BIOCHEMISTRY

SBIA022: PRACTICAL MANUAL 2022

Bioenergetics and Intermediary Metabolism

DEPT. BIOCHEMISTRY, MICROBIOLOGY, & BIOTECHNOLOGY

SCHOOL OF MOLECULAR AND LIFE SCIENCES
FACULTY OF SCIENCES AND AGRICULTURE
UNIVERSITY OF LIMPOPO

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TABLE OF CONTENT
Outline Information
General laboratory and practical rules and regulations
Guidelines for writing scientific reports
EXPERIMENTS
Exp. 1: Fermentation
Part A: Fermentation using various sugars and the influence of
certain inhibitors on fermentation.
Part A1: Fermentation of various sugars.
Part A2: Inhibitors of the Embden-Meyerhof pathway.
Part B: The formation of pyruvate and acetaldehyde during the
fermentation of glucose.
Part B1: Formation of pyruvate from glucose
Part B1.1: Sodium nitropruside test for pyruvate
Part B1.2: 2, 4-dinitrophenylhydrazine test for pyruvate
Part B2: Formation of acetaldehyde from glucose
Exp. 2: Isolation and characterization of Bacterial DNA/ RNA
Exp. 3: Ultraviolet absorbance of DNA – The hyperchromic effect
Exp. 4: Isolation of mitochondria from rat liver.
Part A: Isolation of mitochondria
Part B: Determination of protein
Exp. 5: Reactions of the citric acid cycle; succinate
dehydrogenase, Fumarase and malate dehydrogenase.
Part A: Recovery of mitochondria
Part B: Succinate dehydrogenase assay
Part C: Fumarase assay
Part D: Malate dehydrogenase assay
Practical Test

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 OUTLINE INFORMATION
Module Title Bioenergetics and Intermediary Metabolism
Module Code SBIA022
Department Biochemistry, Microbiology, and Biotechnology
Practical Mr. D.T. Maleka Office 1011 New science
instructors address lab 1st floor
Consultation Times Mon – Fri from 08:00 – 15:00
Preferably by prior arrangement
Practical session Friday 11:10 – 17:00
1st floor New science laboratory
E-mail address [email protected]
Telephone no. 2860
Semester 2nd Semester
Activities 5 Practical experiments
1 Practical test
Emergency no. CONTROL: (015) 268 3498/3521
MANKWENG
HOSPITAL: (015) 267 0330/286 1222
MANKWENG (015) 267 7827
CLINIC:
MANKWENG 0860010111
POLICE STATION:

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GENERAL LABORATORY AND PRACTICAL RULES

1. Students are expected to know and understand the whole experimental

procedure before the commencement of the practical session. Failure to
which, the student(s) will be asked to leave the laboratory immediately until
the next practical session and under these circumstances the student will
forfeit marks allocated to that particular practical.
2. Late coming is totally unacceptable.
3. No student is allowed to leave the laboratory without the permission of the
laboratory supervisor(s).
4. The usage of cell phones and/or other gadgets is prohibited in the laboratory,
unless otherwise allowed by the laboratory facilitator.
5. The medium of instruction in the laboratory is strictly ENGLISH.
6. Students are expected to meet deadlines set for submission of practical
reports failure to which the student will forfeit marks.
7. Always keep the level of your voices very low.
8. Always wear your laboratory coat.
9. Wear closed shoes and never walk barefooted.
10. Never eat, drink, smoke or apply cosmetics in any part of the laboratory.
11. Handle all specimen, chemicals and equipment with care.
12. Always cover any cuts, bites, open sores or wounds with an adhesive
dressing, preferably water-proof.
13. Always wash hands with water after handling all potentially dangerous
materials.
14. Clean any spillages immediately. Students are expected to clean their
working area before leaving the laboratory to avoid penalty.
15. Always label all reagents.
16. Pay careful attention to any safety precautions appearing on the label of a
chemical or reagent container.
17. Handle all radioactive material with absolute care.
18. Report any plumbing and/or electrical faults.
19. Always report any breakages of glassware and equipment immediately.

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 20. Do not remove any laboratory items, e.g: thermometers, magnetic fleas,
electrical appliances, etc., from the laboratory. Any student caught with these
items will be disciplined accordingly.
21. No caps and hats in the laboratory

PLEASE NOTE: Since the practical usually run for 4 to 6 hours, you are advised to
bring a packed lunch that can eat outside during waiting periods or allowed short
breaks.
Students will be required to sign out of the lab and back in on their arrival. If your
absence is noted during the practical and you are NOT signed out, you will be treated
as not having attended the practical at all. Any long absences noted will be dealt with
accordingly.
Submission of practical reports: On the day of submission, ensure that you sign a
submission register. This signature will serve as proof that you have submitted your
report for marking. If you fail to sign your report in, it may be assumed that the report
was never submitted.
Exclusion from practicals: Failure to attend a practical session, submit, or collect a
practical report on a scheduled date and time will only be excused on production of a
medical or death certificate. It is your responsibility to keep all your practical reports
safe until the final marks are processed at the end of the module. These will serve as
evidence should your marks be captured incorrectly on the system.

Complaint handling procedure:

 Address the problem with the lab assistant that marked your report.
 Should you not be satisfied, take the matter up with the senior lab instructors;
Mr.Maleka.
 Should you still not be satisfied, you may escalate the matter to the current
HOD; Prof.Matsebatlela.
 If you did not read or failed to follow the report writing method prescribed, refrain
from lodging ill-conceived complaints. Only students who can show that they
read the instructions, but are failing to understand them, are very much welcome
to seek assistance.

Any and all failure to follow the rules will be asked to leave the laboratory as they
are put into effect for your own safety.

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GUIDELINES FOR WRITING SCIENTIFIC REPORTS

A scientific report must consist of the following sections:

 A cover page which should include the student(s) details and affiliation, group
number, title of the report
 An abstract
 An introduction
 Materials and Methods
 Results
 Discussion
 References
Details of how each of how these sections should be structured follow:

An abstract
The abstract is defined as the summary of the information contained in the report or
document. The abstract usually does not exceed more than 250 words. The abstract
should enable the reader to identify the contents of the document/ report quickly and
accurately. The abstract should define clearly what is dealt with in the document or
report. The abstract should:
 State the principle objectives and scope of the investigation.
 Describe the methodology employed.
 Summarise the results
 State the principle conclusions
The conclusions must be stated three (3) times, in the abstract, in the introduction, and
in the conclusion. The abstract should always be the last to be written, because it
is a summary of the report or document. This does not mean it should be
presented at the end of the report. It must still be in the beginning of the report.

The Introduction
The introduction should supply sufficient background information to allow the reader to
understand and evaluate the results of the present study without the need to refer to
previous publications on the topic. A good introduction must comply with the following
rules:
 It should present, with clarity, the nature and scope of the problem investigated.

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  It should review the relevant literature, in order to orient the reader.
 The method of investigation must be stated.
 The principle results of the investigation should be briefly stated.
However, with specific reference to your experiments (iii) and (iv) must focus on the key
principles of each scientific technique introduced and the importance of your key
findings with respect to the information reviewed (ii)
Information source used/quoted in the introduction and discussion must be
properly references (see reference methods below).

