Part I Lab Manual 2013
Part I Lab Manual 2013
Part I Lab Manual 2013
Department of Biochemistry
Preface
This edition is laboratory manual for first MBBS Biochemistry course. It provides medical students with a laboratory manual of basic biochemistry with a better understanding of the clinical aspect of diseases. Clinical biochemistry is an clinical chemistry with its clinical interpretion in disease detection, understanding cause of disease, decision making in therapeutic monitoring and diagnostics to facilitate the disease treatment and management. The Part I involves the chemistry and determination of chemical analyte levels in body fluids. The Part II involves estimations of metabolites, enzymes and examination of the results to use them in the diagnosis of diseases, screening for susceptibility to diseases and for monitoring the progress of treatment. This manual will enable the student to obtain a better understanding of the analytical techniques, instruments and reagents used in biochemistry and the clinical applications thereof. We are grateful to all the faculty members and staff of the department of Biochemistry for their valuable support and suggestions in the development of this practical manual.
CONTENTS
S. No. Topic 0 1. 2. 3. 4. 5. 6. 7. 8. 9. Brief review of Practical Biochemistry Basic Biochemistry laboratory: Pipetting, Safety principles Hydrogen ion concentration & preparation of buffers Properties of carbohydrate Tests for proteins -I Tests for proteins -II Chromatography Thin layer chromatography (TLC) Electrophoresis of serum proteins Enzyme kinetics Page No. 5 9 17 22 25 29 32 35 42 44 Teacher
Appendix:
1.
1. Multiplying the desired molarity of the solution by the gm. Mol wt. of the solute gives
the number of gms necessary to make 1 liter of solution of desired molarity e.g. Prepare 1 liter of 0.5 M NaCI. 0.5 x 58.5=29.25g. of NaCI in 1 liter of solution.
2. When a volume of solution other than 1 liter is desired the following formula may be
employed.
W = wt. of substance necessary to make 1 liter of desired molarity. V = 1 liter. W1= Wt of substance needed to make the desired volume of some molarity V1= Desired volume in ml. For Example, Make 1500 ml of 2 M NaCI;
W1 = 175.5g of NaCI.
3. Normality : A normal solution contains 1 gram equivalent weight of the solute in one litre of solution. 1 mole HCl, 0.5 mole H2SO4, 0.333 mole H3 PO4 in 1000 ml of solution in water are one Normal solutions. No. of moles x valency = No. of equivalents Molarity x valency = Normality The following equations define the expression of concentrations.
Since Eq. Wt. = Mol. Wt Valency 1. In case of monovalent compounds of elements M=N 2. When valencey is other than one. It must be multiplied by molarity to give the normality. N=M x valency e.g. Normality of 0.05 M H2SO4 N= 0.05 x 2 = 0.1 One milli equivalent (mEq) is 1/1000 of equivalent Mg mEq = --------------Gm. Eq.wt. To convert mg per 100 ml to mEq/liter:
Since,
So,
or
Example:
If serum sodium is 322 mg/100 ml [3220 mg/L]. Atomic wt. of Na = 23, Valency = 1 m Eq/L = (322 x 10 x 1) / 23 = 140 Sodium concentration in plasma is also expressed as mmol/L.
B. Dilution Problems
All the volumetric solutions contain a definite amount of solute in a fixed volume of solution. Whenever a solution is diluted its volume is increased and its concentration is decreased but total amount of solute remains unchanged. Dilutions are usually expressed as one unit of the original solution per total units of final solution. For eg 1:10 dilution required that one unit of concentrated solution be diluted to a total volume of 10 units. To calculate the conc. of a solution multiply by the dilution ratio. If several dilutions are made, multiply them together to arrive at final concentration. e.g. A 10% solution is diluted 1:5 (twice) then conc. of the diluted solution = 10%x1/5x1/5=0.4% Large dilutions can be carried out by doing series of dilutions.
C. Specific Gravity
Specific Gravity is useful in preparing normal solution of liquid. The specific gravity of a solution multiplied by its volume and again by the percentage of material in solution equals the weight of solute in solution. To prepare a normal solution of a liquid mL/liter for 1N= GMW Valency x Sp. gr x conc. (W/V)
To make stronger than 1 N, multiply by appropriate factor to make weaker than 1N dilute appropriately.
E. Problems
1. Make 200ml of 70% (V/V) alcohol form 95% alcohol. 2. How many gms. Of a salt would be required to make each of the following solution. a) 100ml of 10% V/V solution b) 500ml of 5 % (W/V) c) 50ml of 1% (W/V) 3. If 40 g. NaOH is diluted in 1 liter what is conc in terms of : a) Molarity b) Normality c) Percent (V/V) 4. A 1N solution of NaOH is diluted 5:25 then rediluted 3:100 what is the final normality? How many gms of NaOH are present in 100ml of the final solution. 5. Calculate the molarity of conc. HNO3 , Sp.gr. 1.42, conc. 70%
1. Volumetric Equipment
Most clinical chemistry procedure requires accurate measurement of volume. Volumetric equipment should be used with solutions equilibrated at room temp. For accurate work with pipettes and burettes, they should be rinsed out and drained well with some of the liquid to be measured. The filling of pipettes with poisonous corrosive of volatile liquids should be done using a rubber/other pipette aids. Pipettes: Defination: device used for the transfere of a fixed volume of liquid from one container to other They are caliberated to deliver the volume specified.Caliberated glass pipettsare available in various volumes ranging from 1 to 25 ml. TC (To Contain)to contain pipettes are used for volumes lesser than 0.5ml Types 1) Glass pipette are two types A) To Contain (TC)these are used for small volumes(less than 0.5ml).Eg RBC pipettes, WBC pipettes. These calibarations are such that they contain the specific volumes. B) To Deliver (TD)all these pipettes are caliberated to deliver a specific volume. The difference between TC and TD types of pipettes is that the former must be rinsed out after complete delivery in order to wash all the fluids contained in the pipette in to diluents. To Deliver (TD) are two types (1) Blowoutthese pipettes have volume graduation marking extend to the delivery tip of pipettes (Eg: serological pipettes) .Also called direct pipettes. (2) Non blowouthere graduations are not up to the tip. Also called indirect pipettes Othere types of pipettes: 2) Semiautomatic pipettes: These are available in fixed or variable volumes. A) Fixed volumes: These aspirate a fixed volume only. B) Variable volumes: Here we set a fixed volume, then aspirate the sample and deliver the volume. Automated pipettes: It has a digital display where aspiration and delivery of specific volume by means of motar delivery syrings. Pipetting techique1. Pipette must be held in vertical position during adjustment of liquid level to caliberation mark and during delivery
2. for colourless liquid lower meniseus to be read 3. for opaque liquid such as blood top of meniseus to be read. They are principally used for measurement of reagents.
