Biochemistry Laboratory Manual-Combined 2012-2013
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Transcript of Biochemistry Laboratory Manual-Combined 2012-2013
1
BIOCHEMISTRY UNIT
Practical and Information Manual
For Medical Sciences Students
ACADEMIC YEAR
2012/2013
2
BIOCHEMISTRY UNIT LABORATORY PRACTICALS FOR MEDICAL SCIENCES PHASE I
2012/2013
3
CONTENTS PREFACE 4
INTRODUCTORY REMARKS 5
SAFETY AND LABORATORY RULES 7
EXPERIMENT 1 LABORATORY TECHNIQUES 11
EXPERIMENT 2 REACTIONS OF AMINO ACIDS AND PROTEINS 1
EXPERIMENT 3 (A) THIN LAYER CHROMATOGRAPHY OF SERUM LIPIDS 18 (B) SERUM PROTEIN ELCTROPHORESIS (DEMO) 22
EXPERIMENT 4
(A) DEMONSTRATION OF ENZYME SPECIFICITY WITH GLUCOSE OXIDASE AND PEROXIDASE AND THE DETERMINATION OF BLOOD GLUCOSE 26
(B) THE CLASSIFICATION OF SUGARS AND THE DETERMINATION OF
GLUCOSE IN URINE 30 EXPERIMENT 5 CHOLESTEROL DETERMINATION 32
THE BIOCHEMISTRY SYLLABUS
THE BIOCHEMISTRY BOOKLIST
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P R E F A C E
Biochemistry will be taught by PBL as part of an integrated science programme which is
conducted over a period of two and a half years. There will be one of these PBL sessions
per week each one lasting about three hours. In addition to this integrated approach, the
Biochemistry Unit will conduct teaching by the more traditional methods of lectures,
practicals and tutorials.
Lectures: The Unit will give approximately eighty-five lectures over the course of the
first two years. The number of lectures varies from one to four per week depending on the
Block.
Practical: The Unit will aim to conduct four Biochemistry practicals (two per Semester).
For all practical sessions the class will be divided into groups depending on the size of the
class and each group will have session every four weeks. Group assignments and the list of
experiments to be done will be posted on the class bulletin board at the beginning of each
semester.
When no practicals are being conducted the Unit may utilize these sessions for
small group Biochemistry tutorials.
EXAMINATIONS
All Biochemistry examinations will be conducted jointly with other disciplines of Basic
Sciences as part of the Problem Based Learning Program. Biochemistry questions may be
included in spotter examinations.
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INTRODUCTORY REMARKS
This booklet contains the protocols for the practical course, the revised course outline and the Biochemistry booklist. PRACTICALS STUDENTS WOULD NOT BE ALLOWED IN THE LABORATORY IF THEY DO NOT HAVE A LAB COAT OR PROPER FOOTWEAR! The aims of the practical are threefold: (i) to reinforce concepts introduced in the lecture course (ii) to introduce biochemical concepts best taught in a practical setting (iii) to introduce common biochemical techniques As a consequence all students (refer to Exemptions) are expected to perform all of the experiments as designated by the Biochemistry Unit Coordinator. NOTE BOOKS Before the first practical session all students must possess a hard cover laboratory notebook and a permanent marker (for labeling). Please note that you will only be doing four experiments so you do not require a very thick book. All results and working must be entered directly into your laboratory notebook in pen (no
pencils). Recordings made on scraps of paper or loose sheets will not be accepted. Please
ensure that the page containing your results receives the Biochemistry Unit’s stamp at the
end of each practical session. (This will be done by the demonstrator; it is your
responsibility to take your book to him/her).
Students will work in pairs but for each experiment each partner is expected to provide a full practical report or to answer all questions set as the case may be.
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PRACTICAL REPORTS Students will be required to submit short reports for marking. You will be expected to present and process your results, and answer relevant questions as outlined at the end of each procedure. Reports should be no longer than 2 – 3 pages consisting of answers to the questions outlined at the end of each practical. All the reports must contain a copy of your results sheet bearing the Unit’s stamp.
PREPARATION Get into the habit of reading the procedures completely before you come to the laboratory. Try to understand what you are doing, and the rationale for the experiment. You will be required to present flow charts and tables for recording your results for each part of the practicals where relevant at the beginning of the laboratory session. DEMONSTRATORS Demonstrators are in the lab to help you - so ask them questions when you have problems. CODE OF BEHAVIOUR Some of the equipment in the laboratory is sophisticated and delicate. Also some pieces of equipment e.g. centrifuges are potentially lethal if not handled properly. So STUDENTS MUST NEVER: (i) Enter the LAB in the absence of a lecturer or demonstrator. (ii) Attempt to use a piece of equipment until they are familiar with its mode of
operation. (iii) Attempt to use any equipment when it is not directly related to the experiment being
conducted. (iv) PLAY AROUND IN THE LAB. (v) Smoke in the LAB. (vi) Eat in the LAB. (vii) Use their mobile devices in the LAB STUDENTS MUST: (i) Report all accidents immediately – all minor spills should be cleaned up
immediately and MAJOR SPILLS should be reported to the LABORATORY SUPERVISOR.
(ii) Report all broken or faulty equipment (including glassware). (iii) Dispose of broken glassware, syringes and needles into specified bins. (iv) ALWAYS WEAR A LAB COAT WHEN IN THE LAB. (v) Wear safety glasses while working in the laboratory (not provided).
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SAFETY AND LABORATORY RULES
One of the first things any scientist learns is that working in the laboratory can be an exciting experience. However, the laboratory can also be quite dangerous if proper safety rules are not followed at all times. In order to prepare yourself for a safe year in the laboratory, read over and adhere to the following safety rules.
A. Dress Code
1. Laboratory coats should also be worn in the laboratory at all times. 2. Tie back long hair in order to keep it away from any chemicals, burners,
and candles, or other laboratory equipment. 3. Any article of clothing or jewelry that can hang down and touch chemicals
and flames should be removed or tied back before working in the laboratory. Sleeves should be rolled up.