Materials and Methods

The main purpose of this section is to provide enough detail so that a competent worker
can repeat the experiment. Great care should be taken in writing this section because
the cornerstone of the scientific writing method requires that your results must be
reproducible, and for the results to be considered reproducible, you must provide the
basis for repetition of the experiments by others.

 Materials
Materials should include the exact technical specifications and quantities, and source or
method of preparation. Where necessary, the chemical and physical properties of the
reagents used must be stated. Experimental animals, plants and microorganisms
should be identified (1) accurately by genus, species, and strain designations.
 Method
Methods must be presented in chronological order, and should be written out in 3 rd
person past tense. Related methods, however, should be described together.
Whenever necessary, subheadings must be used. When possible, subheadings should
be constructed to match those used in the results section.
 Measurements and analysis
Always state the conditions of incubation of your reactions; i.e. temperatures, and
periods of incubation. Questions such as “how?” and “how much?” should be
precisely answered.

Results
This section serves two objectives:
 First, an overall description of the experiments’ results should be given, taking
care not to repeat the details provided in materials and method.

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  Secondly, the data must be presented. The data can be presented in the form of
figures, graphs or tables which must each be properly labelled.
This means that data cannot just be presented without context; tables, figures and
graphs must be introduced. Legends must always appear at the bottom of your graphs
and figure, where labels must always appear at the top of your tables. The results must
always be presented with crystal clarity. Use the following guide when using tables and
diagrams:
 Contextualize the table/figure by mentioning it in your overall description of the
experiments’ results as mentioned above.
 The table/figure must be given an identity; e.g. Table 1 or Figure 1.
 The table/figure must have a heading/title/name.
 After the table, the keys used in the table must be explained; i.e. explain what +
or B or any shortened writing means.
 For figures, the identity number and heading must come after the figure; i.e. at
the bottom of the figure. E.g. draw the figure/diagram and then label it at the
bottom.

Discussion
The introduction and materials and method are designated to tell why and how you got
the results; the discussion is designated to tell what the results mean.
 Present the principles, relationships, and generalization shown by the results.
You should never recapitulate the results.
 Point out any exceptions or lack or correlation and define unsettled points.
 Show how your results and contrast or agree with previously published work.
 Discuss the theoretical implications of your work and possible practical
applications.
 State your conclusions as clearly as possible, where your evidence will be
summarized to elaborate your conclusive statement.

References
Where facts were obtained from a source, the source must be properly identified.
Different formats are used in referencing; you must use the following format:
 The name date system
 Where only one author is concerned;
E.g. Mbewe (1995) found that…

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Alternatively, state fact then reference,
…Mbewe (1995)
 Where one author published two or more articles in the same year, which are
both/all quoted. Either of the above approaches can be used but the years can
be distinguished using letters, e.g. …Mbewe(1995a). Another reference could be
made to another publication of the same year as follows, …Mbewe(1995b).
Where two or more authors are involved
E.g. Mbewe and Moganedi (1999) found that… Alternately state fact and reference as
follows; …Mbewe and Moganedi (1999).
 The issue of the same authors having published more than once in the same
year and their articles being used is handled as above using letters after the
year, i.e.
…Mbewe and Moganedi (1999a)
…Mbewe and Moganedi (1999b)
Where more than two authors are involved
E.g. Instead of …Mbewe, Moganedi, Crous and Howard (1999), you should write
…Mbewe et al. (1999)

 Listing references
The appropriate format must be used for listing references at the end of your
report. The list of references must appear in alphabetical order. The following
formats must be used depending on the nature of the source/reference.
o Journal articles: Howard, R.L. 1999. The incidence of salmonella
infections among the rural communities of Mamotintane. J. Bacteriol. 120:
23-35.
o Textbooks (One author): Ramasodi, L.M. 1996. Introduction to Medical
microbiology. 4th Ed. Prentice-Hall, Durban, S.A. Pp 45 – 65.
o Textbooks (more than author): Ramasodi, L.L, Mbewe, M, Moganedi,
D.B., and Howard, R.L. 1998. Introduction to Microbiology. 4 th Ed.
Prentice-Hall, Durban, S.A. Pp 45 – 65.

 The et al. notation must never be used when listing references; it is solely
for use in the body of the report.
o Textbooks with authors and editor(s):

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 Referencing a complete book: Van Rensburg, E.L., and Gama, A.D. (ed).
1999. The yeasts and Southern Africa. 2 nd ed. Prentice-Hall, Durban, S.A.
Pp 45-65.
Referencing a chapter or section in an edition: Mogashoa, M.M., and
Moganedi, D.B. 1998. The regulation of amylase genes in
Saccharomyces cerevisiae. In: Proceedings of the 5th International
Conference on Wine Yeasts Biotechnology. Johannesburg. October 25-
30. 1997. Vol 1. Pp. 45-65.

Please note: Different journals and institutions may use different methods of
referencing, the essence of all these formats is however the same. Deviations
from the above formats will not be tolerated and will be severely punishable.
Students are welcome to come to laboratory or facilitator’s office to seek
clarification. However, attention will not be given to students who believe they
deserve more marks, unless they can prove on the basis of this document that
the mark allocation deviates from this guide.

It is always recommended that you always read this guide each time you write
your scientific report. It is not easy to get it right but with practice you will master
the technique.

Mr. DT Maleka

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 EXPERIMENT 1:
METABOLISM: FERMENTATION

INTRODUCTION
Fermentation
The first stage of glucose metabolism in yeast involves the production of pyruvate.
This process, which takes place in a series of reactions with a large number of
intermediates, is known as fermentation or glycolysis. The overall reaction can be
represented as the transfer of two pairs of hydrogen atoms glucose to NAD + (fig.
1.1).

Since the coenzyme is present in only catalytic amounts, it must be deoxidized if the
process is to continue, and NADH + H+ is converted to NAD+ by reaction with
molecular oxygen via the respiratory chain. Under anaerobic conditions, this is no
longer possible and deoxidation takes place when pyruvate is converted to
acetaldehyde, then alcohol as shown (fig. 1.1).

H2O (Respiratory chain) aerobic ½O2

glycolysis
2 NAD + C6H12O6 2NADH2 + 2CH3COCOOH

Anaerobic
(alcohol dehydrogenase)
2C2H5OH 2CH3CHO + CO2

Fig.1.1 The oxidation of glucose in yeast under aerobic and anaerobic

conditions.

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Demonstration of metabolic intermediates
The metabolites, pyruvate and acetaldehyde are normally present at only low
concentrations, so, in order to demonstrate their existences add intermediates in the
pathway. Some means has to be found to prevent the reaction proceeding any
further. This is a general approach used widely in investigating metabolic pathways
and usually involved blocking the enzyme that catalyzes the conversion of the
compound under investigation, by adding an inhibitor.
Alternatively, the physiological conditions can be changed so that the enzyme
operated at well below its maximum activity, or a ‘trapping’ agent can be added
which reacts with the intermediate to form a compound that is not metabolized
further. These latter two approaches are illustrated in the following experiment.