TUTORIAL 1 WASTE DISPOSAL Biomedical waste means any waste, which is genarated during diagnosis, treatment or immunization of human beings or animals or in the research activities. Waste should be segregated at the point of generation and disposed in bags with correct coding. BLACK BAGS: (municipal dump) paper, peels, wrappers, kitchen waste. YELLOW BAGS: (for Incinerator) swabs and item contaminated with blood and body fluids, discarded medicines, gloves etc. BLUE BAGS: (for shredder) syringes, needles and sharps are destroyed in needle destrroyer or discarded in sharp disposal unit containg 1% bleach. All bags and containers must be labelled by using water proof ink or by self adhesive labels. Exercise: RECORD IN YOUR LAB MANNUALS
CONCENTRATION AND INTREPRETATION OF RESULTS -----------------------------------------------------------------------------------------------------------Tube No. Blank Std1 Std2 Std3 Std4 Std5 Test -----------------------------------------------------------------------------------------------------------Optical Density Corrected OD -----------------------------------------------------------------------------------------------------------Calculate the concentration in the test by using graph or formula. Interpretation of results ; Record in Lab mannuals Values obtained with a particular parameter is interpreted as: increased, decreased or within normal (reference) range. Reference values: Values obtained from individuals who are in good health as judged by other clinical and laboratory parameters, after suitable standardization and statistical analysis, under definite laboratory conditions. Normal (Reference) Range: Values within which 95% normal healthy persons fall. The cut off values are set as mean reference value +/- N times standard deviation, of a normal healthy population; where N varies between 1, 2 and 3. Causes/ Etiology of your findings Based on the result give diagnosis/differential diagnosis Any further investigation you would like to do to confirm diagnosis
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A. COLLECTION AND HANDLING OF SPECIMENS The different body fluids that are used for biochemical investigations are given below: Body Fluid WHOLE BLOOD Investigation Performed Blood gases Glucose Urea Enzymes Metabolites Electrolyte Enzymes Metabolites Electrolyte Method of Collection Obtained by arterial or venipuncture; collected with anticoagulants like heparin; Blood with anticoagulants centrifuged at 2000 rpm, the supernatant is plasma Blood collected in plain glass container, without any anticoagulant, centrifuged at 2000 rpm after clotting, the supernatant is serum Directly passed into a glass container, sometimes a catheter is introduced in the bladder
PLASMA
SERUM
URINE
Sugar Proteins Bile salts Pigments Blood steroids CEREBROSPINAL Sugar FLUID Protein Chloride GASTRIC JUICE HCl Blood SEROUS FLUIDS Proteins Serous space. SWEAT Chloride
Lumbar puncture from Subarachnoid space Aspiration by Ryles tube Needle puncture to the(e.g. Pleural, Peritoneal) Soaked into a filter paper
Anticoagulants
Chemical agents that prevent coagulation are routinely used when whole blood or plasma is required. Some of the commonly used anticoagulants are: (1) Heparin (2) Salts of Ethylene diamine tetra acetic acid (EDTA) (3) Oxalates (4) Sodium Fluoride Heparin: It may be considered to be a natural anticoagulant because it is already present in the blood, but in concentrations less than that required to prevent clotting in freshly drawn blood. Heparin prevents coagulation by increasing the activity of antithrombin III, an inhibitor of thrombin. This anticoagulant is used in a concentration of 0.2 mg / ml of blood and since its molecular weight is large, it produces no change in erythrocyte volume. Salts of Ethylene diamine tetracetic acid (EDTA): It is an anticoagulant which acts by virtue of removing calcium ions by chelation. A concentration of 2 mg of the disodium salt/ml of blood is sufficient. Concentrations even greater than this produce no detectable change in erythrocyte volume. Oxalates: Lithium, sodium and potassium oxalates act as anticoagulants by removing calcium ions essential for blood coagulation. Potassium oxalate (K2C204.H20) is commonly used. 1-2 mg of salt / ml of blood is required.
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The disadvantage of the use of oxalate is the alteration of concentrations of plasma components. Shrinkage of erythrocytes results from a water shift from the erythrocytes to plasma. This shift increases with increasing anticoagulant concentration, and if used in the same concentration on a weight basis, all anticoagulants will have this effect inversely proportional to their molecular weight. Aside from the water shift there may be alteration of erythrocyte permeability, which may explain the varied and inconsistent effects of oxalates and other salt anticoagulants on certain plasma constituents. Because of the difficulty, at times, in obtaining satisfactory preparation of heparin commercially, Heller and Paul introduced in 1934, a balanced oxalate mixture for use in hematocrit and sedimentation rate determinations. It consists of three parts by weight of ammonium oxalate, which causes swelling of the erythrocytes, balanced by two parts of potassium oxalate which causes shrinkage. NH4+ & K+ oxalate mixture in the ratio of 3:2, and 2 mg / ml of blood is the required amount. Sodium Fluoride: It is used when blood is collected for glucose estimations. In the erythrocytes (RBC), it specifically inhibits the enzyme enolase of the glycolytic pathway, preventing the consumption of glucose by the RBCs if blood is left standing at room temperature. Though it has a weak anticoagulant action, it is usually combined with another anticoagulant such as potassium oxalate.
