Download Word

The Practice & Communication of Science 2015/2016 Mini-projects
Topics in Biochemistry (Thursday, Friday)
Welcome to the Mini-Projects for the ‘biochemistry’ sessions in USSKAR-30-20, PaCoS. You will be working
in groups of three, and each group will be allocated an exercise by ‘lottery’. Your exercise will cover six
two-hour practical slots (though the last assessment one is three hours long) and will give you the
opportunity to get deeply involved in all the stages of scientific investigation, including background
reading, experimental design, development of methodology and its troubleshooting, data collection, data
analysis and communication of the results of your study via a scientific poster. The timetable for the
exercise is as follows...
Week 26 : Introduction, library work and method development
- Class split into groups; introduction to the Mini-Projects
- Exercises allocated by ‘lottery’
- Background reading and consideration of a risk assessment (laboratory work cannot start without this!)
- Development of methodology; we will be available for guidance, feedback, etc. You need to become
familiar with the subject matter of your particular experiment as this will assist greatly in the design and
execution of your experiments, thereby improving your data, your analysis, your interpretation and
conclusions, your poster, and your marks!
Weeks 28, 30 & 32 : Laboratory (experimental) work
- These three sessions should be devoted to practising the experimental methods and gathering lots of
good experimental data! You may wish to spend most or all of the first session to ‘ironing out the bugs’ in
your methodology before collecting data ‘for real’.
Week 34 : Data analysis & team poster preparation
- See the web-based module support for advice on poster preparation. PowerPoint or Publisher should be
used to produce your team’s poster (as for the Glassbeads experiment), and Word for the abstract
- we will be on hand to help with the data analysis; but ensure you bring your data in a spreadsheet!
Week 38 : Poster presentation and assessment
- The first 15 min will be available to arrange the posters/abstracts for display. Then, each team will be
asked to assess some of the posters. Assessed elements include - overall presentation, appearance, clarity
and content; amount of laboratory work completed and reported; quality of results, consistency,
replication and presentation; discussion and analysis, including statistical analysis and interpretation, and
further work; references cited and employed usefully in the presentation. Further details on the
assessment can be found on the support web-site.
Poster guidelines : Posters should be produced using PowerPoint or Publisher, and abstracts with Word.
One of the most important parts of the write-up is the Abstract which should provide a self-contained
précis of the introduction, methods, results, and conclusions (remember, you normally write the main
sections of the poster, and then précis it to produce the Abstract). In ‘real-life’ Abstracts are usually printed
in the Proceedings of the Meeting; they are usually word-limited (let us say up to 300 words, with one
Figure, if used, equalling 150 words). Posters should have a fairly 'dry' scientific style (no cartoons, etc!),
should be easy to read, and be reasonably self-explanatory. You may recall that the Intro and M&M we
provided for the Glassbeads were too long – try to make yours as concise as possible without interrupting
the ‘flow’ of the story. You will display your posters/abstracts during the first part of the final session; you
are likely to be asked questions about your poster during the last part of the session, on an individual basis
(as preparation for the assessment of your Final Year Project!).
The Practice & Communication of Science 2015/2016 Mini-projects
Each group of 3 students will be assigned one of the following questions …
1. How do the Km values for muscle and heart compare?
2. How do the pH optima for muscle and heart compare?
3. Is oxalic acid an inhibitor of muscle LDH and, if so, what is its Ki?
4. Is oxalic acid an inhibitor of heart LDH and, if so, what is its Ki?
5. Is oxamic acid an inhibitor of muscle LDH and, if so, what is its Ki?
6. Is oxamic acid an inhibitor of heart LDH and, if so, what is its Ki?
The Practice & Communication of Science 2015/2016 Mini-projects
LDH: - Assay and Investigations
Background
Lactate Dehydrogenase (LDH) is a ubiquitous enzyme that exists in various isoforms, depending on its overall role in
the metabolism of the cell in which it is expressed. Heart muscle LDH contains isoenzymes which are richer in H type
subunits, skeletal muscle contains LDH isoenzymes richer in M type subunits.
In this practical we shall be exploring issues such as differences between the H4 and the M4 isoenzymes in relation
to Km, pH-dependence and inhibition by other molecules.
It is worth considering whether any differences in behaviour between the isoenzymes represent an adaptation to
serve the way that particular tissue functions. To appreciate differences in the way pyruvate and lactate are
metabolised by different tissues let’s consider skeletal muscle, heart and brain. In skeletal muscle the reserves of
glycogen are there solely to power contraction, especially while the blood supply is still insufficient to support the
aerobic oxidation of glucose. Because anaerobic glycolysis gives a low yield of ATP, a relatively large amount of
glucose carbons flow into the pathway so concentration of pyruvate and lactate are high. Heart and brain by contrast
are highly aerobic tissues which are normally not allowed to become anaerobic. Aerobic glycolysis in heart muscle
generates pyruvate which is converted to acetyl CoA which then enters the citric acid cycle to be completely oxidised
to CO2 and water. Heart muscle is also well able to utilise lactate, which has diffused into the blood from skeletal
muscle, as fuel. The lactate is converted back to pyruvate and then to acetyl CoA to be efficiently and completely
oxidised as described above (liver on the other hand also takes up lactate but converts most of it back to glucose via
the gluconeogenesis pathway). Brain is an unusual tissue in that it is normally only able to utilise glucose as fuel; this
is converted to pyruvate through aerobic glycolysis and then completely oxidised as in heart. In both heart and brain
the conversion of pyruvate to lactate is a metabolic ‘dead end’, depriving the oxidative pathways of substrate and
serving no useful function beyond regenerating NAD.
The Assay
The activity of LDH is assayed via the following reaction.
Pyruvate
Lactate
Lactate Dehydrogenase
NADH
NAD+
The oxidation of the NADH to NAD+ results in a decrease in absorbance at 340nm allowing the process to be followed
spectrophotometrically.
The Practice & Communication of Science 2015/2016 Mini-projects
Basic Assay Method
Switch on the spectrophotometer, set the wavelength to 340nm. Place a blank, Tris-filled cuvette in the
spectrophotometer and zero using the CAL button. You just need to do this once.
a) For the assay, pipette the following into a cuvette:
 Tris buffer, 100mM pH 7.4
2.7 mL (may differ if adding inhibitors)
 NADH, 6mM
0.1 mL
 LDH purified from heart or skeletal muscle
0.1 mL
b) Mix well with a pastette and place in the spectrophotometer. Select start on the laptop.
c) Add to the cuvette 0.1ml, 30mM sodium pyruvate, pH 7.4; mix well immediately with a pastette and record for 3
min.
d) The easiest way to measure a rate from a recording is to note the absorbances at two points separated by one
minute (make sure the recording is linear over that section) then take one absorbance from the other to give Ab
change/min.
Investigations
We have available to us…