Pyruvate decarboxylase is inactive in slightly alkaline solution, so pyruvate

accumulates and its presence is demonstrated by the reaction with sodium
nitropruside or 2, 4-dinitrophenylhydrazine.

In the second experiment, sodium sulphate is added to the incubation mixture which
‘traps’ the acetaldehyde. The presence of acetaldehyde is shown by the blue colour
produced with sodium nitropruside and piperidine.

EXPERIMENTAL PROCEDURE

MATERIALS
1. Disodium hydrogen phosphate (0.5 mol/1)
2. Potassium dihydrogen phosphate (0.5 mol/1)
3. Yeast suspension (100 g/l in Na2HPO4)
4. Yeast suspension (100 g/l in KH2PO4)
5. Yeast suspension (100 g/l in water)
6. 5% (m/v) of each of the following sugars: glucose, fructose, sucrose, lactose
and galactose
7. Sodium nitropruside (50 g/l, prepare just before use)
8. Ammonium sulphate
9. Sodium sulphite
10. Sodium hydroxide

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11 2, 4 Dinitrophenylhydrazine (saturated solution in 2 mol/l hydrochloric acid)
12. Piperidine (30 g/l aqueous solution)
13. Trichloroacetic acid (100g/l)
14. Water bath
15. 0.02 M Iodoacetic acid
16. 1.0 M Sodium fluoride
17. 1% Sodium metabisulphite
18. Centrifuge tubers

METHODS

PART A: FERMENTATION OF VARIOUS SUGARS AND INFLUENCE OF

CERTAIN INHIBITORS ON FERMENTATION

PART A1: Fermentation of various sugars

1. Add 1ml of an aqueous suspension of yeast cells to 30 ml of a 5% (m/v) solution
of glucose and stir well.
2. Fill small (10 x 75 mm) test tube with the yeast and sugar mixture and use the
remaining portion of the mixture to fill a large test tube.
3. Close the small test tube with a finger, invert the test tube and, working over a
sink, introduce it into the large test tube avoiding the trapping of an air bubble.
4. Pour off the upper cm of yeast suspension and place the test tube in a water bath
maintained at 370C.
5. In a similar manner, prepare test tubes containing yeast suspended in 5%
solutions of fructose, galactose, sucrose and lactose.
6. Incubate the reaction mixture until one of the small test tubes (which serves as a
micro gas jar) is full of gas and note the time taken to fill the test tube. Measure
the height of the gas bubble in each of the other tubes. Taking the rate of gas
evolution from the suspension which first gave a full tube of gas as 100, estimate
the relative rates of fermentation of the other sugars.

NB: In order to test the identity of the gas that has accumulated, carefully
remove the small test tube with the most gas and, keeping it inverted; lower it
into a shallow layer (2-3ml) of saturated Ba (OH) 2 in a 100 ml beaker.

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Note what happens:
(ii) What gas is formed? Explain with suitable reaction equations.
(ii) Record the relative rates of fermentation of the sugars tested.
(iii) Explain your results as fully as possible.

PART A2: Inhibitors of the Embden-Meyerhof pathway

1. As described above pare 3 further tubes containing yeast-glucose suspensions to
which are added respectively:
(i) 1 ml of 0, 02 M Iodoacetic acid solution (MW = 185.75)
(ii) 1 ml of 1.0 M sodium fluoride solution) MW = 41.99)
(iii) 1 ml of a 1% of sodium metabisulphite (Na2S2O5. MW=190.1 which, in
aqueous solution is converted to the bisulphates (NaHS3).
2. Incubate these reaction mixtures together with those set up for experiment 1, and
record the height of the gas (if any) collected in the small test tube. Compare the
rate of fermentation with that of the yeast-glucose suspension without inhibitor.

Tabulate your data and explain your observations.

Calculate how 10 ml of each of the reagents used in this experiment were prepared.

PART B: FORMATION OF PYRUVATE AND ACETALDEHYDE DURING

FERMENTATION OF GLUCOSE

PART B1: Formation of pyruvate from glucose

1. Pipette 5 ml of glucose solution into two boiling tubes (A and B).
2. Add 5 ml of the yeast suspension in the slightly alkaline solution of NaHPO4 to
tube A and 5ml of the yeast suspension in the acid solution of KH2PO4 to tube B.
3. Place the tubes in a water bath at 370C for 1 hr then add 2 ml of trichloroacetic
acid solution to each tube, mix thoroughly, and centrifuge for 10 min at 2 500 x g.
4. Remove the supernatant and test for the presence of pyruvate.

Sodium nitropruside test for pyruvate

1. Add 2ml of boiled supernatant to about 1 cm of solid ammonium sulphate in a
test tube.

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2. Add two drops of freshly prepared sodium nitropruside solution (50 g/l),
thoroughly, and run concentrated ammonia carefully down the side of the tube so
as to form two layers. If pyruvate is present, a green or blue ring forms at the
junction of the two liquids. A transient pink ring at the junction is due to the
presence of thiol groups and this sometimes appears before the
characteristic blue or green colour given by pyruvate.

2, 4-dinitrophenylhydrazine test for pyruvate

1. Add 1 ml of the saturated 2, 4-dinitrophenylhydrazine solution to 2 ml of the
supernatant, mix thoroughly.
2. Place two or three drops of the mixture in a test tube.
3. Add 1 ml of the sodium hydroxide solution (100 g/l).
4. Dilute with water to about 5 ml. A red colour forms if pyruvate is present.

PART B2: Formation of acetaldehyde from glucose

1. Pipette 5 ml of the glucose solution into two tubes C and D.
2. Add 5 ml of the yeast suspension in water to both tubes and 0.5 g of sodium
sulphate to tube D.
3. Mix thoroughly and incubate the two tubes at 37 0C for 1hr.
4. Centrifuge the tube, and add 0,5 ml of freshly prepared sodium nitropruside to
2ml of the supernatant, followed by 2ml of aqueous piperidine and mix. If
acetaldehyde is present then a blue colour is observed.

CALCULATIONS
Calculate the molarities of the 50 g/l sodium nitropruside (MW=298) and 100 g/l
sodium hydroxide (MW=40) solutions respectively.

RESULTS
1. Describe and explain your observations.
2. Answer the following questions:
2.1 During the formation of pyruvate from glucose, what happens to the four
hydrogen atoms released in this reaction?
2.2 Explain why and how NAD+ is recycled under aerobic and anaerobic
conditions respectively.

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 2.3 Name three ways in which glycolysis can be stopped in vitro to study
the metabolic intermediates.

REFERENCE
Plummer, D.T., an Introduction to Practical Biochemistry, McGraw-Hill Book
Company, 1978, pp. 311-313.