Preservation of samples
Alteration in the concentration of a constituent in a stored specimen can result from various processes such as Adsorption on to the glass container Evaporation if the constituent is volatile Water shift due to the addition of anticoagulants Metabolic activities of the erythrocytes & leucocytes (accelerated by haemolysis) Inducing O2 consumption and CO2 production, hydrolysis, glycolysis and finally degradation. Microbial (fungal / bacterial) growth Changes in concentration of volatile substances such as O2 and CO2 are prevented or hindered by collection and storage of samples under anaerobic conditions. The problem of microbial growth appears when the sample is stored for longer than one day either at room or refrigerator temperature. This can be solved by four alternative courses of action: Collection and storage under sterile conditions Freezing of the sample Extreme alteration of pH Addition of an antibacterial agent. Lyophilized samples are stable with respect to many constituents for periods of at least as long as ten years. Samples can be stored at room temperature 18-37oC, refrigerator temperature (4oC) and frozen state (-10oC or lower). Except a few, the lower the temperature, greater is the stability. Further, microbial growth is considerably less at refrigerator temperature than at room temperature and is completely inhibited in the frozen state. Even in the frozen state, however, some components of plasma deteriorate.
Chemical Preservatives:
They can be classified into two groups: Prevention of chemical changes such as glycolysis Prevention of microbial growth.
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Combination of 10 mg Sodium fluoride + 1 mg Thymol / ml of blood. The presence of Thymol effectively controls microbial growth so that non-sterile specimens were stable for all determinations (except non-protein nitrogen) for at least two weeks. Monochlorobenzene and monobromobenzene have also been coupled with fluoride and have been claimed to be superior to thymol. Antibiotics can be used to prevent bacterial growth 1 mg of streptomycin base / 10 ml of blood has been used for preservation of blood for Haemoglobin and Urea determinations. The common preservatives for urine specimen are formaldehyde, thymol, toluene and chloroform. All these act primarily as antimicrobial agents. B. NORMAL VALUES AND INTERPRETATION OF RESULTS
Interpretation of results
Values obtained with a particular parameter is interpreted as: increased, decreased or within normal (reference) range. Reference values: Values obtained from individuals who are in good health as judged by other clinical and laboratory parameters, after suitable standardization and statistical analysis, under definite laboratory conditions. Normal (Reference) Range: Values within which 95% normal healthy persons fall. The cut off values are set as mean reference value +/- N times standard deviation, of a normal healthy population; where N varies between 1, 2 and 3. C. QUALITY CONTROL A major role of the clinical laboratory is the measurement of substances in body fluids or tissues for the purpose of diagnosis, treatment or prevention of disease, and for greater understanding of the disease process. To fulfill these aims the data generated has to be reliable for which strict quality control has to be maintained. Quality control is defined briefly as the study of those sources of variation, which are the responsibility of the laboratory, and the procedures used to recognize and minimize them. Accuracy has to do with how close the mean of a sufficiently large number of determinations on a sample is to the actual amount of substance present and is dependent on the methodology used. Precision refers to the extent to which repeated determination on an individual specimen vary using a particular technique and is dependent on how rigorously the methodology is followed. D. SAFETY Safety is each persons responsibility even in a small clinical laboratory. Even then every clinical laboratory must have a formal safety program. It is a good practice to assign a specific person the title of safety officer with the duties of administering the safety program and keeping it current. It should be ensured that laboratory environment meets the accepted safety standards. Which should include, but not be limited to attention to such items as: 1. Proper labelling of chemicals 2. Types and location of fire extinguishers 3. Hoods that are in good working condition 4. Proper working and grounding of electrical equipment 5. Providing means for proper handling and disposal of bio-hazardous materials including all patient specimen.
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Safety measures to avoid hazards To prevent chemical, electrical and biological hazards following precautions should be followed: 1. Proper storage and use of chemicals is necessary to avoid chemical hazards. Thus knowledge of the properties of chemicals in use and of proper handling procedures greatly reduces dangerous situations. 2. All the electrical equipment should be grounded using three-point plugs and use of the extension cord should be prohibited. 3. Every laboratory should have the necessary equipment to put out a fire in the laboratory, as well as to put out a fire on the clothing of an individual. Easy access to safety showers should be made. 4. Biological Hazards can be avoided by following precautions called universal precautions. All specimens should be treated as if they are potentially infectious. i. ii. iii. Avoid performing mouth pipetting and never blow out pipettes that contain potentially infectious material, for example serum. Do not mix potentially infectious material by bubbling air through the liquid, which leads to aerosol formation. Barrier protection such as gloves, mask and protective eyewear and gowns must be available and used when drawing blood from a patient. This includes removal and handling of all patient specimens. Disposable, non-sterile latex or vinyl gloves provide adequate protection. Wash hands whenever gloves are changed. Facial barrier protection should be used if there is a significant potential for the spattering of the blood or body fluid. Avoid re-using syringes and dispose off needles in rigid containers without touching these, using one-handed technique. Dispose off all sharp objects appropriately. Wear protective clothing, which serves as an effective barrier against potentially infective materials. When leaving the laboratory, protective clothing should be removed. Make a habit of keeping your hands away from your mouth, nose, eye and any other mucous membranes. This will reduce the possibility of infection. Minimize spills and spatters. Decontaminate all surfaces and reusable devices after use with appropriate registered hospital disinfectant. No warning labels are to be used on patient specimens. Before centrifuging tubes, inspect them for cracks. Inspect the inside of caps for signs of erosion or adhering matter. Be sure that rubber cushions are free from all bits of glass. Never leave a discarded tube or infected material unattended or unlabelled. Periodically clean out freezer and dry ice chests to remove broken ampoules and tubes of biological samples. Use rubber gloves and respiratory protection during this cleaning.
xiv. xv.