Two LDH isoenzymes (bovine muscle and bovine heart)
Oxalic and oxamic acids (potential inhibitors of the enzyme)
Buffers of varying pH
Each group of 3 students will be assigned one of the following questions …
7.
8.
9.
10.
11.
12.
How do the Km values for muscle and heart compare?
How do the pH optima for muscle and heart compare?
Is oxalic acid an inhibitor of muscle LDH and, if so, what is its Ki?
Is oxalic acid an inhibitor of heart LDH and, if so, what is its Ki?
Is oxamic acid an inhibitor of muscle LDH and, if so, what is its Ki?
Is oxamic acid an inhibitor of heart LDH and, if so, what is its Ki?
For the above investigations…


Km values can be determined by varying substrate (pyruvate) concentrations.
o Suggested range for the 0.1 ml of stock solutions to add to the assay, 0  30 mM (final conc will
differ, eg 0.1 ml in 3 ml of 0  30 mM stock will give range in cuvette of 0  1 mM)
o Substitute the required pyruvate concentration at step c) of Basic Assay Method
pH optimum can be determined by using buffers of different pHs in the assay (all other components
constant).
o Suggested pHs; 5, 6, 7, 8 and 9
o Substitute the required buffer at step a) of Basic Assay Method
The Practice & Communication of Science 2015/2016 Mini-projects

Potential inhibitors can be investigated by looking at the effects of the presence of a fixed concentration on
the rate of the basic reaction over a range of substrate (pyruvate) concentrations.
o Suggested concs; 30 mM for stock oxalic acid; 30 mM for stock oxamic acid. Note what the final
cuvette concentrations will be when added to the reaction mixture.
o For oxalic/oxamic acid, add 0.1 ml of the stock at step c) of Basic Assay Method (reduce buffer vol to
2.6 ml)
Possible approach to each question …
1. How do the Km values for muscle and heart compare?
a. For each type of enzyme, measure velocity at varying pyruvate concs (7 concs, each x4 for each
enzyme?) (final bath concs for pyruvate of 1, 0.5, 0.3, 0.2, 0.1, 0.05, 0.025 mM?)
b. Double-reciprocal plot to work out Km
c. Compare Km values (paired or unpaired t-tests?)
2. How do the pH optima for muscle and heart compare?
a. For each type of enzyme, measure velocity at a fixed pyruvate conc (need to determine), at pH 5, 6,
7, 8 and 9 (5 pHs, each x6 for each enzyme?)
b. Plots of velocity vs pH?
c. Analyse data with ANOVA?
3. Is oxalic acid an inhibitor of muscle LDH and, if so, what is its Ki?
a. Find a conc of oxalic acid that produces reasonable inhibition (default will be 0.1 ml of 30mM stock)
b. Do 1.a above in the presence and absence of this conc of inhibitor
c. Double-reciprocal plot to work out Km and Ki
d. Compare using paired/unpaired t-tests?
4. Is oxalic acid an inhibitor of heart LDH and, if so, what is its Ki?
a. As above
5. Is oxamic acid an inhibitor of muscle LDH and, if so, what is its Ki?
a. Find a conc of oxamic acid that produces reasonable inhibition (default will be 0.1 ml of 30mM stock)
b. Do 1.a above in the presence and absence of the inhibitor
c. Double-reciprocal plot to work out Km and Ki
d. Compare using paired/unpaired t-tests?
6. Is oxamic acid an inhibitor of heart LDH and, if so, what is its Ki?
a. As above