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 EXPERIMENT 2:
ISOLATION AND CHARACTERIZATION OF BACTERIAL DNA

INTRODUCTION
In this experiment, DNA is isolated from lyophilized E.coli cells. The bacterial cell
wall is disrupted by EDTA treatment in the absence of lysozyme. Sphaeroplasts,
which are produced during EDTA treatment, are lysed with sodium dodecyl sulphate
(SDS), an anionic detergent which binds strongly to proteins. The detergents also

denature proteins. In addition, protein denaturation is promoted at 60 o C during this

treatment. NaClO treatment in the experimental protocol weakens DNA-protein

interactions and consequently aids in the dissociation of protein bound to DNA. A
chloroform-isoamyl alcohol mixture is used for further denaturation and coagulation
of protein, as well as for the extraction of lipids. DNA is precipitated in the form of
fibres using chilled ethanol. DNA, which is collected on a rod by spooling, is
analyzed spectrophotometrically for purity and approximate yield. A diphenylamine-
based quantitation procedure is also used for more accurate determination of yield.
Contaminating RNA and protein are determined by the orcinol reaction and Folin-
Lowry procedure; respectively.

MATERIALS
1. Lyophilized E.coli cells
2. 0.15M NaCl-0.1 EDTA (pH 8.0)
3. 25% sodium dodecyl sulphate (SDS)
4. 6.0M NaCLO4
5. Chloroform:Isoamyl alcohol (24:1 v/v) solution
6. 95% chilled ethanol

EXPERIMENTAL PROTOCOL
ISOLATION OF DNA:
1. Suspend 0.5 g of lyophilized E.coli cells in 20 ml if 0.15 M NaCl-0.1 M
EDTA (pH 8.0) solution in an Erlenmeyer flask.
2. Add 3.0 ml of 25% sodium dodecyl sulphate (SDS) and stir gently.

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 3. Incubate the mixture in 60oC water bath for 15 minutes and then cool to

room temperature.
4. Add 5.0 ml of 6.0 M NaCI04 and stir.
5. Transfer the mixture to centrifuge bottles or tubes and treat with an equal
volume of chloroform: isoamyl alcohol (24:1 v/v) solution.
6. Shake the mixture manually for 15 minutes and centrifuge for 10 minutes
at 3000 rpm.
7. Carefully remove the upper aqueous phase by means of Pasteur pipettes
and transfer it to a 200 ml beaker.
8. Avoid contaminating your sample with the denatured protein which collects
at the interface between the aqueous and organic phases.
9. Gently layer 60 ml of chilled 95% ethanol over the aqueous phase in the
beaker.
10. Immerse a stirring rod through the ethanol layer and up to the interface,
and spool the DNA.
11. Squeeze out at much liquid as possible form the spooled mass by
pressing the rod against the side of the beaker.
12. Store your isolated DNA at -80oC in a labelled 15ml centrifuge tube.

RESULTS
1. Draw up a complete flow chart demonstrating the isolation of DNA. This
flow chart should include the steps taken to isolate the DNA; moreover,
the importance/significance of each step done during the isolation
process should be elaborated.

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 EXPERIMENT 3:
ULTRAVIOLET ABSORBANCE OF DNA-HYPERCHROMIC EFFECT

INTRODUCTION
The strong absorbance of nucleic acids, nucleosides and nucleotides in the
ultraviolet is due to the purines and pyrimidines that can exist in the form of a
number of different resonance structures. These spectra often shift with pH since
changes in pH affect the protonation or deprotonation of the functional groups of the
bases. These changes in the functional groups in turn, affect the types and relative
amounts of possible electronic configurations that a given base can assume.

The ionizations of the functional groups of the bases are shown in Figure1. the
pK values for these groups as well as those for functional groups of nucleosides,
and nucleotides are listed in Table1.note that, in some cases, the reaction entails
the gain (loss) of an added hydrogen atom (basically an association equilibrium,
while in other cases the reaction entails the loss (retention of a ring hydrogen
atom (basically a dissociation equilibrium).

Each base has a characteristic absorption spectrum in the ultraviolet. The

absorbance increases strongly at lower wave lengths (end-absorption) much as
in the case for proteins. Use can be made composition of nucleic acids.
Moreover, since the absorbance of a nucleic acid solution can be considered to
be equal to the sum of the absorbance due to its component nucleotides,
measurements of ultraviolet absorbance (generally at 260 nm) can be used to
calculate nucleic acid concentrations. When DNA is heat denatured, the
ultraviolet absorbance conveniently measured at 260 nm, increases.

This is known as the hyper chromic effect and for double stranded DNA its
amounts to about 40. In the intact native DNA the bases interact strongly with
each other (hydrogen bonding of the complementary, Watson-Crick type base
pairs, as well as hydrophobic stacking interactions of the parallel layers of base
pairs). These extensive base interactions limit the number of different resonance
structures that can be assumed by the bases. When DNA is denatured, these

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interactions between the bases are disrupted as the double helix dissociates into
the single strands. As a result, the bases can now assume a layer number of
resonance structures and hence the ultraviolet absorbance increases. Even in
the denatured, single-stranded, randomly-coiled DNA, there are still some base-
base interactions, though to a much smaller extent than in the highly ordered
double helix. Thus, it is not surprising that when denatured DNA is degraded to
nucleotides the process will yield an additional, though small, hyper chromic
effect.

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Table: 1 pK Values of Bases, Nucleotides and Nucleosides

Compound Pk*

pyr-N imi-N -PO4

-NH2 #1 #3 #7 #9 10 20 -OH
Adenine <1 4.2 4.6 3.2 9.8 0.9 6.3 12.5

Adenosine <0 3.5 4.2 2.2 12.4 0.8 6.3 12.3

5’-AMP 3.8 4.5 2.4 0.7 6.1 12.3

Cytosine 12.2 9.5 1.0 6.4 12.5

Cytidine 9.6 9.2

5’ CMP 9.3 9.5

Guanine 9.4

Guano sine <13

5’ GMP

Uracilic

Uri dine

5’ UMP

*- NH2 - exocyclic amino group

Pyr-N - nitrogen’s of pyrimidine ring

Imi-N - nitrogen’s of imidazole ring

-PO4 - primary (10) an secondary (20) ionization of the phosphate

Group -OH = hydroxyl groups of the sugar moiety

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Blanks indicate either lack of information or absence of ionizable groups

Thus,

Hyper chromic effect (increase in A260)

Native DNA = Denatured DNA = Degraded DNA

(Decrease in A260) Hyper chromic effect

In the present experiment, the DNA will denature by heating and degraded by
means of deoxyribonuclease (DNAse).

Heat denaturation of DNA shows S-type or sigmoidal-type kinetics and, therefore,

indicates the involvement of cooperative interactions between the hydrogen
bonded base pairs. A plot of A260 as a function of temperature is known as a
thermal denaturation profile and is shown in Figure 2.

Frequently one plots the ratio of the absorbance at a given temperature (t o C) to

that at a reference temperature, say 25 oC; that is one plots (to C) 25oC A260/A260
as a function of temperature as shown in Figure2. For precise measurements, the
DNA solution is heated in a stoppered cuvette directly in the spectrophotometer
and allowed to equilibrate at each temperature before the absorbance is
measured. Corrections are then made for the thermal expansion of the solution
which, by itself, would result in a decrease of absorbance (lower effective
concentration of DNA).

Figure 2 Thermal denaturation profile of DNA

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In the present experiment, the thermal denaturation profile will be obtained in a
somewhat different manner.