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3. REFERENCE INTERVAL Normal serum values of some common constituents both in conventional and SI units are given below: Blood Constituents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Protein (total) Albumin Bilirubin (conjugated) Bilirubin (total) Urea Creatinine Uric acid (male) (female) Cholesterol (total) HDL-Cholesterol Triglycerides Sodium Potassium Glucose (fasting) Calcium (total) Phosphorous ALT (SGPT) AST (SGOT) Phosphatase (alkaline) Phosphatase (acid) Amylase Conventional Units 6.7-8.6 g/dl 4.0-5.0 g/dl 0.1-0.4 mg/dl 0.3-1.4mg/dl 20.0-35.0 mg/dl 0.2-2.0 mg/dl 3.1-7.0 mg/dl 2.5-5.6 mg/dl 150.0-250.0 mg/dl 30.0-66.0 mg/dl 30-200 mg/dl 136.0-146.0 mEq/l 3.5-5.0 mEq/l 75.0-100.0 mg/dl 8.7-10.2 mg/dl 2.5-4.3 mg/dl 4.0-13.0 KA Units/dl 1.0-4.0 KA Units/dl SI Units 6.0-7.8 g/dl 3.5-5.0 g/dl upto 3.4mol/dl upto 0.2 mmol/l 3.0-36 mmol/l 17.0-117.0 mol/l 0.18-0.42 mmol/l 0.15-0.37 mmol/l 4.0-6.0. mmol/l 4.0-6.0 mmol/l 0.4-1.75 g/l 136.0-142.0 nnol/l 3.0-4.8 mmol/l 4.0-5.5 mmol/l 2.0-2.5 mmol/l 0.8-1.4 mmol/l 7-44 IU/L 12-38 IU/L 33 - 96 U/l upto 7.4 U/l
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Buffer: The H+ ion concentration varies very little in any biochemical fluid or environment. This variation is arrested by some bases or acids which respectively absorb or donate H+ ions depending on the situation. This phenomenon is called buffering. A buffer has the capacity of resisting the changes in pH (hydrogen ion concentration) of a solution after the addition of small amounts of an acid or an alkali. All weak acids or bases, in the presence of their salts with strong base or strong acid respectively form buffer systems, e.g. carbonic acid/bicarbonate, dihydrogen phosphate/ monohydrogen phosphate, proteins/proteinate. The capacity of the buffer decreases as the ratio deviates from 1. In general buffers should be used at a pH 1 from the pK. If the ratio is beyond 50:1 or 1:50, the system is considered to have lost its buffering capacity. The above mentioned buffers are present in different body fluids which help in the regulation of pH. Buffers are also used in clinical chemistry for enzymatic estimation. In aqueous solutions the pH ranges from 0 to 14. Molar concentration of H+ or OH- ion in pure water is 1 x I0-7mol/L. The ionic product is 1x10-14 mol2/L2. With water, at neutral point: [H+] = [OH-] = 10-7 mol/L. So pH at neutral point is, -log [10-7mol/L], So neutral pH= 7 pH more then 7 indicates that the solution is alkaline pH less than 7 is acidic. pH 0 would be given by 1M HCL.
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pH 14 would be given by a 1M NaOH. Many biochemical substances possess functional groups that are weak acids or weak bases e.g, -COOH, -NH2, phosphates present on proteins, nucleic acids, coenzymes, intermediary metabolites. Their dissociation behaviour influences pH and the structure and function of the entire molecule. The pH of a buffer solution can be calculated by Handerson Hasselbalch equation. (Salt) pH = pKa + Log .. (Acid) where pK = negative logarithm of the dissociation constant K for acid.
Determination of pH A. By using indicator: Indicators are compounds which change in colour with changes in the pH of the solution to which they are added. They are weak organic acids of weak bases. In unionized forms, the indicators show one colour while in their ionized forms (anions or cations) they have a different colour. The colour of a solution in presence of an indicator depends upon the relative proportions of ionized and the unionized forms of the indicator which in turn depends on the hydrogen ion concentration. For such indicator there is a definite pH range in which it is present as mixture of the ionized and unionized forms. In this specific range variations in pH of the solution will bring about visible change in the color of indicator. It is necessary that the effective pH range of the indicator includes the pH of the unknown sample.Do not let the pH paper dry before comparing with indicator. B. By using pH paper: The indicator paper given to you is accompanied by a color chart which shows different colors which the indicator exhibits at different pH values. Take a strip of indicator paper (Wide range) and moisten it with small drop of the solution whose ph is to be determined. Remove the excess fluid adhering to the indicator paper and compare with the color chart of the indicator and thus determine the pH of the solution. Now take a narrow range paper of suitable range and repeat the procedure to get the pH. C. By using universal indicator solution: Place 5.0 ml of the solution in a test tube and 0.1 ml of the universal indicator Mix well and find out the pH by referring to the color chart of the indicator. Determine the pH of biological fluids viz. urine, saliva. Record your observations. B. By using pH meter: The most accurate method for the routine measurement of pH is the pH meter in which a change in [H+] is measured as a change in electrical potential. If a metal rod is placed in a solution of its salts, it acquires potential. If two dissimilar metals are dipped into the solutions of their own salts, the difference in potential can be measured or calculated from the two separate potentials. A standard electrode is thus required against which the potential of all other electrodes can be compared. This is the standard hydrogen electrode, consisting of a platinum rod dipped in an aqueous solution with a given H+ activity in which Hydrogen gas is bubbled continuously at 1 atmosphere pressure. But as this is too cumbersome to be used as a reference electrode for routine use, other secondary reference electrodes of known potential in relation to the standard hydrogen electrodes are used.