4. Sandals, opened toe shoes are not allowed in the laboratory. 5. Some experiments will require that you wear safety goggles (safety
goggles will not be provided).
B. General Safety Rules
1. Read all directions for an experiment several times. Follow the directions exactly as they are written. If you are in doubt about any part of the experiment, ask the lecturer or the demonstrator for assistance.
2. Never perform activities that are not authorized by the lecturer or demonstrator.
3. Never handle any equipment unless you have specific permission. 4. Take extreme care not to spill any material in the laboratory. If spills
occur, ask the lecturer or demonstrator immediately about the proper clean-up procedure. Never simply pour chemicals or other substances into the sink or trash container.
5. Never eat or drink in the laboratory. Wash your hands before and after each experiment.
6. There should be no loud talking or horseplay in the laboratory. 7. When performing a lab, make sure the work area has been cleared of
purses, books, jackets, etc. 8. Know the location and use of all safety equipment (goggles, aprons,
eyewash, fire blanket, fire extinguishers, etc.) 9. Read your assignment before coming to class and be aware of all safety
precautions. Follow directions. 10. Never work alone in the lab.
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C. Heating and Fire Safety
1. Again, never use any heat source such as a candle or burner without wearing safety goggles.
2. Never heat any chemical that you are not instructed to heat. A chemical that is harmless when cool can be dangerous when heated.
3. Always maintain a clean work area and keep all materials away from flames. Never leave a flame unattended.
4. Never reach across a flame. 5. Make sure you know how to light a Bunsen burner. (Your lecturer will
demonstrate the proper procedure for lighting a burner.) If the flame leaps out of a burner towards you, turn the gas off immediately. Do not touch the burner as it may be hot.
6. Always point a test tube that is being heated away from you and others. Chemicals can splash or boil out of a heated test tube.
7. Never heat a liquid in a closed container. The expanding gases produced may blow the container apart, injuring you or others.
8. Never pick up any container that has been heated without first holding the back of your hand near it. If you can feel the heat on the back of your hand, the container may be too hot to handle. Always use a clamp or tongs when handling hot containers.
D. Using Chemicals Safely
1. Never mix chemicals for the "fun of it." You might produce a dangerous, possibly explosive substance. No unauthorized experiments should be performed.
2. Never touch, taste, or smell any chemical (unless instructed by lecturer). Many chemicals are poisonous. If you are instructed to note the fumes in an experiment, always gently wave your hand over the opening of a container and direct the fumes toward your nose. Do not inhale the fumes directly from the container.
3. Use only those chemicals needed in the activity. Keep all lids closed when a chemical is not being used. Notify the lecturer or demonstrator when chemicals are spilled.
4. Dispose of all chemicals as instructed by your lecturer. 5. Be extra careful when working with acids or bases. Pour such chemicals
over the sink, not over your work bench. 6. When diluting an acid, always pour the acid into water. Never pour water
into the acid. 7. Rinse any acids off your skin or clothing with water. Immediately notify
the lecturer or demonstrator of any acid spill. 8. Never pipette by mouth. 9. Be sure you use the correct chemical. Read the label twice. 10. Do not return any excess back to the reagent bottle. 11. Do not contaminate the chemical supply.
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12. Keep combustible materials away from open flames (alcohol, carbon disulfide, and acetone are combustible).
13. Do NOT use the same spatula to remove chemicals from two different containers. Each container should have a different spatula.
14. When you remove the stopper from a bottle, do NOT lay it down on the desk, but place the stopper between your two fingers and hold the bottle so the label is in the palm of your hand so drips won't ruin the label, etc. Both the bottle and the stopper will be held in one hand. Be sure and rinse any drips that might have gotten on the outside of the bottle.
15. Be careful not to interchange stoppers from two different containers 16. Replace all stoppers and caps on the bottle as soon as you finish using it. 17. Mercury spills must be cleaned up immediately. Alert the lecturer or
demonstrator if there is a spill. DO NOT touch the mercury.
E. Using Glassware Safely
1. Glass tubing should never be forced into a rubber stopper. A turning motion and lubricant will be helpful when inserting glass tubing into rubber stoppers or rubber tubing. Your lecturer will demonstrate the proper way to insert glass tubing.
2. When heating glassware, use a wire or ceramic screen to protect glassware from the flame of a Bunsen burner.
3. If you are instructed to cut glass tubing, always fire-polish the ends immediately to remove sharp edges.
4. Never use broken or chipped glassware. If glassware breaks, notify the lecturer or demonstrator and dispose of the glassware in the proper container.
5. Always thoroughly clean glassware before putting it away.
F. Using Sharp Instruments
1. Handle scalpels or razor blades with extreme care. Never cut any material towards you: always cut away from you.
2. Notify your lecturer or demonstrator immediately if you are cut in the laboratory.
G. Electrical Equipment Rules
1. Batteries should never be intentionally shorted. Severe burns can be caused by the heat generated in a bare copper wire placed directly across the battery terminals. If a mercury type dry cell is shorted, an explosion can result.
2. Turn off all power when setting up circuits or repairing electrical equipment.
3. Never use such metal articles as metal rulers, metal pencils or pens, nor wear rings, metal watchbands, bracelets, etc. when doing electrical work.
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4. When disconnecting a piece of electrical equipment, pull the plug and not the wire.
5. Use caution in handling electrical equipment which has been in use and has been disconnected. The equipment may still be hot enough to produce a serious burn.
6. Never connect, disconnect, or operate a piece of electrical equipment with wet hands or while standing on a wet floor.
H. End-of-Experiment Rules
1. When an experiment is completed, always clean up your work area and return all equipment to its proper place.
2. Wash your hands after every experiment. 3. Make sure all candles and burners are turned off before leaving the
laboratory. Check that the gas line leading to the burner is off as well.
