in different foods vary in their nutritional quality because of differences in their amino acid composition. Amino acids are the building blocks of proteins. The quality of dietary protein depends on the pattern of essential amino acids it supplies. The best quality protein is the onw whichprovides essential amino acid pattern very close to the pattern of the tissue proteins. proteins, human milk protein, satisfy these criteria and are classified as high quality proteins and serve as reference protein for defining the quality of other proteins.
Apart fro these proteins, the minimum amount of essential amino acids required by infants are also taken as a reference pattern for defining the quality of proteins. The quality of dietary proteins are computed on the basis of the extent to which its essential amino acid pattern deviates from that of standard reference pattern as found in egg or breast milk. This mode of chemical assessment chemical score does not take into account the digestibility of dietary proteins. Hencebiological methods based on growth or N retention are used to determine the overall quality of a protein.
The proteins of animal foods like milk, meat, fish etc. generally compare well with egg in their essential amino acid composition and are categorized as good quality proteins. They are also highly digestible. Plant proteins on the other hand are of poorer quality since EAA composition is not well balanced and a few EAA deviate much from the optimal level present in egg. For instance that in comparison with egg protein cereal proteins are poor in amino acid lysine. Pulses and oilseed proteins are rich in lysine but they are poor in sulphur containing amino acids. Such proteins individually are therefore incomplete proteins. However, relative insufficiency of a particular amino acid of any vegetable food can be overcome by judicious combination with other vegetable foods which may have adequate level of that limiting amino acid.
The amino acid composition of these proteins complement each other and the resulting mixture will have an amino acid pattern better than either of the constituent proteins of the mixture. This is the procedure normally used to improve quality of vegetable proteins. Thus a protein of cereals, deficient in lysine and pulses with adequate lysine content have a mutually supplementary effect, a deficiency of an amino acid in one can be made good by an adequate level in another, if aboth are consumed together. Thus the habitual diets of vegetarians in India based on cereal and pulse has indeed a rational basis.
Another factor to be considered in assessing the value of the proteins of a food stuff is their digestibility. In general, proteins of uncooked vegetable foods particularly pulses are less digestible than those of animal foods. Often the low digestibility of plant proteins is due to the presence of trypsin inhibitors, which are destroyed on cooking. Soya bean has a powerful trypsin inhibitor which is destroyed only on autoclaving. Excessive heat treatment particularly dry heat treatment should be avoided since it affects the quality of vegetable proteins by making some of the essential amino acids like lysine and methionine unavailable. On excessive heating the lysine in proteins reacts with reducing sugars in foods and renders part of lysine unavailable. Summary Proteins are polymers of varying size that are constructed from 20 different amino acids.
They are structurally and functionally diverse molecules and play important roles in human and other organisms than any other class of compounds. The basic unit of proteins, amino acids, share a general structure composed of a carbon centre surrounded by a hydrogen, a carboxyl group,an amino group and side chain R that differs for each amino acid. All 20 amino acids are white crystalline, high melting solids, soluble in water and insoluble in non-polar organic solvents. At physiological pH values, amino acids exist as dipolar ionic species called zwitterions. All amino acids contain acidic and basic functional groups that can be dissociated; therefore their structures change depending on the pH of their environment.
They also exhibit isomerism. All the amino acids found in proteins belong to L-amino acids. Except glycine, all other amino acids possess asymmetric carbon atoms. The 20 protein amino acids are classified according to the chemical nature of their R groups as aliphatic, aromatic, heterocyclic and sulphur containing amino acids. They undergo chemical reactions due to amino and carboxyl groups. Peptides and proteins are formed by linking the amino acids through peptide bonds. All polypeptide chains have an amino terminus and a carboxyl terminus. Proteins are involved in different functions. Based on function they are classified as enzymes, storage proteins, toxic and structural compounds, secretary and exotic proteins and as antibodies apart from being involved in the activities of transport, muscle contraction and metabolic regulation in biological systems.
They are also classified based on their solubility and composition as simple, compound and derived proteins. Simple proteins include albumins globulins, prolamins, glutelins histones, protamins, and albuminoids. Compound or conjugate proteins constitute nucleoproteins, mucoproteins, chomoproteins, lipoproteins metalloproteins and phosphoprteins. Derived proteins include primary and secondary derived proteins. Conformation of a protein refers to the three-dimensional structure in its native state. The molecular architecture of proteins can be organized into four levels; i primary structure-the order in which amino acids are arranged in a polypeptide chain ii secondary structure-the steric relationship of amino acids that are close to one another on linear sequence iii the tertiary structure defines the complete three-dimensional arrangement in space native conformation iv quaternary structure-the specific manner in which separate polypeptide chains are packed together to form a functional protein aggregate.
Most pure proteins are generally tasteless and odourless. They are colloidal and amphoteric in nature. Most proteins are labile and readily modified by pH, UV radiation, heat and organic solvents. They undergo chemical reactions due to peptide bond and side chain R groups. Which one of the following amino acids is optically inactive? Alanine b. Glycine c. Leucine d. Aspartic acid 2. The reagent that is used for quantitative determination of amino acids is a. Sanger's b. Ninhydrin 3. The protein with quaternary structure is a. Insulin b. Myoglobin c. Hemoglobin d. Keratin 4. You can easily download Fundamentals of Biochemistry: Life at the Molecular Level 5th Edition PDF Free by clicking the link given below. If the link is not responding kindly inform us through comment section.
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20/08/ · Download Fundamentals of Biochemistry: Life at the Molecular Level 5th Edition PDF Free: You can easily download Fundamentals of Biochemistry: Life at the Molecular Fundamentals of Biochemistry PDF 5th Edition By Donald Voet, Judith G. Voet, Charlotte W. Pratt Fundamentals of Biochemistry By voet and voet PDF 5th Edition address the enormous advances in biochemistry, particularly in the areas of structural biology and Bioinformatics, by providing a solid biochemical foundation that is rooted in chemistry to prepare you for the scientific challenges of the future 22/01/ · DOWNLOAD NOW» Voet, Voet and Pratt’s Fundamentals of Biochemistry, 5th Edition addresses the enormous advances in biochemistry, particularly in the areas of 3/04/ · [PDF DOWNLOAD] Fundamentals of Biochemistry: Life at the Molecular Level Author: Donald Voet Pages: pages Publisher: John Wiley & Sons 13/06/ | Fundamentals of Biochemistry PDF: Life at the Molecular Level FIFTH [5th] Edition with PDF (Dimensions: 9 x x inches: Free [ by Judith G. Voet (Author), 30/08/ · Fundamentals of Biochemistry 5th Edition pdf Voet download August 30, Fundamentals of Biochemistry 5th edition is an authentic treatise on biochemistry and ... read more
The aldehyde group of aldoses is oxidized to carboxylic acid. The 20 amino acids found in proteins are referred as the standard or normal or protein amino acids. Peptones are hydrolytic products, which have simpler structure than proteoses. This hint comes from the derivations and equations added in the content. Estimation of starch 8. The controlled removal of methoxyl groups, converting high methoxyl pectins to low-methoxyl pectins, is possible using pectin methylesterases but the reverse process is not easily achieved.
The important members of sucrosyl oligosaccharides are raffinose DP-3stachyose DP-4verbascose DP-5 and ajugose DP ª] edición. The reaction of phenylhydrazine with glucose is shown in figure 2. They enhance the reaction rates a million fold. Lipids — classification, structure and properties. They are soluble in water and are not coagulated by heat.
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