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The most commonly used secondary reference electrode is the calomel electrode consisting of mercury-mercurous chloride in contact with a saturated solution of KCl. Its potential is pH independent. In the pH meter the most commonly used pH dependent unit is the glass electrode. Certain types of borosilicate glass are permeable to H+ but not to other cations and anions. Therefore, if a thin glass membrane separates two solutions of different pH, a potential difference is generated across the membrane, the magnitude of which is given by the equation log Where E = potential, R = gas constant, T = absolute temperature F = Faraday constant, [H+]i = concentration on the inside which is fixed (0.1N HCl), and [H+]o = concentration on the outside (test sample). The voltage measured by such a system is primarily the difference between that of the glass and the reference electrodes (other fixed potentials in the circuit also contribute) and it is linearly related to the pH of the test solution, i.e. [H+]o. The system therefore consists of the glass electrode in contact with the solution to be measured, the calomel reference electrode, a KCl bridge (the KCl should flow slowly into the sample) and the measuring device (meter). These are designed so that pH 7 gives a zero potential. High resistances are used so that very little current is drawn from the circuit (A large current flow could change the ion concentration). Certain precautions have to be observed while using a pH meter. The glass electrode is fragile and must be handled with care. It must not be left to dry. It is usually kept dipped in 3M KCl. The temperature compensation dial must be set before it is calibrated as potential produced is dependent on temperature. The meter must be calibrated first with a standard buffer pH 7, and then with a standard of pH 4 (if the test sample is expected to be acidic ) or with a standard of pH 9 or 10 ( if test is expected to be basic).
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2) Switch on the instrument and wait for 10 minutes. 3) Connect the cleaned electrode the proper terminals. 4) Adjust ZERO control to the pointer read 7 pH. pH measurement 1. Dip the electrode in a buffer of known pH value (usually 4 or 7) and set the TEMP COMPENSATE to the temperature of the Buffer. 2. Keep the SELECTOR in the proper pH range and adjust SET BUFFER to get buffer pH value. 3. Keep the SELECTOR in zero position, clean the electrodes and keep them in the sample. 4. Keep the SELECTOR in the proper pH range and read the value. 5. The electrode should be carefully washed after each pH determination.
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PRACTICAL EXERCISE 2:
Experiment: Preparation of Buffers Procedure: Prepare the following buffer solution: a) In acid range: (Acetate Buffer) Mix 9.2 ml 0.1 N acetic Acid +0.8 ml of 0.1 N. Sodium acetate and check the pH. b) In neutral range : (phosphate buffer) Mix 6 ml of 0.1 M Na2HPO4 + 4ml of 0.1 KH2 PO4 c) In alkaline range: (carbonate buffer) Mix 1.4 ml of 0.2 M sodium carbonate, 1.1 ml 0.2 M Sodium bicarbonate and 7.5 ml of water to get pH of 10. Determine the pH of the solution by one or more of the methods described below. Observation :
pH with wide range pH paper Acetate buffer Phosphate buffer Carbonate buffer
pH with pH meter
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starch iodine complex dissociate. On cooling, colour again appears due to re-formation of complex. Procedure: To 1ml of sugar solution, add a drop of dil. HCL to acdifiy the solution. Add a few drops of iodine solution. Mis and observe the colour. Gently warm the solution and then cool it. Note the change. Results: Tests of Carbohydrate: Carry out the above tests with given solution of carbohydrate and record your observations in the tabular form. Carbohydrate Test A). Molish Test B). Benedicts Test C). Barfoeds Test D). Seliwanoffs Test E). Iodine Test Carbohydrate Test A). Molish Test B). Benedicts Test C). Barfoeds Test D). Saliwanoffs Test E). Iodine Test Carbohydrate: Test A). Molish Test B). Benedicts Test C). Barfoeds Test D). Saliwanoffs Test E). Iodine Test Observation Inference Observation Inference Observation Inference
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Experiment 4
Experiment:
Principle: 1. Colour reactions of Proteins (I) Biuret test: This test is given by substances containing two or more peptide linkages in the molecule. Proteins respond positively since there are pairs of CO-NH groups in the molecule. This is a general test for the detection of proteins. When urea is heated to 180oC, it forms biuret, which in the presence of strong alkali reacts with dilute solutions of copper sulphate to form a violet coloured complex. Proteins and peptides give this reaction. Presence of at least two peptide bonds is essential for the reaction. Proteins give violet or purple colour while proteoses and peptones give light pink colour. Histidine gives a positive Biuret reaction. Two or more CO-NH groups joined directly or through a carbon or nitrogen atom gives this reaction. Absorption maximum of the colored complex is 540 nm. Since the method is based on reaction with peptide bonds, it is an absolute one. The main disadvantage is its lack of sensitivity. It cannot be used to estimate protein less than 1 mg/ml. Amino acids and dipeptides do not give this reaction. Procedure: Take 2 to 3 ml of protein solution in a test tube and add an equal volume of 10% sodium hydroxide. Mix thoroughly; add a few drops of 0.5% copper sulphate solution. A purplish violet colour is obtained. (II) Ninhydrin reaction: This test is given by all compound having free -amino groups e.g., peptide, protein and free amino acids ( except proline and hydroxy proline which are not amino acids but imino acids). Ninhydrins osidases an- amino acid to an aldehyde liberating NH3 and CO2 and is itself reduced to hydrindantin which then reacts with ammonia and another molecule of ninhydrin to form a purple coloured complex called Ruhemanns purple. Failure of purple colour development is indication of absence of -amino acid of group. Procedure: To 1ml protein solution and add 1ml of 0.02% freshly prepared ninhydrin solution .mix and boil. (III) Xanthoproteic reaction: Compounds containing benzenoid ring (e.g. Phenyl Group, C6H5) react with nitric acid to form yellow nitro derivatives which turn deeply coloured on addition of alkali. Only tyrosine and tryptophan respond to the test. Phenylalanine does not respond to the test since it is difficult to nitrate it under the above conditions. Procedure: To 2 to 3 ml of protein solution add 1 ml of concentrated nitric acid and heat. White precipitate is obtained which turns yellow and becomes orange on adding 40% sodium hydroxide (excess). (IV) Millon Test: This reaction is given by substances containing hydroxyl phenyl groups C6H4OH e.g. tyrosine. Procedure: To 5 ml of a dilute solution of protein, add 1 ml of 15% solution of mercuric sulphate in 6N H2SO4. Place the tube in boiling water bath for 10 minutes. Cool the contents in water for 5 to 10 minutes and add 1 ml of 1% sodium nitrite. A deep red colour indicates a positive test. Tyrosine answers this test.
