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LAB MANUAL FOR BIOCHEMSRTY FOR PTU STUDENTS

1. Preparation of standard buffers (citrate, phosphate and carbonate) and measurement of pH. 2. Titration curve for amino acids. 3. Separation of amino acids by two dimensional paper chromatography and gel electrophoresis. 4. Separation of lipids by TLC. 5. Separation of serum proteins by electrophoresis on cellulose acetate. 6. Quantitative estimation of amino acids. 7. Quantitative estimation of proteins. 8. Determination of glucose by means of the enzyme glucose oxidase. 9. Enzymatic hydrolysis of glycogen by alpha- and beta- amylases. 10. Isolation and determination of RNA and DNA. 11. Effect of temperature on the activity of alpha-amylase. 12. Estimation of SGOT, SGPT, Alkaline phosphotase and Bilirubinu in the serum.

Experiment 1
Aim : To prepare standard buffer and measure its pH Reference : Sharma P.K., Dandiya P.C., Pharmaceutical Biochemistry, Vallabh Prakashan, 1st edition, 2006, 263-266. Requirement Apparatus : pH meter Chemical : Standard buffer tablet, potassium dihydrogen phosphate, disodium hydrogen phosphate Theory : Buffers are solutions of weak acids (or bases) together with their conjugate bases (or acids) which diminish the change in pH which would otherwise occur from addition of acid or base. pH = pKa + log [salt] / [acid] Procedure : Preparation of standard buffer solution : Stock solutions: (i) Dissolve 14.2g Na2HPO4 in one litre water (0.1mol/L). (ii) Dissolve 13.6g KH2PO4 in one litre water (0.1mol/L). Mix the volumes of two solutions given below for 10 ml phosphate buffers of different pH values. Na2HPO4 (ml) (0.1mol/L) 0.25 0.50 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 9.50 KH2PO4(ml) (0.1mol/L) 9.75 9.50 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.50 pH at 20 C 5.29 5.59 5.91 6.24 6.47 6.64 6.81 6.98 7.17 7.38 7.73 8.04

3 Measurement of pH : pH meter is put on and allowed to warm. The pointer is adjusted to 0 mV or 7.0 pH position by zero control. Standard buffer solution is taken in beaker. Its temperature is noted and electrodes are dipped in solution. The meter will show the pH of buffer. Set the pointer at exact value of buffer. Bring selector switch again to zero and clean electrodes with water. Now take the pH of unknown solution.

Prepare an appropriate buffer solution by dissolving buffer tablet in distilled water. Place the temperature probe into standard buffer solution. Insert electrode assembly in it and adjust the buffer until the meter reading agrees with known pH of buffer. Remove the electrode assembly, rinse and place it in second buffer solution. Then remove the electrode and rinse with distilled water and place again in first solution to confirm the calibration. Result : pH is noted from pH meter.

Experiment 2
Aim : To perform identification test for a given sample of carbohydrate. Reference : Satyanarayan U., Chakrapani U., Biochemistry, Uppola , 3rd edition, 1617. Requirement Apparatus : Test tube, water bath, holder. Chemical : Molisch reagent, Fehlings solution, Benedicts solution, conc. sulphuric acid, picric acid, sodium carbonate, phenyl hydrazone, sodium acetate, Barfords solution, sample solution. Theory : Molisch's Test (named after Austrian botanist Hans Molisch) is a sensitive chemical test for the presence of carbohydrates, based on the dehydration of the carbohydrate by sulfuric acid to produce an aldehyde, which condenses with two molecules of phenol (usually -naphthol, though other phenols (e.g. resorcinol, thymol) also give colored products) resulting in a red- or purple-colored compound. The Iodine test is used to test for the presence of starch. Iodine solution iodine dissolved in an aqueous solution of potassium iodide reacts with starch producing a purple black color. Benedict's reagent is used as a test for the presence of reducing sugars. This includes all monosaccharides and the disaccharides, lactose and maltose. Even more generally, Benedict's test will detect the presence of aldehydes (except aromatic ones), and alphahydroxy-ketones, including those that occur in certain ketoses. Thus, although the ketose fructose is not. Seliwanoffs test is a chemical test which distinguishes between aldose and ketose sugars. Ketoses are distinguished from aldoses via their ketone/aldehyde functionality. If the sugar contains a ketone group, it is a ketose and if it contains an aldehyde group, it is an aldose. This test is based on the fact that, when heated, ketoses are more rapidly dehydrated than aldoses. In concentrated HCl, ketoses undergo dehydration to yield furfural derivatives more rapidly than do aldoses. These derivatives form complexes with resorcinol to yield deep red colour.

