BIOCHEMISTRY-MIDTERM-1C-AND-1D.pdf

BIOCHEMISTRY LAB - MIDTERM COLLOIDS in which impurities collect after passing out of the blood. Blood The term colloid is derived from Greek, Koli = glue and eidos = proteins a...

BIOCHEMISTRY LAB - MIDTERM COLLOIDS in which impurities collect after passing out of the blood. Blood The term colloid is derived from Greek, Koli = glue and eidos = proteins and other important large molecules remain in the blood. appearance. Colloids are suspensions of particles that are larger 3 TYPES OF MEMBRANES FOR DIALYSIS: than these true solutions but still smaller to settle out by gravity. a. CELLULOSE-BASED MEMBRANE: PES, PAN, and PVDF They cannot be filtered by ordinary filters, e.g. white of eggs, milk, b. REGENERATED CELLULOSE-BASED MEMBRANE plasma. A colloidal system consists of two components— c. COMPOSITE MEMBRANE 1. Dispersed phase and 2. Dispersion phase ADSORPTION The dispersed phase consists of macromolecular solids like Adsorption is a process where a solid is used for removing a proteins and nucleic acids and liquids like oily fats. The dispersion soluble substance from the water. In this process activated phase is the medium in which insoluble materials are dispersed. It charcoal is the solid. may be solid liquid or gas. Activated charcoal is the ideal water filter because it removes SUSPENSOID toxins from the water without stripping the water of salts and When the term lyophobic colloid is used, reference is important minerals. made to a suspensoidal particle wherein water is the dispersing medium but the suspended particles such Methylene Blue ( C3H18ClN3S) is a monovalent cationic dye. MB as metals and/or their oxides have little or no affinity is a basic dye & got many utilities in terms of dyeing of silk, toward water. The stability of a suspensoid is leather, paper & cotton as well as production of ink. The discharge dependent strictly on its electric charge and having lost of MB is a great threat for both toxicological & aesthetical reasons its given charge, precipitation occurs. impede light penetration & are toxic to supply food chain for organisms. Since it has a synthetic origin & complex organic EMULSOID aromatic structure; hence, they are inert & difficult to bio-degrade Lyophilic, when used as a synonym for emulsoid, refers when discharged into the water. to starches and gums which hydrate readily and ”love to loosen”. In other words, they are solvent-loving The various treatment options have been explored and adsorption colloids which have an affinity existing between the technique has been widely welcomed for the removal of dispersed phase and the dispersion medium. methylene blue dye from the effluent. The substance onto which liquid molecules get adsorbed is called adsorbent & the liquid Emulsoids have either a positive or negative electric molecules that get adsorbed onto the adsorbent are known as charge and in some cases none at all (positive charge in adsorbate. The process of adsorption is divided into two acid solutions, negative charge in alkaline solutions). categories depending on type of forces involved between The effect which causes emulsoids to be inherently adsorbent & adsorbate. a) Physiorption b) Chemisorption stable becomes evident when we consider that starches and gums undergo swelling in the presence of water. Hydration of such materials is a natural function, In the process of adsorption onto activated charcoal, the recovery and the forces involved by their nature tend to keep of dyes such as methylene blue relies on several critical factors. the particles from coalescing. Activated charcoal, renowned for its porous structure, exhibits a high adsorption capacity, effectively binding dye molecules from EMULSOID SUSPENSOID solution. Methylene blue adheres to the charcoal primarily Foam Formation Stable Foam Unstable Foam through physical forces such as Van der Waals interactions, Precipitation With Very Stable, Not Unstable, get facilitating reversible binding where the dye can be released when Electrolytes easily coagulated easily coagulated conditions favor desorption. The efficiency of dye recovery by electrolytes by electrolytes depends significantly on the initial concentration of dye in the Reversibility Reversible Irreversible solution and the adsorption capacity of the charcoal. Methylene blue solution when combined with animal charcoal gives a Dialysis colorless filtrate in filtration. Dialysis is a process in which solvent molecules, other small molecules, and hydrated ions pass from a solution through a OSMOSIS AND DIFFUSION membrane. Dialyzing membranes are semipermeable Osmosis membranes with larger pores than osmotic membranes. They hold Osmosis is the spontaneous flow of solvent through a back colloid particles and large molecules but allow solvent, semipermeable membrane from a solution of low concentration