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(V) Sakaguchis arginine reaction: Guanido group of arginine react with alpha napthol and alkali hypobromite to give a red coloured complex. Procedure: To 5 ml of protein solution, add 5 drops of 10% sodium hydroxide and 4 drops of Molischs reagent. Then add 10 drops of bromine water. A carmine red colour is obtained. Compare with blank. (VI) Hopkin-Cole reaction for tryptophan: This reaction is due to the presence of indole ring of the amino acid tryptophan in the protein molecule. Several aldehydes(e.g. formaldehyde) react with the oxidized product of the indole nucleus of trypotophan to give violet coloured complexes (Sulphuric acid with mercuric sulphate is used as oxidizing agent in this reaction). Procedure: To 1 ml of protein solution, add 1 drop of 1:500 formalin and then I drop of 10% mercuric sulphate in 10% suphuric acid. Mix well and gently add 3 ml of concentrated sulphuric acid along the sides of the test tube. Tap gently at the junction of the two layers. A violet ring develops at the junction. (VII) Sulphur test: Cysteine and Cystine amino acids contain sulphur and proteins containing these amino acids make precipitate with lead acetate in alkaline medium. To 3 ml of the protein solution, add an equal volume of 40% sodium hydroxide and heat for 2 minutes. Cool, and acidify with dilute HCI. Then add 1 ml of 5% lead acetate solution and warm. A grey or black precipitate indicates a positive test. Cysteine and cystine give positive test while methionine does not. Perform the colour reaction and precipitation with the given solution of protein and record your observation. Results: Test Observation Date: Inference
I) Biuret test II) Ninhydrin reaction III) Xanthoproteic test IV) Millon test V) Sakaguchis test VI) Hopkin-Cole test VII) Precipitation by heavy metals VIII) Precipitation by TCA
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Results: Precipitation Tests 1. Precipitation of proteins: (A) By zinc sulphate (B) By TCA Observation
Date Inference
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A.PAPER CHROMATOGAPHY
Paper chromatography is very simple and convenient method of separating a mixture of a number of constituents of similar nature but having different physical properties. The separation is carried out on pieces of filter paper and therefore, the technique is referred to as paper chromatography. In case of paper chromatography, a small quantity (2-25 ml) of the mixture to be analyzed is placed as a spot on a piece of filter paper and the solvent is allowed to travel up the paper (which is called ascending chromatography) or down the paper (in which case it is
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called descending chromatography). In this process, the mixture is separated into its individual components, which occupy different positions on the chromatogram depending on the distance traveled by each component. If the constituents are colored, the separation is immediately apparent. But in most of the cases, the chromatogram will have to be sprayed with or dipped in a reagent, which reacts with the compounds to be separated, to give color. Amino acids can be sprayed with Ninhydrin. The chromatogram on warming shows purple spots. In principle, the paper serves as an inert support for an aqueous stationary phase (which saturates the paper) over which flow a water immiscible organic solvent (the mobile phase). In such a system, the solutes undergo a continuous redistribution between the two phases. The position to which, a given solute moves up the paper, is therefore, largely dependent upon its partition co-efficient. Under standard conditions of solvent composition, temperature and type of paper, the distance traveled by each individual component of the mixture applied to the paper, is characteristic of that component, and is expressed by Rf (relative fraction) Rf = Distance traveled by a solute (center of spot) --------------------------------------------------------------Distance traveled by solvent (front)
In practice, known standards are run simultaneously on adjacent lanes so that by comparing the Rf values, the unknown spots can be identified. Difficulty of identification arises if the two components have the same Rf value. This can be solved by doing a two dimensional chromatography (separating along one direction and then perpendicularly).
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Experiment: IDENTIFICATION OF AMINO ACIDS BY PAPER CHROMATOGRAPHY MATERIALS REQUIRED: 1) 2) 3) 4) Whatman filter paper (no. 1). Standard amino acid solutions: (A) Cysteine (B) Glutamic acid (C) Leucine (D) A mixture of unknown amino acids. Development solvent: N-butanol: Acetic acid: Water = 4:1:2. Spraying reagent: 0.1% Ninhydrin in acetone or N-Butanol.
PROCEDURE: Take a piece of Whatman filter paper (No. 1; size about 5 x 15cm) and draw a horizontal line (using a pencil) about 2 cms away from the edge of the paper. Do not touch the paper with bare hands or make it dirty or wet. Keep the paper on a piece of ordinary filter paper or clean brown paper. Put 4 pencil marks along the line, about 1 cms, apart from each other. Apply one tiny drop (1-2l) of each of the amino acid solutions (at each point) with the help of a capillary tube at the first three points (only one amino acid on one point). Then apply the solution containing mixture of amino acids at the fourth point. Let the spots dry. Put the paper (chromatogram) in the chromatography chamber, which is already equilibrated with the solvent. Then place the solvent (mixture of n-butanol, acetic acid and water) in the chromatography chamber. Leave the chamber closed and allow the chromatogram to run, taking care to see that the solvent does not run off the paper. Take the paper chromatogram out of the chamber and allow it to dry in air, after marking with a pencil, the position upto which the solvent front has traveled. Spray the dried paper with ninhydrin solution and let it dry for few minutes. Keep the paper in the oven at 45oC for 20 minutes. Each amino acid will give a purple color. Spot mark each spot with pencil and compare the Rf values of standard amino acids and thereby identify the amino acids in the given mixture. INTERPRETATION: Paper chromatography is particularly well suited for identification of reducing sugars or amino acids excreted in urine. Paper chromatography also permits excretory products such as catecholamines and tryptophan metabolites to be separated from interfering substances so that they can be identified by specific color reactions. The renal threshold for amino acid is lowered in pregnancy, increasing the number of amino acids appearing in the chromatogram. Histidine is frequently seen; phenylalanine, lysine and tyrosine may also occur. Newborn babies have greater excretion of amino acids than adults and premature babies show marked renal aminoaciduria. The adult pattern is reached by six months. The pathological aminoacidurias have been classified into renal aminoaciduria and overflow aminoaciduria resulting from increased plasma levels. Typical of renal aminoacidurias are cystinuria in which a congenital defect leads to excretion of increased amount of the basic amino acids, cystine, lysine, ornithine and arginine, Fanconis syndrome, in which nearly all the common amino acids are excreted in excess, and the aminoaciduria due to tubule cell poisons such as heavy metals or Lysol. In overflow aminoaciduria, the plasma amino acid levels are high or normal depending on their thresholds, without primary disease. These may be acquired, secondary to other conditions. In cirrhosis and other chronic liver diseases, generalized aminoaciduria occurs. In malignant disease of bone and bone growth, hydroxy prolinuria occurs. They may also be caused by an inborn error of metabolism, congenital disorders due to enzyme defects e.g. phenylketonuria, alkaptonuria, maple syrup urine disease, etc.