I. Other Safety Rules
1. Do not use hair spray or hair mousse during or even before coming to laboratory class. These are highly flammable and might cause automatic ignition when in close proximity to a heat source.
(http://www.sanbenito.k12.tx.us/teachers/science_safety/Safety_And_Lab_Rules.html)
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EXPERIMENT 1
LABORATORY TECHNIQUES
The aim of this experiment is to allow students to become familiar with some basic
operations that are routinely performed in a biochemistry laboratory. The procedures include
centrifugation, volumetric measurements, dilutions, spectrophotometry, and pH
measurement. The concept of buffer action is also introduced.
A. Preparation of Mitochondria from Calf Liver
In biochemistry is it often necessary to isolate specific cell organelles for
investigation of particular biomolecules, enzymes, metabolic pathways etc. Such
separation or fractionation of cell components is accomplished by differential
centrifugation after the cells have been broken, by subjecting the tissue to one of
several cell-rupturing procedures.
PROCEDURE
A 10% (w/v) liver homogenate in 0.25M sucrose, 0.001M EDTA, pH7.2 has been
prepared for you.
i. Obtain approximately 20 ml of homogenate and add equal amounts into 2
plastic tubes for centrifugation. Make sure that the tubes are balanced and
counter poised when placed in the centrifuge.
ii. Centrifuge at 2300 rpm (750Χg) for 10 min. Carefully remove the tubes at
the end of the spin and immediately pour off the supernatants (S1) into 2
new centrifuge tubes. Do not transfer any of the loose fluffy layer on top
of the pellet (P1).
iii. Balance the tubes as in (i), above. You may use some sucrose medium to
add weight to one of the tubes. Centrifuge at 5400rpm (4000Χg) for 10
min. Pour off the supernatants (S2) into a single test tube and save. Note
the appearance of the pellets (P2) i.e. crude mitochondria.
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iv. Add 8.0 ml of cold sucrose to each of the P2 pellets and resuspend with a
glass rod. Balance the tubes and centrifuge at 5400rpm (4000Χg) for 10
min. discard the supernatants (S3). Note the appearance of the pellets (P3)
i.e. mitochondria.
N.B. Cleaner mitochondria may be obtained by further resuspensions
and re-centrifugation.
B. Spectrophotometry: Beer-Lambert Law.
The measurement of absorbance is the final step in many quantitative
determinations in the laboratory. The compound p-nitrophenol, in the
dissociated form i.e. alkaline conditions, absorbs in the visible range of the
EM spectrum with an absorption maximum centered at 405nm (λmax).
PROCEDURE
i. Collect approximately 10 ml of solution N, a 0.30 mM p-nitrophenol
solution and prepare a stock solution (O). To prepare O: Take 8 ml of
solution N and make up the volume to 40 ml using 0.02M NaOH.
ii. Using your stock solution O, prepare the dilutions X2 to Xs below using
graduated pipettes and repeat the dilutions using automatic pipettes.
Prepare between 2 ml to 5 ml of each dilution.
a. Χ2: a one in two dilution i.e. -1 part O: 1 part 0.02M NaOH
b. Χ3: one in three dilution i.e. -1 part O: 2 parts 0.02M NaOH
c. Χ4: one in four dilution i.e. - 1 part O: 3 parts 0.02M NaOH
d. Χ5: one in five dilution i.e. - 1 part O: 4 parts 0.02M NaOH
iii. Zero the spectrophotometer at 405nm using 0.02 M NaOH as the blank.
iv. Record the absorbances for the 2 sets of dilutions and for solution O.
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C. Buffer Action
In biological systems constant pH is essential for most cellular processes. The
maintenance of this narrow range of pH is accomplished by buffer systems, which
resist the changes in pH that would otherwise occur in metabolism. A buffer
solution is characterized by the pK value of the weak acid (base) and the ratio of
conjugate base to acid as shown by the Henderson-Hasselbalch equation.
pH = pK + log [conjugate base]
[conjugate acid]
PROCEDURE
i. Use the 4 vials supplied as follows:
Tubes 1 and 2 containing 10 ml each of distilled H2O and tubes 3 and 4
containing 10 ml each of any one of solutions A, B, C, or D ( these are already
measured for you) to be tested for buffering capacity.
Draw a table as shown and use it to record your results.
Tube pH Before pH After
1
2
3
4
ii. Measure the pH of tubes 1 and 2. Record. Add 0.1 ml of 0.2M HCl (to
tube 1), mix well, and record the pH. Add 0.1 ml of 0.2M NaOH to tube 2,
mix well, and record the pH.
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iii. Measure the pH of tube 3 and 4. Record. Add 0.1 ml of 0.2 M HCl (to
tube 3), mix well, and record the pH. Add 0.1 mL of 0.2M NaOH to tube
4, mix well, and record the pH.
iv. Obtain results for the three other solutions from other members of the
class (eg. if your solution was A, obtain results for solutions B,C and D).
The compositions of the test solutions are:
A. 500mL of 0.1M NaH2PO4 added to 500 mL of 0.1M Na2HPO4.
B. 500mL of 0.1M HCl added to 500 mL of 0.2M Na2HPO4.
C. 50mL of 0.1M acetic acid added to 950mL of 0.2M sodium acetate.
D. 50mL of 0.1M NaH2PO4 added to 50mL of 0.1M Na2HPO4 and diluted to
1L with H2O.
15
EXPERIMENT 2
REACTIONS OF AMINO ACIDS AND PROTEINS
This experiment illustrates some of the general reactions of amino acids and proteins
which are important in the study of protein chemistry.