5 Procedure : Iodine solution: Add a few crystals of iodine to 2% potassium iodide solution till the colour becomes deep yellow. Fehlings reagent A: Dissolve 34.65 g copper sulphate in distilled water and make up to 500 mL. Fehlings reagent B: Dissolve 125 g potassium hydroxide and 173 g Rochelle salt (potassium sodium tartrate) in distilled water and make up to 500 mL. Benedicts qualitative reagent: Dissolve 173 g sodium citrate and 100 g sodium carbonate in about 500 mL water. Heat to dissolve the salts and filter, if necessary. Dissolve 17.3 g copper sulphate in about 100 mL water and add it to the above solution with stirring and make up the volume to 1 L with water. Barfoeds reagent: Dissolve 24 g copper acetate in 450 mL boiling water. Immediately add 25 mL of 8.5% lactic acid to the hot solution. Mix well, Cool and dilute to 500 mL. Seliwanoffs reagent: Dissolve 0.05 g resorcinol in 100 mL dilute (1:2) hydrochloric acid. Bials Test: It is specific for pentoses. They get converted to furfural. In the presence of ferric
ion orcinol and furfural condense to yield a coloured product.

The reactions of carbohydrates are given in Table: Experiment

Molischs Test Add two drops of Molischs reagent (5% 1-naphthol in alcohol) to about 2 mL of test solution and mix well. Incline the tube and add about 1 ml of concentrated sulphuric acid along the sides of the tube. Iodine Test Add a few drops of iodine solution to about 1 mL of the test solution. Appearance colour of deep blue This indicates the presence of starch in the solution. The blue colour is due to the formation of starch-iodine complex.

Observation
A red-cum-violet ring appears at the junction of the two liquids.

Remarks
The colour formed is due to the reaction of alpha-naphthol with furfural and/or its

derivatives formed by the dehydration of sugars by

concentrated sulphuric acid. All carbohydrates react

positively with this reagent.

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Fehlings Test A, add 1 mL of Fehlings solution B and a few drops of the test solution. Boil for a few minutes. Formation of yellow or The blue alkaline cupric

To 1 mL of Fehlings solution brownish-red precipitate.

hydroxide present in Fehlings solution, when heated in the presence of reducing sugars, gets reduced to yellow or red cuprous oxide and it gets precipitated. Hence, formation of the coloured precipitate indicates the presence of

reducing sugars in the test solution. Benedicts Test To 2 mL of Benedicts reagent add five drops of the test solution. Boil for five minutes in a water bath. Cool the solution. Barfoeds Test add about 2 mL of Barfoeds reagent. Boil it for one minute and allow to stand for a few minutes. Seliwanoffs Test To 2 mL of Seliwanoffs reagent add two drops of test solution and heat the mixture to just boiling. Bials Test To 5 mL of Bials reagent add 2-3 mL of solution and warm gently. When bubbles rise to Appearance of green colour or precipitate. It is specific for pentoses. They get converted to furfural. In the presence of ferric ion orcinol and furfural condense Appearance of deep red colour It is a timed colour reaction specific for ketoses. Formation of brick-red Only monosaccharides answer this test. Since Barfoeds Formation of red, yellow or Depending green colour/precipitate. on the sugar

concentration yellow to green colour is developed.

To 1 mL of the test solution precipitate.

reagent is weakly acidic, it is reduced only by

monosaccharides.