hydrated ions, and small molecules to pass through. The passage to one of higher concentration. Physiological activities such as of these ions and small molecules through such membranes is absorption from the gastrointestinal tract, and fluid interchange in called dialysis. Dialysis can be used to separate small particles various compartments of body (e.g. between plasma and RBC) follow the principle of osmosis. from colloids. A mixture containing water, ions, small molecules, and colloid DIFFUSION THROUGH GELATIN particles is placed inside a bag made from a dialyzing membrane. Molecules or particles also diffuse in water and other liquids, but Water flowing around the bag carries away ions and small diffusion alone, without the effects of convection currents and molecules that pass through the membrane. Water molecules gravity, is very difficult to demonstrate in liquids (see Elster, 2014). move through the membrane in both directions, but the colloid This is because convection currents, which are movements of a particles remain inside the bag. liquid within another liquid, are created by simply adding a dye solution to the liquid. Besides forming convection currents, the A similar technique is used to clean the blood of people suffering higher density of the dye solution compared to water and other from kidney malfunction. The blood is pumped through tubing liquids causes it to gradually (or quickly) sink when it is added to made of a dialyzing membrane. The tubing passes through a bath these liquids. These movements of the dye in the liquid are not caused by diffusion. Because these movements and their effects  BIOCHEMISTRY LAB - MIDTERM are large compared to the diffusional movement of the dye, it is difficult to measure movement due only to diffusion by adding a dye solution to a liquid. Diffusion is faster for small molecules, e.g., Potassium Permanganate, than larger ones, such as Methylene Blue. DYE MOLECULAR WEIGHT Potassium Permanganate 158g/mol Methylene Red 269.3g/mol Phenolphthalein 318.31g/mol Methylene Blue 319.85g/mol Diffusion times are dependent on temperature – they are faster in warm agarose (gelatin) than room temperature test tubes, and slower in test tubes. PROTECTIVE COLLOIDS When a large amount of hydrophilic colloids carrying opposite QUALITATIVE TEST FOR PROTEINS AND AMINO ACIDS charges is added to hydrophobic colloids, these get adsorbed on Proteins are made up of amino acid residues joined by peptide the hydrophobic particles and form a protective layer around it. bonds. Due to their polypeptide structure and different amino acid This adsorption layer prevents the precipitating ions reaching the residues, protein reacts with a variety of reagents to form colored sol particles and hence preventing the coagulation. products. These tests, known as colour reactions of proteins, are An entire colloids are thermodynamically stable and behaves like of importance in qualitative detection and quantitative estimation hydrophillic colloid. of proteins, and of their constituent amino acids in body fluids and other biological materials. The colloid that helps to stabilize other colloids is called as a protective colloid. Proteins and amino acids used in different experiments: 1. Egg albumin is an egg protein, which is soluble in water. SURFACE TENSION 2. Casein is the major protein in milk. It is a The interior molecules of a homogeneous liquid are equally phosphoprotein with phosphate groups attached to the attracted in all directions by surrounding molecules. They are free hydroxyl groups or serine and threonine residues. It is to move in all directions. But the molecules in the surface of the deficient in cysteine. liquid are attracted downward and sideways but not upward 3. Gelatin is formed from collagen, the connective tissue (except for the little attraction of air molecules). protein, by boiling with water. It is a rich source of As a result, the molecules of the surface are not so free to move. amino acid glycine. It is deficient in tyrosine, They are held together and form a membrane over the surface of tryptophan, and cysteine. the liquid. 4. Metaproteins, proteoses, and peptones are partially hydrolysed products of proteins like albumins and Therefore, when finely powdered sulfur or other non-wetting globulins. Albumin has relatively low molecular weight. powders are sprinkled upon water, they do not sink but are Gelatin, metaproteins, proteoses and peptones are suspended on the surface. derived proteins. A great part of the energy required to convert a liquid into a gas is NEUTRAL AA ACIDIC AA BASIC AA essential to overcome surface tension and drag the molecules free Glycine Aspargine ASPARTIC ACID LYSINE from the surface of the liquid. There is also an interfacial tension Serine. Proline GLUTAMIC ACID ARGININE which is biochemically very important, especially in