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Advantages of TLC: Greater speed, better resolution, greater sensitivity and separation of hydrophobic substances viz. lipids, which are difficult to separate on paper. Compared to the various advantages, a few minor disadvantages have been found with TLC (1) Difficulty in recording and preserving (2) Greater cost of plates compared to paper. Experiment: SEPARATION OF SERUM LIPIDS BY TLC: PROCEDURE: (1) Glass plates are coated uniformly with a thin layer of absorbent (silcagel-G). The plates are generally dried and activated for a short time at 105 to 120oC. The sample is applied as a single spot with a micropipette in the form of a solution in a non-polar solvent. Selection of proper solvent depends on the type of separation desired. (2) After spotting the sample, the plate is placed vertically in the chromatographic tank, which has been saturated with solvent mixture. Two solvent systems are employed solvent system 1: n-Hexane; diethyl: ether: glacial acetic acid (60:4:1 vol/vol). Solvent 2: n-Hexane : Diethyl ether : Glacial acetic acid (90:10:1 vol/vol). The plates are first developed upto 7 cm height in solvent system 1, air-dried and subsequently developed upto 15 cm in solvent system 2. (3) After the run for a specific time (usually 2-4 hrs.), the plate is taken out, air-dried and spots outlined by exposure to iodine vapor. (4) Draw a diagram of the pattern obtained. Usually 6 spots can be visualized for phospholipids, diglyceride, free cholesterol, free fatty acid, triglyceride and esterified cholesterol.
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(ii)
(iii)
(iv)
Procedure: In this experiment, a mixture of three different molecules ranging in molecular size form 376 Da to 2000 KDa, is separated. (i) The column is fixed vertically to a stand. (ii) The column is equilibrated with 4 ml of gel filtration buffer and the buffer is allowed to drain completely. (iii) 0.2 ml of the sample is loaded on the column and allowed to sink in it. (iv) 0.2 ml of buffer is then added and allowed to flow out. (v) Buffer is then added to the column continuously to allow all the coloured biomolecules to elute out. (vi) The coloured fractions are collected in different tubes.
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Interpretation: (i) The colored component is blue dextran, which has a molecular weight of 2000KDa. These are very large molecules that exit fast from the column and are collected as the first fraction. (ii) The brownish red colored component is hemoglobin and has a molecular weight of 64500 Da. They are of intermediate size and require an average time to exit. (iii) The pink colored component is Vitamin B12 , having a molecular weight of 376Da. These are very small molecules that permeate the pores of the beads in the column and are retained for a longer time. They therefore, take a long time to exit the column and are collected as the third fraction.
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D.AFFINITY CHROMATOGRAPHY
Principle: It exploits the unique property of extremely specific biological interaction to achieve separation and purification. Theoretically, it is capable of going absolute purification, even from complex mixture in a single process. The performance of affinity chromatography is determine by comparing the specific activity of protein before and after purification. The material to be isolated is capable of binding reversibly to a specific ligand (example concavlin A for HRP, Heparin for lipoproteins) i.e. attached to an insoluble matrix. M + L Ligand ML Complex
Macromolecule Applications
1. Purification of enzymes example Horse Radish Peroxidase 2. Purification of Nucleotides, Nucleic acids example mRNA is routinely isolated by selective hybridization on poly (U) sepharose by exploiting its poly (A) tail 3. Isolation of membrane receptors 4. Purification of Immunoglobins 5. Separtion of whole cells, cell fragments Procedure 1. Set up conventional chromatographic column 2. A complex mixture containing the specific compound to be purified was added to the immobilized ligand 3. A compound will bind to the ligand, all other compound was washed away 4. The require compound was recovered by the displacement from the ligand 5. Estimation of proteins was done according to the any preferable method 6. Calculate specific activity Specific activity is defined as number of units of enzyme activity present per milligram of the protein. = Enzyme Activity Protein concentration mol/ min/mg
7.
Calculate enzyme activity One unit of enzyme activity is that amount of enzyme which transforms 1 micromole of substrate into product /min.