Ninhydrin Reaction
The most common means of detecting amino acids is by reaction with Ninhydrin
(triketohydrindene hydrate). Ninhydrin, which is a very strong oxidizing agent, reacts
with α –amino acids, to decarboxylate the amino acid, producing a blue-purple
compound, CO2, H20 and an aldehyde. Proline gives a yellow colour. Under controlled
conditions, the reaction is used for quantitative estimation of amino acids, but is not as
sensitive as newer methods that yield flourescent products.
PROCEDURE
To 1 ml of neutral glycine solution (1g/L) in a test tube add five drops of Ninhydrin
solution #1. Place in a boiling water bath for 5 minutes to develop the blue-purple
reaction complex. Do x6 50% serial dilutions of the stock (1g/L) glycine solution and
repeat the test to determine the approximate limit of detection of this qualitative test.
For the quantitative test, pipette 0.70 ml each of 0.5mM solutions of glycine, tyrosine,
and proline in separate test tubes. Also set up a blank tube with 0.70ml of distilled H2O.
Add 0.8 ml of Ninhydrin solution #2, mix well, and place the tubes in a boiling water
bath for 15 minutes.
Cool the tubes and add 4ml of 50 % aqueous n-propanol to each tube. Mix and leave at
room temperature for 10 minutes.
Read the absorbance of the amino solutions against the blank at 570nm and at 440nm for
proline (you will have to blank the instrument at 570nm and 440nm).
16
Quantitative Determination of Protein
The Folin-Lowry assay is very sensitive and it is one of the most commonly used
procedures for determining protein concentration. The protein solution is first heated with
alkaline copper solution. Folin-Ciocalteu’s reagent contains phosphotungstic and
phosphomolybdic acids and produces a blue-green colour, the intensity of which depends
on the tyrosine and tryptophan residues in the protein.
I. Prepare a standard curve using 0.2, 0.4, 0.6, 0.8 and 1.0 ml of the standard protein
solution (20mg/100ml). Make up all volumes to 1.0ml with distilled H2O. Set up a
blank tube with 1.0ml of H2O. Into separate tubes pipette 1.0ml each of the two
unknowns.
II. Mix (just before using) 50ml of Reagent X (2% Na2CO3 in 0.1M NaOH) with
1.0ml of Reagent Y (0.5% CuSO4 in 1% sodium citrate).
III. Add 4 ml of the prepared solution to all tubes, mix and leave at room temperature
for exactly 10 minutes.
IV. Add 0.5 ml of Folin-Ciocalteu’s reagent to all tubes, mix well, and leave standing
at room temperature for 30 minutes.
V. Read the absorbances of all tubes against the blank at 750nm.
Protein Precipitation and Denaturation
A wide variety of chemicals, including certain organic acids, concentrated neutral salts,
heavy metal ions and certain organic solvents, can precipitate proteins. In many cases, the
precipitation results in irreversible denaturation of the protein.
i. With a small measuring cylinder, put approximately 3 ml of protein solution
(5mg/ml) into 4 test tubes. To each add only one of the following:
a. A few drops of 0.1M CuSO4.
b. 2ml of 10% trichloroacetic acid.
c. 2ml of saturated (NH4)2SO4 solution.
d. 10ml of cold ethanol.
ii. Place the tubes, except the one with ethanol, in a boiling water bath for 5 minutes.
Note the results.
17
TREATMENT AND WRITE UP OF RESULTS
1) What is the role of 0.25M sucrose as the medium for the fractionation process?
2) List the major components that are present in (a) pellet P1, and (b) supernatant S2.
3) Define the Beer-Lambert Law and briefly explain why Absorbance has no units.
4) Plot a graph of absorbance vs. concentration for both sets of dilutions (use the
concentrations calculated from your dilutions, do not use the concentrations obtained
by using Beer-Lambert law). Which dilution appears to be more accurate? Comment
on the spread of values.
5) Tabulate the data for the pH measurements for all solutions. State whether the test
solutions did or did not exhibit buffering capacity. Explain your observations based
on the composition of the solutions.
6) List the major buffer systems in the blood of mammals and show the equations for
each system?
7) Mention the importance of the Nihydrin test in clinical or research laboratory?
8) Plot a graph of your standard curve for the Folin-Lowry assay. Determine the
concentration of your two unknowns and report your answer in mg/ml.
9) Name the important chemical method available to determine total protein in the body
fluids
10) What is denaturation of proteins? Mention a few denaturing agents used in the
laboratory
11) What happens to the structural organization of protein when it is denatured?
12) Mention the importance of the denaturing process in the research laboratory
REFERENCE:
1. Tietz Textbook of Clinical Chemistry, Third Edition. Carl A. Burtis and Edward R.
Ashwood, eds. Philadelphia, PA: WB Saunders,
2. Manipal Manual of Clinical Biochemistry, Third edition. S Nayak, Jaypee medical
Publishers,
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EXPERIMENT 3
A. THIN LAYER CHROMATOGRAPHY OF SERUM LIPIDS
Along with proteins and nucleic acids, lipids form the other major macromolecule of
biological systems. There are many classes of lipids and lipids perform a diversity of
functions. One group of lipids, the triacylglycerides, constitute the major energy stores of
many organisms, while another group, the phospholipids, form the main component of
plasma membranes. Cholesterol, another important lipid, is also a component of membranes
and is the precursor of all of the steroid hormones.
The Lipids of serum can readily be separated into classes by thin layer chromatography. The
solvent used in this experiment separates fatty acids, triglycerides and cholesterol esters into
three distinct spots, but individual members of these three groups are not separated. Free
cholesterol runs separately; phospholipids remain on the base-line.
Serum is treated with a mixture of ether and ethanol, which strips the lipids from the
proteins to which they are bound in serum. The proteins are precipitated and the lipids
remain in the solvent. After filtration, the solution is evaporated to dryness, and the lipids
dissolved in a small volume of chloroform. This solution is used for thin layer
chromatography.
The spots are located with iodine vapour which dissolves in the lipid material giving a
brown colouration.