After heating the DNA solution at a given temperature, the solution is cooled
rapidly and then allowed to warm up to room temperature. The rapid cooling
serves to maintain the DNA in its denatured form and to prevent renaturation
(annealing, reformatting of the double helix) which occurs upon slow cooling. By
making absorbance measurements in this way, one measures the extent to which
the denaturation, occurring at the higher temperature, has not been reversed
upon cooling. In other words, the measurement reflects the extent to which the
thermal denaturation represents a reversible or an irreversible process.

The highly ordered structure of DNA accounts for its great stability to heat
denaturation. As a result, disruption of this structure only occurs at temperature
of about 80-90 0C. By analogy with the melting out of the DNA and the
temperature corresponding to the midpoint of this transition is known as the
melting out temperature, or Tm.

The transition of the DNA from double helix to random coil will be followed in this
experiment by measurements of ultraviolet absorbance. The transition can also
be followed by measurements of viscosity, or optical rotation.

The Tm then, is the temperature at which one half of the maximum absorbance
change (hyper chromic effect) is observed. The Tm depends on the base
composition of the DNA, the greater the guanine and cytosine content of the
denaturation and, hence, the higher its Tm. This is due to the fact that a GC pair is
linked via 3 hydrogen bonds while an AT pair is linked via 2 hydrogen bonds.
Thus the greater the (G+C) content of a DNA, the greater the number of
hydrogen bonds holding the two strands together and, hence, the greater the
thermal stability of the DNA.

By analyzing a large number of different DNA’s (isolated from various sources)

and plotting base composition [in terms of mole % of (G+C) as a function of T m, it
has been shown that a straight-line relationship is obtained. The equation for this
line is Tm = 69.3 + 0.41 (G+C) for DNA solutions containing 0.2M sodium ions

13
and 0.015M citrate (pH 7.5); (G+C) – mole % (G+C). Thus is possible to measure
the hyper chromic effect of an unknown DNA solution and to determine its base
composition, in terms of mole % (G+C), from the above equation.

PROCEDURE
Absorption Spectrum
1. Determine the ultraviolet absorption spectrum for DNA (0,03 mg/ml,
RNA) 0,03 mg/ml), and the enzyme lysozyme (0,6 mg/ml). All three
solutions are made up in 0,03M Tris buffer, pH 7,5. Measure the
absorbance against a buffer blank at 10 nm intervals from 220 to 320 nm
in the ultraviolet spectrophotometer. If the spectrophotometer cannot be
zeroed at 220 nm, begin at a longer wave length.

Thermal Denaturation
1. Set up 18 x 150 mm test tubes containing 2,0 ml of DNA solution (0,05
mg DNA/ml of 0,15M NaCl) and add 2.0 ml of borate buffer (0,05M, pH
8,5) to each tube. Cover each tube with a large marble.
2. Prepare a blank containing 2,0 ml of 0,15M NaCl and 2,0 ml of the
borate buffer. The blank is kept at room temperature.
3. Place the tubes (except the blank) in water baths as shown in Table 2.
Use as many commercial baths as are available. For the remaining
temperatures set up your own water baths in beakers and maintain the
temperature to within D 0,5 o C. for tubes 9 and 10 use a boiling water
bath. Keep the tubes as upright as possible to avoid accidental falling off
of the marbles.
4. Maintain the tubes at the indicated temperatures for 10 minutes.
5. At the end of the 10 minutes incubation period, remove the tubes from
the baths. Plunge tubes 1 through 9 into an ice-water bath, swirl, and keep
them in the ice-water bath for 10 minutes.
6. Allow tube 10 to cool slowly at room temperature for renaturation
(annealing) of the DNA.
7. Remove tubes 1 through 9 from the ice-water bath and let them warm
up to room temperature.

14
 8. Measure the absorbance of tubes 1 through 9 at 260 nm against the
buffer blank. Measure the absorbance of tube 10 likewise, but just before
the end of the laboratory period allowing as much time as possible for
renaturation to occur. Record the time allowed for renaturation.

Table: 2 Thermal denaturation of DNA

Tube number 1 2 3 4 5 6 7 8 9

Temperature (o C) 25 50 60 70 80 85 90 95 100

(room temp)

Enzymatic Digestion
1. Measure the hyper-chromic effect arising from DNAse digestion by
utilizing what is known as a difference spectrum.
2. Set up two small test tubes. To each tube add 3,0 ml of Tris buffer
(0,03M, pH 7,5, containing 0,15M NaCl and 0,03M MgCl2) and 0,5 ml of
DNA solution (0,5 mg/ml of 0,15M NaCl).
3. To tube 1 add 0,5 ml of 0,1M MgCl2 and to tube 2 add 0,5 ml of DNAse
(200 +g/ml of 0,1M MgCl2).
4. Keep tube 1 at room temperature and incubate tube 2 for 30 minutes in

a water bath at 37 oC.

5. After incubation, transfer the solutions to cuvettes and measure the

absorbance of tube 2 at 260 nm by zeroing the instrument on tube 1.

15
 EXPERIMENT 4
ISOLATION OF MITOCHONDRIA FROM BEEF LIVER

INTRODUCTION
Many of the biochemical processes that generate chemical energy for the cell take
place in the mitochondria. These organelles contain the biochemical equipment
necessary for fatty acid oxidation, di-and tri-carboxylic acid oxidation, amino acid
oxidation, electron and transport and oxidative phosphorylation. In this experiment, a
mitochondrial fraction will be isolated from rat liver. The mitochondria will be
analyzed for protein content and then stored. During your next practical session the
mitochondria will be analysed for succinate dehydrogenase, fumarase and malate
dehydrogenase activity.

Mitochondrial structure and function

Mitochondria are intracellular centre for aerobic metabolisms. They are cell
organelles that are identified by well-defined structural and biochemical properties. In
morphological terms, mitochondria are relatively large particle that are characterized
by the presence of two membranes, a smooth outer membrane that is permeable to
most important metabolites and an inner membrane that has unique transport
properties. The inner membrane is highly folded, which serves to increase its surface
area. The structure of a typical mitochondrion is shown in fig.2.1.

Fig 2.1: Structure of the mitochondrion.

16
Biochemically, mitochondria are characterized by the presence of the proteins and
enzymes of respiratory metabolism, many of which are present in the folded inner
membrane. Hence, this is the location for the processes of electron transpose,
oxidative phosphorylation and others. The enzymes involved in the tricarboxylic acid
cycle, fatty acid oxidation and amino acid metabolism are present in the matrix
region.

Because of the biological significance of these intracellular particles, biochemists

have devised several methods for the isolation of mitochondria. Most isolation
methods take advantage of the relatively large size of mitochondria. In animal cells
only the nucleus is larger. The standard method for the preparation of a
mitochondrial fraction is a differential centrifugation of homogenized tissue. It should
be pointed out that only rarely done one isolate and characterize a preparation of
pure mitochondria. Rather, the isolated sub cellular fraction is a mitochondrial
fraction, which indicates that it contains mitochondria as the major components.

Other cellular components that may be present are lysosomes; cell fragments
nuclear fragments and microbodies (peroxisomes). The purity of the fraction
depends on the source of the extract and the methods chosen for isolation.

