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St. Augustine
Practical and Information Manual for courses
MDSC 1001, MDSC 1101, MDSC 1102, OPTM 1062
Academic Year:
(2016-2017)
BIOCHEMISTRY UNIT
DEPARTMENT OF PRECLINICAL SCIENCES
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Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016
BIOCHEMISTRY UNIT
DEPARTMENT OF PRECLINICAL SCIENCES
FACULTY OF MEDICAL SCIENCE
Date (dd/mm/yy)
Course
Student Name
Student ID number
Experiment number :
Experiment name
Marks Obtained
Comments
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Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016
CONTENTS
PREFACE
INTRODUCTORY REMARKS
EXPERIMENT 1
LABORATORY TECHNIQUES
12
EXPERIMENT 2
19
EXPERIMENT 3
30
EXPERIMENT 4
REFERENCES
CHOLESTEROL DETERMINATION
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Biochemistry Unit
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PREFACE
Biochemistry will be taught by PBL as part of an integrated basic science
programme which is conducted over a period of two 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
There will be approximately eighty-five Biochemistry lectures over the
course of the first two years. The number 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 a session
about once every four weeks. Group assignments and the list of
experiments to be done will be posted on the class bulletin board and on
Myelearning at the beginning of each semester.
When no practicals are being conducted the Unit may utilize these sessions
for small group Biochemistry tutorials.
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Biochemistry Unit
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Examinations
Laboratory
practical
assignments
will
contribute
towards
students
INTRODUCTORY REMARKS
This booklet contains the protocols for the practical course, the revised
course outline and the Biochemistry booklist.
Practicals
The aims of the practical are threefold:
(i)
(ii)
setting
(iii) to introduce common biochemical techniques
As a consequence all students (refer to note on Exemptions) are expected
to perform all of the experiments as designated by the Biochemistry Unit
Coordinator.
Laboratory note books
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2015-2016
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 Units 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 and to answer all of the questions
in the treatment of results and write-up section associated with each
practical exercise.
Laboratory 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 unde r treatment of results and write-up section
found at the end of the description of each procedure. Reports must
consist of (i) a cover page (page two of this manual), (ii) a copy
of your signed and stamped results and (iii) the answers to the
questions given at the end of each lab.
The deadline for submission of each report will be advertised. Plagiarism
and other forms of cheating are treated very harshly; whether the source of
materials is the Internet or a colleagues report. If you are discovered plagiarizing you
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Biochemistry Unit
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will lose marks. For information regarding the university regulations on plagiarism
please
refer
to
http://sta.uwi.edu/resources/documents/Exam_Regulations_Plagiarism.pdf.
Preparation
Study the experimental procedures completely before your laboratory
session. You will be required to present flow charts and tables (for
recording your results), for each of the practicals, at the beginning
of the laboratory session. Try to understand what you are doing, and the
rationale for the experiment. Designing the flow charts and tables prior to
the laboratory session ensures that you study and think about the various
aspects of the experiment before you perform it.
(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)
(v)
Students must:
(i)
(ii)
(iii)
(iv)
(v)
(vi)
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.
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C.
D.
G.
H.
End-of-Experiment Rules
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(http://www.sanbenito.k12.tx.us/teachers/science_safety/Safety_And_Lab_Rules.html)
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.
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A.
i.
ii.
Centrifuge at 2300 rpm (750g) for 10 min. Carefully remove the tubes at
the end of the spin and immediately pour off the supernatants (S 1) into 2 new centrifuge
tubes. Do not transfer any of the loose fluffy layer on top of the pellet (P 1).
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 (4000g) 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.
iv.
Add 8.0 mL of cold sucrose to each of the P 2 pellets and resuspend with a
glass rod. Balance the tubes and centrifuge at 5400rpm (4000g) 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 recentrifugation.
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 (max).
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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.
iii.
iv.
Record the absorbances for the 2 sets of dilutions and for solution
O.
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.
conjugate base
pH = pKa + log10 conjugate acid
PROCEDURE
i.
pH Before
pH After
ii.
Measure the pH of vials 1 and 2. Record. Add 0.1 mL of 0.2M HCl (to vial
1), mix well, and record the pH. Add 0.1 mL of 0.2M NaOH to vial 2, mix well, and record
the pH.
iii.
Measure the pH of vial 3 and 4. Record. Add 0.1 mL of 0.2 M HCl (to vial
3), mix well, and record the pH. Add 0.1 mL of 0.2M NaOH to vial 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.
TREATMENT OF RESULTS AND WRITE-UP
(1)
(25 marks)
What is the role of 0.25M sucrose as the medium for the fractionation process?
(2 marks)
________________________________________________________________
________________________________________________________________
________________________________________________________________
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(2)
List the major components that are present in (a) pellet P 1, and (b) supernatant
S2.
(2 marks)
(a)______________________________________________________________
(b)______________________________________________________________
(3)
(i)
(2 marks)
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
(ii)
(1 mark)
________________________________________________________________
________________________________________________________________
(4)
cm-1. Use this value to calculate the concentration of solution O. (show your
(3 marks)
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(5)
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). Attach the graph to your report.
(4
marks)
(6)
(i) Tabulate the data for the pH measurements for all solutions in the space
below.
(2
marks)
(ii) State whether each of the test solutions did or did not exhibit buffering
capacity.
(2 marks)
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
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(7)
List the major buffer systems in the blood of mammals and show the equations
for each system?
(3 marks)
________________________________________________________________
________________________________________________________________
________________________________________________________________
EXPERIMENT 2
REACTIONS OF AMINO ACIDS AND PROTEINS
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This experiment illustrates some of the general reactions of amino acids and proteins
which are important in the study of protein chemistry.
(A)
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 amino acids, to decarboxylate the amino acid, producing a bluepurple 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 fluorescent
products.