PROCEDURE
(1) Preparation of Serum: Obtain about 10 mL of whole blood from two members of
the class. Allow to clot for about 15 minutes in a centrifuge tube. Burst the fibrin clot
and centrifuge at 6000 r.p.m. for five minutes, then remove the serum (supernatant)
with a Pasteur pipette.
19
NOTE: Each side of the bench will do the following preparation and both groups
on that side will spot from the resulting lipid solution.
(2) Preparation of Serum Lipids: Pour 20 mL of a diethyl ether-ethanol mixture
(ethanol: diethyl ether - 3:1) into a 100 mL conical flask. Transfer in 1.5 mL serum
from a pipette. Cork and shake rapidly for 1 min. Allow to stand for 30 mins and
then filter through a fluted filter paper into a 50 mL conical flask or beaker.
NOTE: You can proceed to spot the marker solutions during this thirty- minute
wait.
Evaporate just to dryness in the fume cupboard on an electric hot plate or water bath
(no naked flames). Allow to cool and add 0.5 mL chloroform and swirl to dissolve
the lipids. Spot on the thin layer plate within a few minutes or the chloroform will
have evaporated. (See spotting instructions below).
(3) Preparation of the Tank: (one per 2 pairs of students). Make sure the lid is
properly greased and well fitted. Pour in the necessary quantity of solvent (hexane:
diethyl ether: glacial acetic acid, 80:20:1 by volume). Put two filter-paper liners into
the tank against one large wall, replace the lid, and tip the tank so that the lining
papers become well soaked in solvent. Return the tank to the vertical and leave to
equilibrate. The paper liners are necessary with this extremely volatile solvent, to
ensure saturation of the vapour phase.
(4) Spotting: Marker solutions of lipids in chloroform are provided as follows:
Cholesterol 4%, cholesterol oleate 2%, phospholipids 2%, olive oil 1% oleic acid
1%. The 20 cm square plates will have been coated with silica gel and dried at
110OC. Be very careful not to damage the solid layer, which is extremely fragile.
20
Place a plate flat on the bench. Prop up a ruler so that it passes over the plate without
touching it, and is parallel to and about 2 cm away from one of the edges, which is
fully coated with solid. Make a small scratch at each edge of the plate to mark the
position of the ruler, which will be the base line. Then make 8 small scratches at the
very bottom of the plate, so that they will be submerged in solvent during the run, but
will serve to indicate the position of the spots across the plate. These marks should be
2 cm apart, with the outer marks not less than 2 cm from the edges of the solid layer.
Keeping the ruler in position, apply the spots to the plate. Use a capillary pipette held
against the ruler, in the positions indicated by the scratches at the bottom of the plate.
Take great care to touch the plate very lightly with the capillary, or the silica will be
rubbed away. (A small hole in the solid at the point of application can be tolerated).
Keep the diameters of the spots to less than 5 mm. When applying several drops to
one spot, allow to dry between applications.
Apply spots as follows:
(1) Olive oil (0.5%) 50 µL
(2) Oleic acid (0.5%) 50 µL
(3) Serum extract 50 µL
(4) Serum extract 100 µL
(5) Serum extract 200 µL
(6) Cholesterol oleate (1%) 50 µL
(7) Cholesterol (2%) 50 µL
(8) Phospholipid (1%) 50 µL
(5) Chromatographic Development: Arrange one paper liner against each large wall
of the tank, and insert two plates, facing inward, with the glass of each plate resting
against the liner. Replace the lid quickly and allow to develop for 20 min. by which
time the solvent should have traveled about three quarters of the way up the plates.
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(6) Staining of Spots: Remove the plate, place horizontally and allow to dry. Stand the
plate in a dry tank containing iodine crystals, in the fume cupboard, for 15 min.
Remove the plate, place flat on the bench and at once mark the positions of the
spots by scratching their outlines with a pin. The spots fade rapidly as the iodine
evaporates, so work quickly and start with the fainter spots.
TREATMENT OF RESULTS AND WRITE UP
1. Calculate Rf values for the different lipid classes under the conditions of your
experiment.
2. Which groups of lipids are detectable in your serum?
3. Taking into consideration the apparent intensities of the spots, and the
concentrations of the marker solutions used, comment on the approximate amounts
of the different lipid classes found in serum.
4. What are some of the other biological functions of lipids?
22
B. SEPARATION OF SERUM PROTEINS BY AGAR GEL ELECTRO PHORESIS This is a very common technique used in clinical and research laboratories and is used for
the separation of charged particles. Biological materials such as aminoacids, peptides,
proteins, nucleic acids posses ionisable groups and hence exist as charged molecules in
solutions, either as cations (+vely charged) or anions (– vely charged) depending upon
the pH of the medium. Even typical non-polar substances such as carbohydrates can be
given charges by derivatisation, for example as borates or phosphates.
These charged particles move in an electric field. i.e. cations towards cathode (– vely
charged electrode) and anions towards anode (+vely charged electrode). So it is obvious
that the molecules having similar charges move in the same direction. But because of the
difference in their molecular mass the extent to which they move differs. Hence the
difference in Charge: Mass ratio (C/M) forms the basis for the differential migration of
particles in an applied electric field. And this forms the general principle of
electrophoresis.
Procedure: i) Slide preparation: About 1.3ml of warm (60°C) Agar solution (100mg/10ml of
barbitone buffer) is delivered through pipette on a slide uniformly at room
temperature and allowed to solidify.
ii) Chamber saturation: Twenty minutes before starting the experiment, both the buffer
tanks of the electrophoretic chamber (figure 2.5.1) are filled with equal volume of
barbitone buffer (pH-8.6) and kept closed to saturate the chamber with solvent
vapours.