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the surface cool under the tap. to yield a coloured product. Osazone Test To 0.5 g of phenylhydrazine hydrochloride add 0.1 g of sodium acetate and 10 drops of glacial acetic acid. To this mixture add 5 mL of test solution and heat on a boiling water bath for about half an hour. Allow the tube to cool slowly and examine the crystals under a microscope. Glucose, fructose and mannose produce needleshaped yellow osazone crystals, whereas lactosazone is mushroom shaped. Maltose produces flower-shaped crystals. The ketoses and aldoses react with phenylhydrazine to produce a phenylhydrazone which in turn reacts with another two molecules of phenylhydrazine to form the osazone.

Observation : Note the various changes occurring after completion of reaction. Result : According to observation write down the result whether test is positive or negative.

Experiment 3
Aim : To perform identification test for a given sample of protein. Reference : Sharma P.K., Dandiya P.C., Pharmaceutical Biochemistry, Vallabh Prakashan, 1st edition, 2006, 316. Requirements : Test tube, dropper, pipette, beaker Chemicals : Conc. nitric acid, chlorophenol, acetic acid. Theory : Heller's test: Proteins get denatured when acid is added and this forms a white coagulum which is slightly yellow in colour because of nitro- derivatives of proteins given by aromatic amino acids. Heat coagulation test: Coagulation is a result of irreversible denaturation of proteins like albumin and globulins. Procedure : Heller's test: In 3ml conc. Nitric acid add 3 ml of protein solution. White ppt. appears at junction of two fluids indicating that protein is present.

Heat coagulation test: Fill 2/3 of test tube with protein solution. Add 4-5 drops of chlorophenol red and mix. A purple colour develops. Add 1% acetic acid until colour changes to faint pink. Incline the test tube and heat the upper part and a dense coagulum is formed.

Observation: Note the various changes occurring after completion of reaction. Result : According to observation write down the result whether test is positive or negative.

Experiment 4
Aim : To separate amino acids by thin layer chromatography. Reference : Sharma P.K., Dandiya P.C., Pharmaceutical Biochemistry, Vallabh Prakashan, 1st edition, 2006, 270-273, 275. Requirements : Glass slide , beaker Chemicals : Amino acids, n-propanol, Butanol, glacial acetic acid, ninhydrin reagent, silica gel. Solvent system : n-butanol : glacial acetic acid : water :: 12 : 5 : 3 Ninhydrin Solution : 250 mg ninhydrin in 100 ml acetone. Theory : Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose (blotter paper). This layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.

10 Procedure 1.Prepare slurry of stationary phase and apply on glass plate. 2. Activate the air dried plates by keeping in oven at 110o C for 1 hr. 3.Apply the sample using micropipette. Saturate the TLC chamber and place the glass plate into it and the solvent is allowed to run. 4. Plate is air dried and spray ninhydrin solution. 5. Heat the plates in oven at 110o C for 10 min.and calculate the Rf value. Calculation : Rf = Distance travelled by analyte from origin Distance traveled by solvent front from origin Result : Separate different amino acids according to their Rf values.

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Experiment 5
Aim : To separate amino acids by paper chromatography. Reference : Sharma P.K., Dandiya P.C., Pharmaceutical Biochemistry, Vallabh Prakashan, 1st edition, 2006, 270-273, 275. Requirements : Whatman filter paper no.1, beaker Chemicals : Amino acids, n-propanol, Butanol, glacial acetic acid, ninhydrin reagent, silica gel. Solvent system : n-butanol : glacial acetic acid : water :: 12 : 5 : 3 Ninhydrin Solution : 250 mg ninhydrin in 100 ml acetone. Theory : Paper chromatography is an analytical chemistry technique for separating and identifying mixtures that are or can be colored, especially pigments. This can also be used in secondary or primary colors in ink experiments. This method has been largely replaced by thin layer chromatography, however it is still a powerful teaching tool. Two-way paper chromatography, also called two-dimensional chromatography, involves using two solvents and rotating the paper 90 in between. This is useful for separating complex mixtures of similar compounds, for example, amino acids.