the process of Phenylalanine HISTIDINE adsorption. This tension lies at the boundary between immiscible Alanine. Glutamine liquids, e.g., oil drops emulsified in water. The tension is due to Threonine Isoleucine unequal attraction of the film molecules as compared with the Tyrosine molecules in the interior of the liquid. Valine Cysteine Factors Affecting Surface Tension Tryptophan 1. Temperature: Surface tension decreases with the increase in Leucine temperature. Methionine 2. Dissolved Substances: a) Most inorganic salts slightly raise surface tension of Aliphatic amino acids Hydroxy amino Sulfur-containing water although potassium permanganate lowers it. Glycine acids amino acids b) Organic substances usually lower surface tension. Alanine Threonine Cysteine Soaps and bile salts are most effective in this respect. Valine Serine Methionine c) Alkalis increase surface tension but ammonia lowers it. Leucine Tyrosine Strong mineral acids also decrease surface tension. Isoleucine d) In liquid-liquid and solid-liquid systems, dissolved Aromatic amino acids Dicarboxylic acid Diamino acids substances generally lower interfacial tension. Phenylalanine and their amides Lysine Tyrosine Glutamic acid Arginine Physiological Importance of Surface Tension: Tryptophan Glutamine (amide of Histidine Surface tension is involved in the process of digestion; because glutamic acid) bile salts reduce the surface tension of lipids and thus assist Aspartic acid emulsification. As a result, the surface area is increased which favors lipase activity on lipids.  BIOCHEMISTRY LAB - MIDTERM Aspargine (amide of aspartic acid) CO-NH is the peptide proteoses purple Imino acids or heterocyclic amino acids – Proline linkage in biuret. At least and peptones dark two peptide bonds in the pink colour molecule are required indicating that for a positive test. albumin/ globulins have largest number Individual amino acids of peptide linkages and dipeptides will not and peptones the answer this test. least. Nindhydrin a-amino acid + ninhydrin All a-amino acids → aldehyde + give purple colour. hydrindantin + NH3 + The imino acids, CO2; proline and hydrindantin + NH3 + hydroxyproline give ninhydrin → Ruhemann’s yellow colour. Test Principle Positive purple + 3H2O The solubility of amino The coloured acids and proteins is Proteins do not give a complex is known as largely dependent on the true colour reaction; but Ruhemann’s purple. solution pH. N-terminal amino group The structural changes in of a protein can react Glutamine and an amino acid or protein with ninhydrin to asparagine produce Solubility test that take place at produce a faint blue brown colour. different pH values alter colour. the relative solubility of the molecule. Xanthoproteic Yellow colour is due to Yellow precipitate the formation of nitro In acidic solutions, both derivatives of benzene Colour of precipitate amino and carboxylic ring containing amino and the solution groups are protonated. acids (tyrosine change to orange In basic solutions, both and tryptophan), the groups are colour turns orange due deprotonated. to ionization when alkali is added. In neutral aqueous solutions, amino acids All proteins usually act as amphoteric mol- respond ecules. For example, an to this test. amino acid with a neutral side chain contains two This reaction is also the charges: one positive, basis of yellow stain in due to the protonation of skin by nitric acid. the amino group, and Nitration of one negative, due to the phenylalanine dissociation of the under these conditions carboxylic acid proton. normally does not take This double ionic form of place. Acidity/ an amino acid is the Alkalinity zwitterionic form. Hopkins-Cole Mercuric sulphate cause Violet ring at the Test mild oxidation of indole junction of two Amino acids are group of tryptophan, liquid layers due to essentially soluble in which condenses with an indole ring. water. Their solubilities aldehyde to give the in water, dilute alkali and colored complex. dilute acid vary from one compound to the other Gelatin, poor in depending on the tryptophan, does not structure of their side give the test. chains. Biuret Test The reaction is so named The colour varies Sakaguchi Test Guanidino groups in Bright red colour since biuret (NH2-CO- depending on the for Guanidine arginyl residues of due to NH-CO-NH2) formed by number of peptide Group proteins react with the a- guanidium group the condensation of two linkages: (Reaction of naphthol. (Arginine present) molecules of urea when albumin/ globulin Arginine) heated. give violet This test is given by albumin, globulin and  BIOCHEMISTRY LAB - MIDTERM gelatin as it contains precipitate normal arginine. proteins of the gastro Lead Sulfide Organic sulphur in Black or brown PPT intestinal wall. Raw egg Test (Test for cysteine and cystine are is sometimes used as an Sulphur- released