23
iii) Slide placement and Wick connection: The slide prepared is kept in the chamber and
connected to buffer by means of filter paper strips (Wicks) as shown in the figure
2.5.1.
iv) Sample application: A small filter paper strip (Whatman No.3, 1x5 mm) soaked in
the serum sample is kept on the mounted slide perpendicular to the length of the
slide close towards the cathode and the chamber is closed.
v) Application of Electric field:
The electrophoretic chamber is connected to a power pack (equipment used to alter
or adjust the required current or voltage values). The power pack is switched on and
current is adjusted so that 3 mAmp current flows through each slide. The process
has to be carried out for about 90-120 minutes.
vi) Fixing of proteins: The slides are kept immersed in the absolute alcohol at 4°C for
about 30 minutes to prevent the diffusion of separated proteins.
vii) Staining: The slides are dried and kept immersed in the Amidoschwartz 10B dye for
about 2-3 minutes.
viii) Destaining: The stained slides are washed with 3% acetic acid to clear the
background and to see the protein bands clearly.
Finally the slides have to be dried. These slides can be preserved for a long time.
Since the electrophoretic pattern of serum proteins in certain diseases vary markedly
from a normal pattern it is of great diagnostic significance in several conditions like
nephrosis, liver disease, multiple myeloma, agamma-globulinemia and others.
24
25
RESULTS WRITE UP:
1. Give the reasons for: decreased serum albumin and increased alpha-2 macroglobulin in
nephrotic syndrome
2. What is multiple myeloma? Name the protein band appears during electrophoresis
3. Mention the different types of electrophoresis available to separate and identify proteins
4. Can we use this technique to separate hemoglobin? If yes in which conditions we can use
Reference: 1. Tietz Textbook of Clinical Chemistry,Carl A. Burtis and Edward R. Ashwood,
eds. Philadelphia, PA: WB Saunders,
2. Manipal Manual of Clinical Biochemistry, Third edition, Shivananda Nayak,
Jaypee Medical publishers, New Delhi,
26
EXPERIMENT 4
AIM:
I. DEMONSTRATION OF ENZYME SPECIFICITY WITH GLUCOSE OXIDASE
AND PEROXIDASE AND THE DETERMINATION OF BLOOD GLUCOSE LEVELS.
II. THE CLASSIFICATION OF SUGARS AND THE DETERMINATION OF
GLUCOSE LEVELS IN URINE.
Experiment I: Demonstration of Enzyme Specificity with Glucose Oxidase and Peroxidase.
PRINCIPLE :
Glucose oxidase specifically oxidizes β-D-glucopyranose to the lactone of gluconic acid in
the presence of oxygen.
Glucose oxidase
β-D-Glucose + H2O + O2 D-gluconic Acid + H2O2
When the enzyme peroxidase is included in the reaction mixture, the peroxidase substrate
can be oxidized by the "indicator reaction". The oxygen liberated oxidises a weakly
coloured hydrogen donor DH2 (O-dianisidine) to a coloured derivative, D.
H2O2 + DH2 Peroxidase
2H2O + D
The conditions of this reaction are so arranged that the oxidation of glucose go to
completion. Thus, by using these coupled enzyme reactions, the amount of the easily
measured compound, the dye, can be used to quantify the amount of
27
D-glucose oxidized, by direct proportionality. The absorption spectrum of the dye formed
from o-dianisidine has a broad peak centered around 450 nm.
In this experiment the concentration of glucose in blood will be determined. Also the
specificity of the enzyme will be demonstrated.
METHOD A : Determination of blood glucose using standard graph
REAGENTS:
The enzyme cocktail consist of:
Glucose oxidase 12.5 mg
Peroxidase 4.0 mg
O-dianisidine dihydrochloride (1% in ethanol) 0.5 ml
Add 0.5 M sodium phosphate buffer pH 7.2 to make 100 ml
PROCEDURE
Treatment of Blood sample:
Pipette 0.1 ml of blood into a centrifuge tube containing 1.5 ml distilled water. Add 0.2 ml
Ba (OH)2 solution (4.7%), mix well and then add 0.2 ml ZnSO4 solution (5%).
Prepare a reagent blank by pipetting 1.6 ml water into a centrifuge tube. Add 0.2 ml
Ba(OH)2 solution (4.7%), mix well and add 0.2 ml ZnSO4 solution (5%).
Mix thoroughly and centrifuge for 5 minutes at 500g. The supernatant should be clear.
Pipette 0.3 ml of supernatant to a clean dry semi-micro test tube add 0.2 ml distilled water
and finally 2.0 ml enzyme cocktail (prepare in duplicate T1 and T2). Incubate at 37°C along
with your standard curve samples.
Prepare a standard curve by pipetting 0.0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of the 0.3mM
glucose solution provided (do not use the 0.1M glucose solution) into clean dry semi-
micro test tubes. Add distilled water to make the final volume in each tube 0.5 ml. Add 2.0
ml enzyme cocktail to each tube and incubate at 37°C for 45 minutes. Along with your
samples to follow (see table provided).
28
Perform the experiment as follows: Reagents Tube (Volume in ml) B S1 S2 S3 S4 S5 S6 T1 T2 L F Glucose solution(0.3mM) 0.0 0.1 0.2 0.3 0.4 0.5
Supernatant 0.3 0.3
Lactose(0.3mM) 0.5
Fructose(0.3mM) 0.5
Reagent blank 0.3
Distilled water (ml) 0.2 0.5 0.4 0.3 0.2 0.1 0.2 0.2
Enzyme Cocktail 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Mix and incubate for 45 minutes at 37. C
Mix and Read the absorbance at 450 nm B= Blank T= Test, S= standard, L= Lactose, F=Fructose
Plot the standard curve of absorbance (Y axis) versus concentration of glucose in mmol
(x-axis).
Use the standard graph to determine the concentration of glucose in your sample
of blood in mmol/l then express your results in mg/dl
CLINICAL SIGNIFICANCE:
Normal ranges (plasma): Fasting glucose: 60-110mg%
Post-prandial glucose: 90-140 mg%
Random glucose: 90-150 mg%
• The increase in blood glucose is called as hyperglycemia.