Procedure : 1.Apply the sample using micropipette. Saturate the chamber and place the paper into it and the solvent is allowed to run. 2. Paper is air dried and spray ninhydrin solution and calculate the Rf value.

12 Calculation : Rf = Distance travelled by analyte from origin Distance traveled by solvent front from origin Result : Separate different amino acids according to their Rf values.

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Experiment 6
Aim : To separate lipids by thin layer chromatography. Reference : Sharma P.K., Dandiya P.C., Pharmaceutical Biochemistry, Vallabh Prakashan, 1st edition, 2006, 278-279. Requirements : Glass slide , beaker Chemicals : Petroleum ether, diethyl ether, glacial acetic acid, silica gel, sulphuric acid (50%v/v) Solvent system : Petroleum ether : diethyl ether : glacial acetic acid :: 80 : 20 : 1 Theory : Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose (blotter paper). This layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.

14 Procedure: 1. Prepare slurry of stationary phase and apply on glass plate. 2. Activate the air dried plates by keeping in oven at 110o C for 1 hr. 3. Apply the sample using micropipette. Saturate TLC chamber and place the glass plate into it and the solvent is allowed to run. 4. Plate is air dried and spray sulphuric acid solution. 5. Heat the plates in oven at 110o C for 10 min.and calculate the Rf value. Calculation : Rf = Distance travelled by analyte from origin Distance traveled by solvent front from origin Result : Separate different amino acids according to their Rf values.

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Experiment -7
Aim : To determine casein in the milk. Reference : Sharma P.K., Dandiya P.C., Pharmaceutical Biochemistry, Vallabh Prakashan, 1st edition, 309,311. Requirements : Flask, beaker Chemicals : Acetic acid 10%, KOH 0.1N, HCl 0.1N, Phenolphthalein indicator Theory : Casein (from Latin caseus, "cheese") is the name for a family of related phosphoprotein proteins (S1, S2, , ). These proteins are commonly found in mammalian milk, making up 80% of the proteins in cow milk and between 60% and 65% of the proteins in human milk. Casein has a wide variety of uses, from being a major component of cheese, to use as a food additive, to a binder for safety matches. As a food source, casein supplies essential amino acids, carbohydrates and two inorganic elements, calcium and phosphorus. Procedure: 1. Take 10 ml milk in 200 ml flask and add 75 ml of distilled water and 1.0-1.5 ml of 10% acetic acid. 2. Mix content vigorously. Filter the ppt. and wash with cold water to remove acetic acid. 3. Transfer ppt. and paper in flask, add 75-80 ml of neutral water, 10 ml of 0.1N KOH and a few drops of phenolphthalein. 4. Stopper the flask and shake vigorously and titrate alkaline casein solution with 0.1N HCl until all red colour disappears. Calculation : Substract the corrected acid value from 10ml of alkali used to give % of casein in milk. % Casein = 10 (actual value + check value) Check value = 0.2 - 0.3 Result : Write down the % casein present in milk.

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Experiment 8
Aim : To estimate level of glucose in blood serum. Reference : Sharma P.K., Dandiya P.C., Pharmaceutical Biochemistry, Vallabh Prakashan, 1st edition, 2006, 294-295. Requirements : Beaker, test tube, syringe, Centrifuge, Colorimeter. Chemicals : Blood sample, glucose estimation kit. Theory : Enzymatic method yields maximum specificity for glucose estimation. Glucose can be measured by its reaction with glucose oxidase, in which gluconic acid and hydrogen peroxide are formed. Hydrogen peroxide than reacts with an oxygen acceptor, such as ortho-dianisidine, phenylamine-phenazone or any other chromogenic oxygen acceptors, in a reaction catalysed by peroxidase to form a colour. Procedure
Sr.No. Reagents Blank Standard Test tube

1. 2. 3. 4.