The general procedure for preparation of a mitochondrial fraction consists of

the following steps:
1. Select and procure a suitable organ or other biological material Mitochondrial
fractions have been prepared from many animal organs including liver, heart,
muscle and brain as well as many plant tissues including spinach, avocado
and yeast.
2. The tissue is minced or ground and suspended in sucrose buffer. The choice
of buffer conditions is critical in order to avoid loss of protein and other
components that may leak from the mitochondria. Cytochrome c is one of the
easiest of mitochondrial proteins to dislodge, and some is almost always lost.
The buffer should be isotonic or hypotonic with a low ionic strength. Sucrose
has been found to be an ideal buffer component because mitochondria are
especially stable and remain intact under these conditions.

17
3. Homogenise the tissue. This is carried out with a glass-Teflon homogenizer or
a blender. This procedure gently breaks open the cell and allows the release of
the sub cellular organelles into the buffer. The mechanical disruption of cells
should be gentle so the fragile mitochondria are not damaged.
4. Centrifuge the homogenate at low speeds. The heavier particles such as
whole and fragmented cells, nuclei and nuclear fragments and membrane
fragments are sedimented during this step
5. Centrifuge the supernatant from step 4 at higher speed; this step sediments
lighter particle including mitochondria. Two distinct layers are usually observed
in the pellet: (a) a loosely packed, fluffy upper layer, which consists of
damaged mitochondria and this sometimes called light mitochondria and (b) a
dark layer that consists of heavy mitochondria.
6. The heavy mitochondrial fraction is suspended in buffer and homogenized for
a brief period in a glass-Teflon homogenizer to complete cell lysis.
7. The mitochondrial fraction is then obtained by centrifugation at relatively high
speeds (15 000 to 20 000 rpm; 25 000 x g).
8. The pellet from step 7 now consists primarily of heavy mitochondria
suspended in sucrose buffer. Typical yield are approximately 1 mg of protein
per gram of starting minced tissue.

Several variations of this procedure may be found in the literature. Often,

homogenization is facilitated by adding a proteolytic enzyme to the original
suspension in step 2, but the proteolysis must be carefully controlled to avoid
degradation of mitochondrial enzymes. The various methods lead to mitochondrial
fractions of different quality and biochemical characteristics. The choice of methods
depends to a great extent on what is to be done with the mitochondria.

This experiment describes the preparation of a mitochondrial fraction from rat liver.
Liver is an excellent choice of tissue because isolated mitochondria are stable and
most enzyme activities remain high for a long period of time. The preparation
described in this experiment is suitable for the study of characteristic enzymatic
activity, electron transport and oxidative phosphorylation.

18
EXPERIMENTAL PROCEDURE

MATERIALS
1. Beef liver
2. Isolation medium:
0, 25 M sucrose containing 10-3 M EDTA and 0, 01 M Hepes, adjusted to pH
7,4 with KOH
3. Crushed ice
4. Pasteur pipettes
5. 10% Sodium deoxycholate
6. Bovine serum albumin (BSA (, 10 mg/ml
7. Biuret reagent
8. Dissection kit

METHOD
PART A: Isolation of mitochondria
1. The mitochondria may be prepared from either rats or mice or beef liver. The
animals may be fed at libitum (i.e. without restrictions). Livers from animals
starved for 16 hours weigh approximately 30% less than those from fed animals;
mainly die to the depletion of liver glycogen.
2. All glassware used in this experiment must be thoroughly cleaned and finally
rinsed with distilled water that has been redistilled in an all-glass still, and then
boiled to free it of absorbed carbon dioxide. Work in the cold room or use ice
buckets to keep all solutions cold.
3. Stun the animal with a sharp blow at the back of the head, at the base of the skill.
Immediately decapitate the animal, using a knife or large scissors and let the
blood drain into the sink (this is known as exsanguination).
4. Remove the liver rapidly (speed is more critical than clean direction), rinse it with
ice-cold isolation medium, blot the liver lightly to remove excess liquid and
transfer it to a pre-weighed 250 ml beaker.
5. Weigh the liver, then cut it into small pieces and add ice-cold isolation medium
(10ml of isolation medium per 1.0 g of liver).
6. Transfer portions of the mixture to a pre-chilled Potter-Elvehjem homogenizer.
Keep the tube of the homogenizer in crushed ice during the operation. Move the

19
 tube up and down while turning for a total of 4-6 times (i.e. make 4-6 passes) till
the liver is dispersed. The time for each pass (up or down stroke) should be
approximately 5 seconds.
7. Pool the homogenization in a pre-chilled Waring blender may be used if a Potter-
Elvehjem homogenizer is not available.
8. Transfer the homogenate to pre-chilled centrifuge tubes and centrifuge for 15
minutes at 660 x g in the cold) 0-40C.
9. Carefully remove the supernatant and transfer it to ice-cold centrifuge tubes.
Discard the pellet of nuclei and cell debris.
10. Centrifuge the supernatant for 15 minutes at 7 000 x g in the cold (0-40C).
11. Discard the supernatant and very gently suspend the pellet in ice-cold isolation
medium (5.0 ml of isolation medium per 1.0 g of liver). Press the particulate
matter with a stirring rod or a rubber policeman against the wall of the tube or
homogenize it very gently, using a small, pre-chilled Potter-Elvehjem
homogenizer.
12. Centrifuge the suspension for 15 minutes at 7 000 x g at 0-40C.
13. Discard the supernatant.
14. Carefully rinse off the fluffy layer above the pellet with isolation medium. Discard
this washing.
15. Very gently suspend the pellet in ice-cold isolation medium (see step 10). Use
2.5 ml of isolation medium per 1.0 g of liver.
16. Repeat steps 11-13.
17. Suspend the pellet washed mitochondria (see step 10) in ice-cold isolation
medium (1.0 ml of isolation medium per 1.0 g of liver) and store the preparation in
crushed ice.

PART B: Determination of protein

Before the mitochondrial fraction can be biochemically characterised, the protein
content must be measured. The Biuret method will be described although the Lowry
or Bradford methods may be used.
1. Obtain five small test tubes and set up the protein assay according to table 13.1
Tubes 1 and 2 contain two different concentrations of mitochondrial protein.
Tubes 3 and 4 are duplicates of a standard protein, bovine resume albumin.
Tube 5 is used as a blank for the spectrophotometer. The purpose of sodium

20
 deoxycholate is to disrupt the mitochondria and release the protein material into
solution.
2. Add the appropriate amount of each of the first four reagents (sucrose solution,
mitochondria, sodium deoxycholate and BSA) to each of the five test tubes. If the
solutions are not clear, add 10% sodium deoxycholate with graduated pipettes.
Be sure you note the exact amount of deoxycholate added. Do not add more
than a total of 0.4 ml.

Table 13.1: Preparation of tubes for the Biuret protein assays of the mitochondrial
fraction (all quantities are in ml).