• The blood glucose level is increased in uncontrolled diabetes mellitus.
• The increased levels are also seen due to hyperfunctions of anterior pituitary and adrenal
cortex.
29
• In hyperthyroidism, fasting blood sugar level may be normal but there is a pronounced
hyperglycemia in the fed state.
• The decrease in blood glucose is referred to as hypoglycemia (< 40 mg %).
• This condition is seen in insulin secreting tumors of the beta cells of pancreas.
• Occasionally it is encountered in renal diabetes.
• Over dosage Insulin also causes hypoglycemia
TREATMENT OF RESULTS AND WRITE UP
1. Plot the standard curve of absorbance (Y axis) versus concentration of glucose in mmol (x-axis).
2. Use the standard graph to determine the concentration of glucose in your sample of blood in mmol/l then express your results in mg/dl
3. Explain the term hyperglycemia with a specific example 4. List the chemical and enzymatic method(s) that are available for determination of
glucose in blood. 5. Why is an enzymatic method preferred for blood glucose estimation? 6. List any two enzymatic methods which are commonly used in laboratories of Trinidad
and Tobago to determine blood glucose 7. What happens if vacutainer does not contain fluoride? Mention the action of fluoride 8. Which hormone deficiency leads to diabetes mellitus? 9. How do you differentiate diabetes mellitus from diabetes insipidus? 10. What are the two types of diabetes mellitus? 11. List the three major clinical complications of diabetes mellitus 12. What is glycated hemoglobin and mention its clinical importance
30
Experiment II : The Classification of Sugars and the Determination of Glucose in Urine.
INTRODUCTION :
Carbohydrates may be classified as either reducing or non-reducing sugars. The reducing
sugars have a free aldehyde or ketone group or a potentially free aldehyde group as in the
hemi-acetal forms present in the cyclic molecule. These monosaccharides reduce alkaline
solutions of copper (Cu2+), with the formation of a coloured (usually brick-red) precipitate
of cuprous oxide (Cu2O). Benedict's solution contains cupric sulphate (CuSO4), sodium
carbonate (Na2CO3) and sodium citrate.
The Benedict's test is utilized in the detection and quantitation of monosaccharides, such as
glucose, in biological fluids e.g. urine. You may recall that patients suffering with diabetes
mellitus have abnormally high blood glucose levels, which may be detected in their urine.
PROCEDURE
Carry out Benedict's test on 0.1 M solutions of the glucose, fructose, lactose and sucrose
solutions provided. Examine the sensitivity of the test, in the case of glucose, by diluting the
0.1 M glucose solution provided to give solutions of concentrations 0.05 M, 0.025 M, 0.01
M, 0.001 M and 0.0001 M. Carry out Benedict's test on each of these diluted solutions.
Obtain a sample of urine and carry out the Benedict's test. Do not dilute the urine sample.
The Benedict's test
Pipette 2.0ml of Benedict's reagent into a test tube. Add 2.0ml of the test solution mix well
and place in a boiling water bath for 5 minutes. Let the tube cool slowly (do not place under
running water). A green, red or yellow precipitate is indicative of a positive reaction.
31
TREATMENT OF RESULTS AND WRITE UP
1. Record and briefly explain your results. 2. Name the nonreducing sugar which does not give a positive Benedict’s test and
why? 3. List the metabolic actions of insulin in regulating the blood glucose? 4. From the sensitivity test, can you estimate the threshold concentration of glucose
required to give a positive Benedicts test 5. What is the renal glycosuria and mention its causes 6. Give the renal threshold value for glucose? 7. What other substances (other than carbohydrates), which may be present in urine,
are able to reduce Benedict's reagent? 8. Explain the following with specific examples a) Gestational diabetes b) Spot test 9. What is the glucose tolerance test and list the significance of the test? 10. Write the brief procedure of oral glucose tolerance test (GTT) 11. Mention the conditions in which the oral GTT is replaced with intravenous 12. How do you differentiate normal GTT from abnormal GTT?
REFERENCES
1. Clinical Chemistry, 3rd Edition, Marshall, W.J., J.B. Lippincott and Company,
London. pp 167-179. 1993
2. Manipal Manual of Clinical Biochemistry, Third edition, Shivananda Nayak,
Jaypee Medical publishers,New Delhi
32
EEXXPPEERRII MM EENNTT 55
AAIIMM:: DDeetteerrmmiinnaattiioonn ooff SSeerruumm Cholesterol [Zak’s ferric chloride method]
NOTE: For this lab two volunteers are required to donate 5mL of blood each. Volunteers
are required to come to the lab for 1:40 pm
INTRODUCTION
Cholesterol is a very low water soluble lipid found in blood. It is one of the major
constituents of plasma lipoproteins and it exists in two forms; as free cholesterol and
esterified to some long-chain fatty acids, which enhances its hydrophobicity.
Cholesterol can be obtained from the diet or manufactured in the liver from acetyl CoA
by endogenous synthesis. It plays a number of important roles such as, being a major
structural component of cell surfaces and intracellular membranes and serving as a
precursor of the bile acids and steroid hormones.
PRINCIPLE Cholesterol in acetic acid reacts with ferric chloride and sulfuric acid to produce a red
color. The absorbance of the red color is read in a photoelectric colorimeter at 540 nm
(green filter).
The proteins in serum are precipitated with ferric chloride-acetic acid reagent. Equal
volumes of protein-free filtrate containing cholesterol, a standard and blank containing
ferric chloride-acetic acid reagent are separately treated with sulphuric acid and optical
densities are read.
Specimen: serum
33
REAGENTS:
1. Glacial acetic acid
2. Ferric chloride (0.05 %): Dissolve 50 mg of ferric chloride in 100 ml acetic acid.
Store in a brown bottle and it is stable for one month.