Glucose reagent Glucose standard Serum sample Distilled water

1 ml 0.01 ml

1 ml 0.01 ml -

I ml 0.01 ml -

Mix them and incubate at 370 C for 20 min. Read the absorbance of standard and test against blank at 540 nm. Calculation : Plasma glucose (mg/dl) = reading of test x 100 reading of standard Result : Write down the concentration of glucose in blood.

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Experiment 9
Aim : To perform the quantitative estimation of proteins in serum using Biuret method. Reference : Sharma P.K., Dandiya P.C., Pharmaceutical Biochemistry, Vallabh Prakashan, 1st edition, 2006, 296. Requirements : Beaker, test tube, syringe, Centrifuge, Colorimeter. Chemicals : Blood sample, protein estimation kit. Theory : Copper in alkaline solution reacts with peptide linkages of amino acids in protein producing a violet colour which is measured colorimetrically. Procedure : Sr. No. 1. 2. 3. 4. Reagents Biuret reagent Protein standard Serum sample Distilled water Blank 1 ml 0.01 ml Standard 1 ml 0.01 ml Test tube I ml 0.01 ml -

Mix them and incubate at 370 C for 10 min. Read the absorbance of standard and test against blank at 540 nm. Calculation : Serum Total Protein (g/dl) = reading of test x 6 reading of standard Result : Write down the concentration of proteins in serum.

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Experiment 10
Aim : To estimate SGOT level in serum. Reference : Sharma P.K., Dandiya P.C., Pharmaceutical Biochemistry, Vallabh Prakashan, 1st edition, 2006, 297. Requirements : Beaker, pipettes, test tube, syringe, Centrifuge, Colorimeter. Chemicals : SGOT estimation kit Theory : Serum aspartate aminotransferase (AST) also known as serum glutamic oxalacetic transaminase (SGOT) is a tissue enzyme that catalyzes the exchange of amino and keto groups between alpha amino acids and alpha-keto acids. AST is widely distributed in tissue principally cardiac hepatic muscle and kidney. Injury to these tissues results in the release of the AST (SGOT) enzyme to general circulation. AST catalyzes the following reaction. L-Aspartate + 2-Oxoglutarate ----- Oxalacetate + L-Glutamate In the present method a diazonium salt is used which selectively reacts with the oxalacetate to produce a color complex that is measured with colourimeter.. Procedure : Reagent Substrate reagent Deionised water Sample Standard 0.1 ml 0.1 ml 0.1 ml Blank 0.5 ml Standard 0.5 ml control 0.5 ml Test 0.5 ml

Mix and incubate at 370C for 60 min. SGOT colour reagent Sample 0.1ml 0.5 ml 0.5ml 0.5ml 0.5ml

19 Mix and incubate at 370C for 20 min. Add 3ml of alkali reagent to each test tube. Read the absorbance against distilled water at 505 nm. Calculation : SGOT (U/L) = Absorbance of test - Absorbance of std x Conc. of std (160 U/L) Absorbance of std - Absorbance of blank Result : Write down the SGOT level in serum.

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Experiment -11
Aim : To estimate SGPT level in serum. Reference : Sharma P.K., Dandiya P.C., Pharmaceutical Biochemistry, Vallabh Prakashan, 1st edition, 2006, 297. Requirements : Beaker, pipettes, test tube, syringe, Centrifuge, Colorimeter. Chemicals : SGPT estimation kit Theory : Alanine transaminase or ALT is a transaminase enzyme. It is also called serum glutamic pyruvic transaminase (SGPT) or alanine aminotransferase (ALAT). ALT is found in serum and in various bodily tissues, but is most commonly associated with the liver. It catalyzes the two parts of the alanine cycle. It catalyzes the transfer of an amino group from alanine to a-ketoglutarate, the products of this reversible transamination reaction being pyruvate and glutamate. glutamate + pyruvate -ketoglutarate + alanine Procedure : Reagent Substrate reagent Deionised water Sample Standard 0.1 ml 0.1 ml 0.1 ml Blank 0.5 ml Standard 0.5 ml control 0.5 ml Test 0.5 ml

Mix and incubate at 370C for 30 min. SGPT colour reagent Sample 0.1ml 0.5 ml 0.5ml 0.5ml 0.5ml

Mix and incubate at 370C for 20 min.