as inorganic S2- antidote for mercury containing ions which form lead poisoning. Amino Acids) sulphide as follows: Acid Test The precipitation of a Precipitate R-SH + 2NaOH → ROH (TCA) protein in the presence + Na2S + H2O Test with of acid reagents is Na2S + (CH3COO)2Pb Trichloroacetic probably due to the → PbS + 2CH3COONa acid (TCA) formation of insoluble salts between the acid Methionine does not anions and the give this test as the positively charged sulphur group in this protein particles. These amino acid is in thioether precipitants are only linkage, which is difficult effective in acid to break, and not solutions. released by treatment with NaOH. Precipitation Tungstic acid, Hager’s reagent by Alkaloidal phosphotungstic acid, (Picric Acid) – Yellow Albumin and keratin will Agents trichloroacetic acid, Crystalline answer this test, but picric acid, sulphosalicylic Precipitate casein (containing acid and tannic acid are methionine) will not. powerful protein Tannic Acid – Buff Sodium Sodium nitroprusside Red/Intense Purple precipitating agents. Colored Precipitate Nitroprusside reacts with the thiol These acids lower the pH Test group of the cysteine of the medium, when under alkaline condition proteins carry net to yield an intense positive charges. purple colored compound, which fades These protein cations are after few minutes. electrostatically complexed with The nitroprusside test is negatively charged ions specific to cysteine, the to form protein- only sulfhydryl group tungstate, protein- amino acid containing picrate, etc. and thick cysteine (-SH). In the flocculant precipitate is presence of excess formed. ammonia, that group reacts with Alkaloidal reagents nitroprusside. include Esbach's reagent Precipitation of Proteins: ( picric acid + citric acid ), The precipitation of a protein occurs in a stepwise process. The sulphosalicylic acid, addition of a precipitating agent and steady mixing destabilizes the metaphosphoric acid, protein solution. tannic acid, phosphotungstic acid & Mixing causes the precipitant and the target product to collide. trichloroacetic acid. Enough mixing time is required for molecules to diffuse across the Heller’s test Heller’s test is a A white ring at the fluid. biochemical test zone of contact Precipitation Heavy metal salts usually White Precipitate performed to detect indicates a positive By Salts of contain Hg2+, Pb2+, proteins in a sample by test. Heavy Metals Ag1+, Tl1+, Cd2+ the denaturation of and other metals with those by the addition of high atomic weights. strong acids. Heller’s test Since salts are ionic, they usually uses disrupt salt bridges in concentrated nitric acid proteins. The reaction of for the denaturation of a heavy metal salt with a proteins. The test is protein usually leads to performed for clinical an insoluble metal purposes to detect protein salt. abnormal proteins in biological fluids, Salts of iron, copper, zinc, including urine. lead, cadmium and mercury are toxic, Robert’s Test Albumin precipitates in A white ring at the because they tend to the presence of zone of contact  BIOCHEMISTRY LAB - MIDTERM Magnesium Sulfate and indicates a positive an inorganic salt like strong acid test. ammonium sulphate is (concentrated nitric added to a solution of acid). protein, it decreases concentration of water The principle of this test molecules available for is based on the stabilizing the protein precipitation of protein solution and the protein and formation of white is consequently compact ring using precipitated. The concentrated Nitric acid process is known as (HNO3). “salting out”. Precipitation Proteins in solution form Precipitate By Alcohol hydrogen bonds with Solubility of a protein water. Organic solvents depends on ionic like acetone, ether or concentration of the ethanol when added to a medium. Therefore, the protein solution in water, presence of very small reduce the concentration quantities of salts will of water molecules increase the solubility of available for keeping the a protein by diminishing proteins in solution and protein-protein thus decrease the interaction. This is called number of hydrogen “salting-in. bonds. Acid/Base One end of the protein molecule has free The dielectric constant of amino group, while the the medium is also other end has free COOH reduced causing group. aggregation, In acid solution, the precipitation and NH2 groups accept H+ denaturation of proteins. ion and This denaturation does present as NH3+ (cation). not occur to some Therefore protein in acid proteins at low solution will be positively temperature. charged. By Heating The principle in this test In alkaline pH, the is also based on COOH groups donate H+ precipitation. Proteins ion and when exposed to heat in are present as COO– acidic conditions (anion). Hence, proteins undergo structural in alkaline solution are denaturation