PROCEDURE
Place 1 mL of neutral glycine solution (1g/L) in a test tube. Prepare six (6) 50%
serial dilutions of the neutral glycine solution (1g/L). Add five drops of Ninhydrin
solution #1 to all tubes. Place all tubes in a boiling water bath for 5 minutes to
develop the blue-purple reaction complex. 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).
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(B)
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(C)
PROCEDURE
i.
a.
b.
c.
d.
ii.
Place the tubes, except the one with ethanol, in a boiling water bath
for 5 minutes. Note the results.
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(20
(3 marks)
________________________________________________________________
________________________________________________________________
________________________________________________________________
(2)
(2 marks)
________________________________________________________________
________________________________________________________________
(3)
Plot a graph of your standard curve for the Folin-Lowry assay. Determine the
concentration of your two unknowns in mg/mL.
report.
(6 marks)
________________________________________________________________
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________________________________________________________________
________________________________________________________________
________________________________________________________________
(4)
Define what is meant by the denaturation of proteins, and state any two
(4 marks)
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________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
(5)
(4 marks)
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
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EXPERIMENT 3
(A) DEMONSTRATION OF ENZYME SPECIFICITY WITH GLUCOSE
OXIDASE AND PEROXIDASE AND THE DETERMINATION OF BLOOD
GLUCOSE LEVELS.
(B) THE CLASSIFICATION OF SUGARS AND THE DETERMINATION OF
GLUCOSE LEVELS IN URINE.
(A)
OXIDASE AND
PEROXIDASE
AND
THE
DETERMINATION
OF
-D-Glucose + H2O + O2
Peroxidase
2H2O + D
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experiment
the
concentration
of
be demonstrated.
Clinical significance of blood glucose
Normal ranges (plasma):
Fasting glucose:
60-100mg%
Post-prandial glucose:
90-140 mg%
Random glucose:
90-150 mg%
adrenal cortex.
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
PROCEDURE
The enzyme cocktail consist of:
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Glucose oxidase
12.5 mg
Peroxidase
4.0 mg
0.5 mL
100 mL
clear.
Assay
Perform the assay according to the following table.
Reagents
Glucose
(0.3 mM)
Supernata
nt
Lactose
(0.3 mM)
Fructose
(0.3 mM)
Reagent
blank
Distilled
water
Enzyme
cocktail
Tube (mL)
S4
S5
S6
0.3 0.4 0.5
B
-
S1
0.0
S2
0.1
S3
0.2
T1
-
T2
-
L
-
F
-
0.3
0.3
0.5
0.5
0.3
0.2
0.5
0.4
0.3
0.2
0.1
0.2
0.2
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
Fructose
Pipette 0.3 mL of supernatant to a clean dry semi-micro test tube and
add 0.2 mL distilled water (prepare in duplicate; T1 and T2).
standard curve by
Prepare a
enzyme
cocktail
to each of the eleven (11) tubes and incubate at 37C for 45 minutes. Mix by
vortex and read the absorbance at 450 nm.
Plot the standard curve of absorbance (y-axis) versus concentration of
glucose in mmol (x-axis).
concentration of glucose
(20 marks)
(6.0
marks)
(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.
Show
your
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Biochemistry Unit
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calculations in
(4.0 marks)
(3)
Name the two enzymatic methods which are commonly used in laboratories
to
(1.0 mark)
________________________________________________________________
________________________________________________________________
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Biochemistry Unit
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(4)
(1.0
mark)
________________________________________________________________
________________________________________________________________
Briefly describe the purpose of sodium fluoride in blood collection
tubes, where
(1.0 mark)
________________________________________________________________
________________________________________________________________
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(5)
(0.5 marks)
_________________________________________________________________
List three mechanisms by which this hormone regulates blood glucose.
(1.5 marks)
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
(6)
(1
mark)
_________________________________________________________________
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_________________________________________________________________
(3
marks)
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
(7)
(1 mark)
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
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(B)
in Urine.
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
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
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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.
(15
marks)
(1)
(7.0 marks)
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
(2)
Name the non-reducing sugar which does not give a positive Benedicts test and
explain why?
(2.0 marks)
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
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(3)
(2.0 marks)
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
(4)
(1.0 mark)
______________________________________________________________
______________________________________________________________
(5)
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(6)
(1.0 mark)
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
EXPERIMENT 4
Serum Lipids
Lipids are a very diverse group of biomolecules that carry out a wide range of
bodily function. They are insoluble in water and consequently need to be
transported in plasma in association with various lipoprotein particles.
Plasma lipoproteins can be separated into five major classes: chylomicrons,
very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL),
low-density lipoproteins (LDL), and high-density lipoproteins (HDL).
Each
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PROCEDURE
Reagents provided:
1.
2.
3.
4.
the blood to
pipette.
Standard
100L
Sample X
100L
mple
Enzyme
1 mL
1 mL
1 mL
cocktail
Vortex and incubate for 10 min at room temperature
Read absorbances at 500nm against the blank
The amount (mg/dL) of total cholesterol, triglycerides and HDLcholesterol in each sample can be calculated based on the change in
absorbance and the concentration of analyte in the standard.
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Sample calculation:
sample
A sample
A standard
standard =
mg/dL
(20
marks)
1.
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Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016
2.
of VLDL
below.
(3 marks)
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2015-2016
3.
List the lipoproteins which constitute a lipid profile and briefly discuss
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4.
List any four clinical conditions in which you may find high blood
cholesterol.
(2 marks)
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
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5.
(3 marks)
6.
(2
marks)
________________________________________________________________
________________________________________________________________
________________________________________________________________
REFERENCES:
1.
Chemistry and
2.
3.
Biochemistry:
An
Illustrated
Colour
Text,
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