21 Add 3ml of alkali reagent to each test tube. Read the absorbance against distilled water at 505 nm.

Calculation : SGPT (U/L) = Absorbance of test - Absorbance of std x Conc. of std (160 U/L) Absorbance of std - Absorbance of blank

Result : Write down the SGPT level in serum.

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Experiment 12
Aim : To estimate Alkaline Phosphatase level in serum. Reference : Sharma P.K., Dandiya P.C., Pharmaceutical Biochemistry, Vallabh Prakashan, 1st edition, 2006, 299-301. Requirements : Beaker, pipettes, test tube, syringe, Centrifuge, Colorimeter. Chemicals : Alkaline Phosphatase estimation kit Theory : Alkaline phosphatase (ALP, ALKP) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. Procedure : Reagent Working buffered substrate Distilled water 1.5 ml 1.5ml 1.5ml 1.5ml Blank 0.5 ml Standard 0.5ml Control 0.5ml Test 0.5ml

Mix and incubate at 37C for 3 min. Serum Reagent 3 0.05 ml

-

0.05ml -

Mix well and incubate at 37C for 15 min. Reagent 2 Serum 1ml 1ml
-

1ml 0.05ml

1ml
-

Mix well and measure optical density of all the test tubes against distilled water at 510nm. Calculation : Serum Alkaline Phosphatase (KA units/ 100 ml) = Abs of test - Abs of std x 10 Abs of std Result : Write down the Alkaline Phosphatase level in serum.

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Experiment -13
Aim : To estimate Billirubin level in serum. Reference : Sharma P.K., Dandiya P.C., Pharmaceutical Biochemistry, Vallabh Prakashan, 1st edition, 2006, 298-299. Requirements : Beaker, pipettes, test tube, syringe, Centrifuge, Colorimeter. Chemicals : Billirubin estimation kit Theory : Bilirubin is the yellow breakdown product of normal heme catabolism. Heme is found in hemoglobin, a principal component of red blood cells. Bilirubin is excreted in bile and urine, and elevated levels may indicate certain diseases. It is responsible for the yellow color of bruises, the yellow color of urine (via its reduced breakdown product, urobilin), the brown color of faeces (via its conversion to stercobilin), and the yellow discoloration in jaundice. Procedure : Reagent Total bilirubin reagent Sodium nitrite reagent Distilled Water Sample 0.5 ml 0.5 ml 0.5 ml 0.5 ml Blank 1 ml Test 1 ml

Mix well reagents and wait for 30 sec before next addition. Then add 0.15 ml of serum sample to each test tube.mix well and incubate at 370C for 5 min and note the absorbance at 540nm against distilled water. Calculation : Total Billirubin (mg/dl) = Abs of test - Abs blank x 10 Abs of std Result : Write down the Total Billirubin level in serum.

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Experiment -14
Aim : To estimate Total Serum Cholesterol. Reference : Sharma P.K., Dandiya P.C., Pharmaceutical Biochemistry, Vallabh Prakashan, 1st edition, 2006, 301-302. Requirements : Beaker, pipettes, test tube, syringe, Centrifuge, Colorimeter. Chemicals : Cholesterol estimation kit Theory : Serum is treated with ferric chloride - acetic acid reagent to precipitate proteins. The protein free supernatant is treated with conc. sulphuric acid. A reddish purple colour is developed which is measured colorimetrically at 560nm0 Procedure : Reagent Cholesterol reagent Cholesterol Std Sample 0.1 ml 0.1s ml Blank 1 ml Standard 1 ml Test 1 ml

Mix well and incubate at 370C for 5 min and note the absorbance at 560nm. Calculation : Total Serum Cholesterol (mg/ 100ml) = Abs of test x 200 Abs of std Result : Write down the Total Serum Cholesterol.