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E. ION-EXCHANGE CHROMATOGRAPHY
This form of chromatography relies on the attraction between oppositely charged particles. It is frequently chosen for the separation and purification of proteins, peptides, nucleic acids, polynucleotides and other charged molecules mainly because of its high resolution and capacity. Principle: The stationary phase consists of fixed charges on a solid support. These charges can be either negative or positive. Hence there are two types of ion-exchangers: cation and anion exchangers. Cation exchangers: possess negatively charged groups and will attract positively charged cations. These are also known as acidic ion-exchangers because their negative charges result from ionisation of their acidic groups. Strong cation exchange resins contaion sulphonic acid groups SO3-, while weak ones contain carboxylic acid groups COO-. Eg: Carboxymethyl cellulose. Anion exchangers: have positively charged groups that will attract negatively charged anions. They are also described as basic ion-exchangers as positive charges generally result from the association of protons with basic groups. Strong anion-exchange resins have N+(R1R2R3) and weak ones have N(R1R2). Eg: Diethylaminoethyl cellulose Ion-exchange mechanism consists of five distinct steps: 1. 2. i) Diffusion of the ion to the exchanger Diffusion of the ion through the matrix structure of the exchanger Dependent on the degree of cross-linkage of the exchanger and concentration of the solution.
ii) Controls the rate of the whole process. 3. 4. 5. Exchange of ions at the exchange site Diffusion of the exchanged ion through the exchanger to the surface. Selective desorption by the eluent and diffusion of the molecule into external eluent i) Selective desorption of the bound ion is achieved by changes in pH and/or ionic concentration. Procedure: 1) Solution containing the protein of interest (lysozyme in egg white) is applied to the ion-exchanger. 2) Protein binding to the ion-exchanger is dependent on the net charge on the protein which in turn is dependent on the pH of the solution and the ionic strength of the mobile phase. Lysozyme has a pI of 10.5. Hence, it is positively charged at pH below 10.5. At pH 7.0 (Phosphate buffer) it binds to the negatively charged cation exchanger (CM-cellulose). Wash buffer (pH 9.0) is used to remove proteins that have pI below 9.0.
3)
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4)
Bound protein is eluted out from the stationary phase by changing the pH or by increasing the concentration of cations, which compete with positively charged groups of lysozyme for binding sites on the column. Extent of purification is determined by estimating its specific activity before and after purification and quantitatively expressed as fold purification. Enzyme activity of lysozyme is determined using the bacterium Micrococcus luteus by estimating its specific activity. The initial suspension of bacteria is cloudy, absorbing light strongly at 450nm. As lysozyme breaks down the bacterial cell wall due to osmotic shock, the breakdown products dissolve and the solution becomes clearer.
5)
6)
One unit of lysozyme is defined as the amount of lysozyme that will produce a decrease in absorbance at 450nm of 0.001 absorbance units/minute. 7) Protein concentration of lysozyme is estimated by UV absorbance at 260nm and 280nm. 8) Estimation of Fold purification:
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The above mentioned equation shows that as the reaction proceeds, substrate concentration [S] will decrease and product concentration [P] will increase. In general, enzyme catalyzed reactions can be thought to proceed in two steps. In the first step, enzyme (E) and substrate (S) bind, reversibly to form an enzyme-substrate complex (E.S). In the second step the product (P) forms and is released from the enzyme, usually this step is irreversible, and E and P do not combine to give E.S, at an appreciable rate. This process is depicted more concisely, in Eq.1 above.
Enzyme Unit:
1 unit of enzyme activity is that amount of enzyme which transforms one micro mole of substrate into product per minute.
Turnover Number: It is the number of substrate molecules converted to product per molecule
of enzyme per minute.
The kinetics of many enzyme catalysed reactions is described by the MichaelisMenten equation, which is as follows:
Eq 2
Vo = Initial velocity of the reaction,
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[S] Km
= Concentration of the substrate expressed as moles/Litre. = Michaelis-Menten constant for the particular enzyme.
Let us consider the reaction represented by Eq.1 again. According to the law of mass action, as the concentration of the substrate is increased the reaction will shift towards right. A stage is reached where all the enzyme molecules will get saturated with the substrate and there will be no further increase in the velocity of the reaction on adding more and more substrate. The maximum velocity is then attained and is called the Vmax When a graph is plotted between [S] and the velocity of the reaction a rectangular hyperbola is obtained as shown below ( Fig.1).
An important numerical relationship that can be developed from Michaelis-Menten equation when Vo is exactly one half of Vmax is; Eq. 3
This represents a very useful and practical definition of Km : Km is defined as the substrate concentration at which the initial velocity (Vo) is half of maximal velocity (Vmax). It has the units of substrate concentration. We can rewrite the Michaelis-Menten equation to get a much more useful form of the curve called the Double Reciprocal or Lineweaver Burke Plot in the following way:
Eq. 4 If 1/Vo is plotted on Y axis and 1/[S] on the X-axis then eq.4 is an equation of a straight line with Km/Vmax as its slope and 1/Vo as Y-intercept. Since both Vo and S are plotted as
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their reciprocals on the Y and x-axes, respectively, it is called Double Reciprocal Curve (Lineweaver-Burk plot). When 1/[S] = 0, then 1/V0 = 1/ Vmax = AO in Fig 2. and when, 1/V0 = 0, 1/[S] = - 1/Km = BO in Fig 2
Double reciprocal presentation of has the great advantage of allowing a more accurate determination of Vmax which can only be approximated from a simple plot of Vo versus [S]. Double reciprocal plot offers an easy way of determining whether an enzyme inhibitor of competitive, non-competitive or mixed.
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Mixed Inhibition:
Mixed inhibition refers to a combination of two different types of reversible enzyme inhibition competitive inhibition and uncompetitive inhibition. The term 'mixed' is used when the inhibitor can bind to either the free enzyme or the enzyme-substrate complex. In mixed inhibition, the inhibitor binds to a site different from the active site where the substrate binds. Mixed inhibition results in a decrease in both Vmax and Km. A special kind of mixed inhibition where the affinity of the enzyme is same for both the substrate and the inhibitor is known as non competiive inhibition. In the special case where noncompetitive inhibition occurs, in which case Vmax is reduced but Km is unaffected. This is very unusual in practice.
Uncompetitive-inhibition:
Uncompetitive inhibition takes place when an enzyme inhibitor binds only to the complex formed between the enzyme and the substrate (the E-S complex) and inactivates it. This reduction in the effective concentration to the E-S complex increases the enzyme's apparent affinity for the substrate (Km is lowered) and decreases the maximum enzyme activity (Vmax), as it takes longer for the substrate or product to leave the active site. Uncompetitive inhibition works best when substrate concentration is high. An uncompetitive inhibitor need not resemble the substrate of the reaction it is inhibiting.
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