Reagent Tube
1 2 3 4 5
Sucrose Herpes isolation solution 0.5 0.5 0.5 0.5 0.5
Mitochondrial fraction 0.5 0.25 - - -
10% Sodium deoxycholate 0.2 0.2 0.2 0.2 0.2
Bovine serum albumin - - 0.1 0.1 -
H2O 0.3 0.55 0.70 0.70 0.80
Biuret reagent 1.5 1.5 1.5 1.5 1.5

3. Add sufficient water to each tube so that the total volume of all liquids is 1.5 ml.
Table 13.1 is set up assuming that 0.2 ml of deoxycholate solution is sufficient. If,
for example, tube 1 required a total of 0.4 ml of deoxycholate, then only 0,1 ml of
water should be added to the tube.
4. Add 1.5 ml of biuret reagent to each tube and mix well by inverting several times
while holding a piece of hydrocarbon foil over the opening or vortex mixing.
5. Incubate the tubes for 15 min at 370C.
6. Measure and record the absorbance at 540 nm of tubes 1-4 using tube 5 to
adjust the A540 to 0.0 absorbance.
7. If the absorbance readings of tubes 1 and 2 are greater than twice the A 540 of
tubes 3 or 4, repeat the assay using less mitochondrial fraction in tubes 1 and 2.
Be sure the new assays contain a total of 3.0 ml of liquids in each tube.

21
8. If necessary, dilute the mitochondrial fraction to 1 mg protein with ice-cold
isolation medium and store at -200C until your next practical session

REPORT

(a) Preparation of the mitochondrial fraction

Prepare a flow chart of the procedure followed in the isolation of mitochondria. Briefly
explain the purpose of each step.

(b) Determination of protein

Calculate the protein concentration in the mitochondrial fraction using equation 1.
Where:
Cstd Cunk
------- = -------

Astd Aunk (1)

Astd = absorbance at 540 nm of the standard bovine serum albumin (tubes 3

and 4)
Aunk = absorbance at 540 nm of the protein in the mitochondrial fraction
Cstd = concentration of standard bovine serum
Cunk = concentration of protein in the mitochondrial fraction

The biuret assay is linear up to protein concentrations of about 2 mg/ml.

REFERENCES
1. Boyer, R.F. Modern Experimental Biochemistry, pp. 489-490 and pp. 495-497.
2. Stenesh, J. Experimental Biochemistry, pp. 226-228.

22
 EXPERIMENT 3.
REACTIONS OF THE CITRIC ACID CYCLE; SUCCINATE DEHYDROGENASE,
FUMARASE AND MALATE DEHYDROGENASE

INTRODUCTION
Succinate dehydrogenase
The enzymes Succinate dehydrogenase (EC 1.3.99.1), fumarase (EC 4.2.1.2), and
malate dehydrogenase (EC 1.1.1.37) catalyse the last three reactions of the citric
acid cycle.

Succinate dehydrogenase has a molecular weight of about 97 000 and is tightly

bound to the inner mitochondrial membrane; it apparently constitutes an integral part
of that membrane. The enzyme is a glycoprotein, containing FAD as the prosthetics
group. The isolloxazine ring of the FAD is covalently linked to a histidine residue in
the enzyme. The reaction catalyzed by Succinate dehydrogenase is the following:

Succinate + FAD → Fumarate + DADH2 (1)

The biochemical standard free energy change ( G0') for the reaction is
approximately zero.
In addition to the flavin group, the enzyme contains four iron atoms and four organic
sulfides; it does not contain a heme. It is, therefore, classified as an iron-sulfur (FeS)
protein or as a non-herms iron protein.
The reaction of succinate dehydrogenase is assayed qualitatively in this experiment
in the presence of several artificial electron acceptors that are listed in fig. 3.1.

23
Normally, the electrons from FADH2 are transferred directly to the Fe3+ atoms of the
enzyme (E) and from there ultimately to oxygen via the electron transport system. In
this experiment, the electrons are transferred to the added artificial electron
acceptors.

The reduction of these electron acceptors can be observed visually, for example:

E-FADH2 + Methylene blueox → E-FAD + Methylene bluered + H+ (2)

(Blue) (Colourless)

The reaction can, of course also be studied quantitatively by measuring the

absorbance changes in a spectrophotometer.

24
Malonate is a classical example of a competitive inhibitor; the inhibition is due to the
structural similarity of malonate with the enzyme substrate, succinate. Cyanide is an
uncompetitive inhibitor of the enzyme.

Fumarase
Fumarase is an oligomeric protein, composed of four subunits and has a molecular
weight of about 2000 000. The physicochemical properties of fumarase have been
studied in great detail. The enzyme catalyses the reaction:

Fumarate + H2O → L - Malate (3)

The biochemical standard free energy change for the reaction (ΔG0') is -0.9
kca/mole.
The fumarase reaction represents stereo specific trans addition of H and OH so that
only the L-isomer of malate is produced. Since L-malate is optically active while
fumarate is not, the reaction can, in principle, be followed by measurements of
optical rotation. The sensitivity of this measurement is, however, very low and the
enzyme is commonly assayed by measuring the decrease in ultraviolet absorbance
at 300 nm. This is based on the fact that fumarase absorbs strongly at 300 nm while
the absorbance of L-malate is negligible at that wavelength.

Malate dehydrogenase
The last reaction of the citric acid cycle is catalyzed by the enzyme malate
dehydrogenase. This is a pyridine-linked dehydrogenase (molecular weight about
70 000) for which NAD+ serves as a cofactor. Two forms of the enzyme exist;
mitochondria and one in the cataplasm; both require
NAD+. The mitochondrial enzyme is the important one in the operation of the citric
acid cycle. The reaction catalyzed by malate dehydrogenase is the following:

L-Malate + NAD+ → Oxaloacetate + NADH + H+ (4)

25
The biochemical standard free energy change (ΔG0') for the reaction is +7.1
kca/mole. The enzyme is stereospecific with respect to L-malate and the transfer of
the hydrogen to the pyridine ring of NAD+. The reaction is endergonic as written, but
proceeds readily in vivo because of the rapid removal of the product, NADH. The
assay of in this experiment measures the reverse, exergonic, reaction whereby
oxaloacetate is converted to L-malate. The reaction is conveniently followed by
measuring the decrease in absorbance at 340 nm due to the conversion of NADH to
NAD+.

EXPERIMENTAL PROCEDURE

MATERIALS
1. Mitochondrial fraction isolated in experiment 13
2. Crushed ice
3. Isolation medium (as in experiment 13)
4. 0, 1 M Phosphate buffer, pH 7, 2
5. 0, 1 M Succinate
6. 0, 1 M malonate
7. 0, 1 M NAD+ (prepared fresh)
8. 0, 02% Methylene blue
9. 0, 02% Dichlorophenol indophenol (DCIP)
10. 0, 02% N-methyl phenazinium methylsulphate (PMS)
11. 0, 01 M Potassium ferricyanide (K3Fe (CN) 6)
12. 0, 1 M KCN
13. Mineral oil
14. Parafilim
15. 0, 04 M Phosphate buffer pH 7, 3
16. 0,017 M Potassium fumarate in 0,04 M potassium phosphate buffer, pH 7,3
17. 0, 25 M Phosphate buffer, pH 7, 4
18. 0, 0076 M Oxaloacetate, pH 7, 4
19. 0, 0015 M NADH, pH 7, 4

26
METHOD

PART A: Recovery of Mitochondria

1. Use the mitochondria isolated in experiment 2. Let your frozen sample thaw at
room temperature.

PART B: Succinate dehydrogenase assay

2. Set up eight 13 x 1000 mm test tubes as shown in table 2.1. Pipette everything
except the mitochondrial suspension.

Caution: KCN is toxic: maybe fatal if swallowed. Upon contact with acid, poisonous
HCN gas is evolved. Phenazine methosulfate is an irritant; K3Fe(CN)6 is harmful if
swallowed.