3. Sulfuric acid, AR grade
4. Stock cholesterol standard solution: 100 mg / 100 ml acetic acid, keep in a cool,
dark place and stable for one month.
5. Working cholesterol standard solution (0.1 mg/ml): dilute 10 ml stock solution to
100 ml with 0.05% ferric chloride reagent
PREPARATION OF SERUM
Obtain 5mL of whole blood each from 2 members of the class. Allow the blood to clot
for about 15 minutes in a centrifuge tube. Burst the fibrin clot and centrifuge at 6000
r.p.m. for 5 minutes, then remove the serum (supernatant) with a Pasteur pipette. This
will be carried out by a demonstrator.
PROCEDURE Treatment of Serum:
Pipette 0.1 ml of serum and 3.9 ml of ferric chloride-acetic acid reagent into a centrifuge
tube. Cover the mouth of the tube with a piece of Parafilm and mix well with a vortex
mixer. Let stand for 10 minutes for the proteins to flocculate. Centrifuge at 3000rpm for
10 minutes.
34
Perform the experiment as follows: Reagents Blank (B) Standard (S) Test ( T1) Test (T2) Supernatant (ml) 1.0 1.0 Ferric chloride-acetic acid 1.0 ml
Working standard (0.1 mg/ml) 1.0
Sulphuric acid (ml) 1.0 1.0 1.0 1.0
Mix and incubate for 20 minutes at room temperature Read the absorbance at 540 nm Note: Take necessary precautions when pipetting concentrated sulphuric acid CALCULATION:
dardSofAbsorbance
TestofAbsorbance
tan X 0.1 x 4 = mg of cholesterol in 0.1 ml serum
X 1000 = mg of cholesterol / 100 ml serum (mg/dl) CLINICAL SIGNIFICANCE: � Normal serum cholesterol ranges from 150- 220 mg%
� The increased level of cholesterol in serum is called hypercholesterolemia. This is
seen in: nephrotic syndrome, obstructive jaundice, myxoedema, glomerulonephritis,
coronary artery thrombosis and angina pectoris.
� Decreased level is called as hypocholesterolemia and seen in hyperthyroidism,
pernicious anemia, malabsorption syndrome, acute infections and hemolytic jaundice.
35
TREATMENT OF RESULTS AND WRITE UP
1. From the results, how much cholesterol was there in 100 ml of serum?
2. Express the results for cholesterol in molar terms.
3. Name the enzyme which helps in the esterification of cholesterol
4. List the tests under lipid profile and mention one clinical significance of its usage
5. Which sample is suitable for cholesterol determination?
6. What is the normal range of values for cholesterol concentration in serum or
plasma?
7. What fraction of cholesterol is esterified in blood?
8. Draw the formula of cholesterol and name the steroid ring present in it.
9. Apart from plasma, where is cholesterol found in the animal body?
10. List the clinical conditions in which we find high blood cholesterol
11. Which lipoprotein has high cholesterol content?
12. Name the bad and good cholesterol of the human body and give the reason for it
13. Mention the four conjugated bile acids which you find in the human bile
14. List the compounds formed from cholesterol in the human body?
15. Name the precursor and the regulatory enzyme of cholesterol biosynthesis
16. Mention the technique through which you can separate lipoproteins and which
lipoprotein contains high cholesterol
REFERENCES:
2. Manipal manual of Clinical Biochemistry, Third edition, Shivananda Nayak,
Jaypee Medical publishers.
3. Tietz Textbook of Clinical Chemistry, Carl A. Burtis and Edward R. Ashwood, eds.
Philadelphia, PA: WB Saunders,
36
THE BIOCHEMISTRY BOOKLIST NAME/AUTHOR PUBLISHERS Students Should Own 1. Harper's Biochemistry Prentice Hall Inter. Granner, Mayes, Murray, Rodwell (Latest Ed.) Recommended For Reference 1. Textbook of Biochemistry John Wiley & Sons Inc. with Clinical Correlations T M Devlin (latest edition) 2. Biochemistry by Diagrams Canoe Press E. Morrison 3. Biochemistry A Case Oriented Approach C V Mosby Conway, Montgomery & Spector (5th Ed.) (St Louis, 1990) 4. Biochemistry for the Medical Sciences John Wiley & Sons Inc E A Newsholme & A R Leach (1983) 5. Biochemistry W H Freeman L Stryer (5th Ed.) (1995)
6. Essentials of Biochemistry, Jaypee Medical publishers,
S Nayak New Delhi, India
7. Biochemistry Saunders College Publishers R.H. Garrett & C.M. Grisham (1999) Harcourt Brace College (2nd Ed.) 8. Lippincott’s Illustrated Reviews Biochemistry J.B. Lippincott Company Champe & R.A. Harvey (Latest Ed. Philadelphia) 9. Elements of Medical Genetics Churchill Livingstone A E Emery & R F Mueller (7th Ed.) (1988)
37
10. Biochemistry Illustrated Churchill Livingstone P N Campbell and A L Smith (1988) (2nd Ed.) 11. Membranes and Their Cellular Blackwell Scientific Function Publication J B Finean, R Coleman and R H Michel (3rd Ed. or later) 12. Biochemistry (Board Review Series) Harwal Publishing D B Marks (1994) 13. Immune Recognition IRL Press M J Owen (1988) 14. Metabolic and Nutritional Blackwell Scientific Diseases of Cattle Payne 15. Animal Nutrition Longman McDonald, Edwards & Greenhalgh (4th Ed.) 16. Recombinant DNA Scientific J Watson, M Gilman, American Books J Witkowski, M Zoller (2nd Ed.)(1993) 17. Genes VII Oxford University Press B Lewin 7th edition (January 2000) 18. Principles of Gene Manipulation Blackwell Publishers RW Old, SB Primrose 6th Edition 2002
DENTAL 16. Basic and Applied Dental Biochemistry Churchill Livingstone Williams & Elliot (4th Ed.) (1989)