leading to negatively charged. aggregation. When a protein solution is So, proteins are heated in a boiling water ampholytes acting both bath, the proteins get as donors and acceptors coagulated and loose of H+ ion. their biological activity, e.g. boiling of eggs. The best example is casein, which forms a Albumin and globulin are flocculent precipitate at easily coagulated by heat its isoelectric pH 4.6; and near or at their redissolves, in highly isoelectric point. On acidic or alkaline addition of acetic acid, solutions. there is a decrease in pH. When pH approaches the When milk is curdled, the isoelectric pH of casein forms a white albumin/globulin, curd, because lactic acid coagulation occurs produced by the spontaneously since the fermentation process solution is pre-heated. lower the pH to the This is called Heat and isoelectric point of acetic acid test. casein. Casein is Salt Generally proteins can Precipitate precipitated from milk Denaturation be precipitated by the and the supernatant is addition of salts. When called whey.  BIOCHEMISTRY LAB - MIDTERM Alcohol Alcohol are capable of Precipitate other in the molecular weight and the amount of phosphorus Denaturation engaging in groups they contain. Casein exists in milk as the calcium salt and intermolecular hydrogen calcium caseinate. The white color of milk is due to this salt and bonding with protein emulsified lipids. molecules, disrupting intramolecular hydrogen Principle: bonding within the Most proteins show minimum solubility at their isoelectric point protein. and this principle is used to isolate the casein by adjusting the pH of milk to (4.5-4.8) its isoelectric point. Casein is also insoluble in DENATURATION OF PROTEINS ethanol and this property is used to remove unwanted fat from The three-dimensional conformation, the primary, secondary, the preparation. Milk is present at a higher pH than the isoelectric tertiary, and even in some cases quaternary structure is point of casein, so to precipitate the casein acetic acid is added characteristic of a native protein. Hydrogen bond, ionic bond and drop by drop until the isoelectric point is reached. Note that if hydrophobic bond stabilize the structure to maintain its excess CH3COOH is added the precipitate redissolves. conformation in space. This conformation can upset and disorganized without breakage of any peptide linkage, only by the The isoelectric point of a protein(I.E.P.): is the pH at which the rupture of ionic bond, hydrogen bonds and hydrophobic bond molecule is electrically neutral(net charge is zero). When a protein which stabilize the structure. This is called denaturation. is charged e.g. with a –ve charge, the –ve molecules repel each other and thus remain in the solution and do not precipitate. But Denaturation of proteins leads to: when these molecules are neutral at the I.E.P. they tend to Unfolding of natural coils of native protein. precipitate due to the absence of the repelling forces and Decrease in solubility and increase in precipitability. protein's high molecular weight. Loss of biological activities, (e.g. enzyme activity) and antigenic properties. Increased digestibility. Denaturing Agents Denaturation is brought about by certain: – Physical agents: Heat, Ultraviolet rays and ionizing radiations can denature proteins. – Chemical agents: Acids, alkalies and certain acid solutions of heavy metals, e.g. mercury, lead, detergents; organic solvents like alcohol, acetone, etc. denature proteins. – Mechanical means: Vigorous shaking or grinding leads to denaturation of the protein. Examples of Denatured Protein Cooked meat or boiled egg, milk paneer, etc. Significance of Denaturation Digestibility of native protein is increased on denaturation by gastric HCI or by heat on cooking. Denaturation causes unfolding of native polypeptide coil so that hidden peptide bonds are exposed to the action of proteolytic enzyme in the gut. It also increases reactivity of certain groups. Denaturation property of a protein is used in blood analysis to eliminate the proteins of the blood (deproteinization of blood). Coagulation Denaturation may, in rare cases be reversible, in which case the protein refolds into its original native structure, when the denaturing agent is removed. However, most proteins, once denatured, remain permanently disordered and are called irreversible denaturation or coagulation, e.g. coagulated egg white of boiled egg. Isolation of Casein from Milk There are three kinds of proteins in milk: casein, lactalbumin and lactoglobulin. They are complete proteins because they contain all amino acids essential for building blood and tissue. Casein is the main protein in milk and is present at a concentration of about 35 g/l. Casein is a phosphoprotein. It is not a single compound, it is a heterogeneous mixture of α, β, κ− caseins which differ from each