Table 3.1: Succinate dehydrogenase assay.

Tube number
Reagent 1 2 3 4 5 6 7 8
0.1 M Phosphate
buffer pH 7,2 (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.1 M Succinate (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.1 M Malonate (ml) - - - - - - 0.4 -
0.01 M NAD+ (ml) 0.2 - - - - - - -
0.02% Methylene - - -
blue (ml) 0.5 0.5 0.5 0.5 0.5
0.2% Dichlorophenol - - - - - - -
indophenol (ml) 0.5
0.02% Phenazine - - - - - - -
methosulfate (ml) 0.5
0.01 M K3Fe(CN)6 - - - - - - -
(ml) 0.2
0.1 M KCN (ml) - - - - - - - 0.2
Water (ml) 0.2 0.4 0.4 0.4 0.4 0.7 - 0.2

27
 Isolation medium - - - - - - -
(ml) 0.4
Mitochondria (ml) 0.4 0.4 - 0.4 0.4 0.4 0.4 0.4

3. Initiate the reaction by the addition of the mitochondria. Mix well and immediately
place 0.5 ml of mineral oil over tubes 1,2,3,7 and 8 that contain methylene blue.
4. The reduced form of methylene blue rapidly deoxidized by air: hence, the addition
of mineral oil.
5. Let the tubes stand at room temperature for 30 minutes and examine them at 3 -
minute intervals.
6. Record both the time and the nature of any colour changes. Use a scale of 1-5
assign a relative colour intensity of each tube.
7. Seal the tubes with Parafilim and shake vigorously. Note whether the original
colours are restored, indicating reoxidation of the methylene blue by molecular
oxygen.

PART C: Fumarase assay

1. Carefully pipette the reagents shown in table 14.2 into two quartz cuvettes with a
pipette.
2. Cover cuvette 1 with parafilim and mix gently by inversion.

Table 3.2 Fumarase assays.

Cuvette Number
Reagent 1 2
0.04 M Potassium phosphate buffer, pH 7.3 (ml) 3.4 -
0.017 M Potassium fumarate in 0.04 M
potassium phosphate buffer, pH 7.3 (ml) - 3.4
Isolation medium (ml) 0.1 -

3. Set the wavelength of the spectrophotometer at 300 nm, and zero the
instrument against air. Place the cuvette in the reference compartment.
4. Now add 0.1 ml mitochondrial to cuvette 2, mix immediately by inversion and
place the cuvette sample compartment of the spectrophotometer.

28
5. Measure the change in absorbance of cuvette 2 at 15 second intervals for 2
minutes and then at 1 minute intervals for an additional 5 minutes.
6. The enzymatic activity is calculated from the initial, linear, part of the curve,
obtained by plotting absorbance as a function of time. Use a molar extinction
coefficient of 41.2 for fumarate. That is:

1M
E = 41.2; E = 41.2M-1 cm-1
1cm λ 300nm
300 nm

An enzyme unit (U) is defined as that amount of enzyme causing the loss of 1.0
micromole of fumarate per minute under the given assay conditions. Specific activity
= units/mg mitochondrial protein.

PART D: Malate dehydrogenase assay

1. Carefully pipette the reagents shown in table 3.3 into two quartz cuvettes. Do not
scratch the optical surfaces of the cuvette with a pipette.

Table 3.3: Malate dehydrogenase assay.

Reagent Cuvette Number
1 2
0.25 M Phosphate buffer pH 7.4 (ml) 0.3 0.3
0.0076 M Oxaloacetate, pH 7.4 (ml) 0.1 0.1
0.0015 M NADH, pH 7.4 (ml) - 0.1
Water (ml) 2.55 2.45

2. Add 0.05 ml mitochondria to cuvette 1 only. Cover the cuvette with parafilim and
mix gently by inversion.
3. Now add 0.05 ml mitochondria to the other cuvette, mix immediately by inversion
and place it in the spectrophotometer.

29
4. Measure the change in absorbance of cuvette 2 and 15 second intervals for 2
minutes and then at 1 minute intervals for an additional 3 minutes. Plot
absorbance (A340) as a function time.
5. The enzymatic activity is calculated from the initial, linear rate of the decrease in
absorbance. This decrease should not exceed 0.04 per minute. If it does, the
experiment should be repeated, using a mitochondrial suspension that has been
further diluted with isolation medium.
6. An enzyme unit (U) is defined as that amount of enzyme catalyzing the
conversion of 1.0 micromole of oxaloacetate to L-malate per minute under the
above assay conditions. The molar extinction coefficient of NADH is 6,220. That
is:
1M
E = 6,220; E = 6,220 M-1 cm -1
1 cm 340
340 nm
Specific activity = units/mg mitochondrial protein.

REPORT
Your report should include the following:

(A) Succinate dehydrogenase assay

Tube number (Table 1) Time (min) Colour change (Scale 1-5)
1
2
3
4
5
6
7
8

Briefly discuss your observation for each tube.

30
(B) Fumarase assay
Time (sec) A300
0
15
30
45
60
75
90
105
120
180
240
300
360
420

Attach a plot of A 300 (ordinate) as a function of time in seconds (abscissa).

Determine the enzymatic activity from the initial slope:
Time interval selected for calculation of the initial slope............................ Sec to
......................... sec
Corresponding A300
=.................................................................................................................
Change in fumarate concentration ................................................ μmoles/ml
Initial slope.................................................. (µmoles/ml) per min
Number of U/assay tube ...............................................
Number of U/ml of mitochondrial suspension .....................................................
Protein concentration of mitochondrial fraction ................................................. mg/ml
Amount of mitochondrial protein used in assay...................................... Mg
Specific activity =........................................ U/mg mitochondrial protein

31
(c) Malate dehydrogenase assay
Time (sec) A340
0
15
30
45
60
75
90
105
120
180
240
300

Attach a plot of A340 (ordinate) as a function of time in seconds (abscissa).

Determine the enzymatic activity from the initial slope:

Time interval selected for calculation of the initial slope .................................... ..........
sec to
................................ Sec
Corresponding ΔA340 =........................................
Change in NADH concentration............................. M
Change in NADH concentration ............................. µmoles/ml
Initial slope............................................ (µmoles/ml) per min
Number of U/assay tube...................................................
Number of U/ml mitochondrial suspension..................................................
Protein concentration of mitochondrial fraction......................................... mg/ml
Amount of mitochondrial protein used in assay......................................... Mg
Specific activity =.................................... U/mg mitochondrial protein

REFERENCE
1. Stenesh, J. Experimental Biochemistry, pp. 409-420.

32