TECHNISCHE UNIVERSITÄT BRAUNSCHWEIG
INSTITUTE FOR BIOCHEMISTRY AND BIOTECHNOLOGY
DEPARTMENT FOR BIOINFORMATICS AND BIOCHEMISTRY
Chair Prof. Dr. Dietmar Schomburg
Energy metabolism
Experimental details for the
practical course BM02
2011
TU Braunschweig, Biocenter
TU Braunschweig, Biocenter
Dr. Kerstin Schreiber
Ph.D. students
Dr. Eva Jordan
Spielmannstr. 7 | Room 047/048
Spielmannstr. 7 | Room 047
38106 Braunschweig
38106 Braunschweig
Tel. (0531) 391-8353/8356
Tel. (0531) 391-8354
E-mail: [email protected],
[email protected]
1
Table of contents
Time schedule.......................................................................................................................................3
1 General informations for the practical course...................................................................................4
1.1 Seminar topics............................................................................................................................4
1.2 Safety in the laboratory..............................................................................................................4
1.3 Structure of the protocols...........................................................................................................5
1.4 Continuative literature...............................................................................................................7
2 Metabolome analysis via GC-MS.....................................................................................................8
2.1 Metabolome analysis.................................................................................................................8
2.1.1 Overview............................................................................................................................8
2.1.2 Gas chromatography-Mass spectrometry.........................................................................10
2.1.3 Derivatisation of substances in biological samples..........................................................12
2.1.4 Identification of the metabolites.......................................................................................12
2.2 Introduction - Corynebacterium glutamicum..........................................................................14
2.3 Schedule for metabolome analysis..........................................................................................15
2.3.1 Destruction of the cells and extraction of metabolites.....................................................16
2.3.2 Necessary preparations.....................................................................................................17
2.3.3 Experimental procedure...................................................................................................19
2.4 Evaluation................................................................................................................................22
2.4.1 Analysis of the raw data...................................................................................................22
2.4.2 Statistical analysis and interpretation of the results.........................................................23
3 Growth phenotype of C. glutamicum: carbon metabolism.............................................................24
3.1 Growth phenotype....................................................................................................................24
3.2 Phenotype MicroArrays...........................................................................................................24
3.3 Goals and principle of the experiment.....................................................................................27
3.4 Experiment...............................................................................................................................27
3.4.1 Investigation of the carbon metabolism of C. glutamicum WT and ptsG::tnIS6100 by
Phenotype MicroAssays............................................................................................................27
3.4.2 Investigation of the carbon metabolism of C. glutamicum WT and ptsG::tnIS6100 by
shake flask cultivations.............................................................................................................29
4 Enzyme kinetics...............................................................................................................................32
4.1 Enzymes...................................................................................................................................32
4.2 Enzyme kinetics.......................................................................................................................33
4.3 Enzyme inhibition....................................................................................................................37
4.4 Photometric activity tests.........................................................................................................40
4.5 Alcohol dehydrogenase from Saccharomyces cerivisiae.........................................................41
4.6 Goals and principle of the experiment.....................................................................................42
4.7 Recording of the absorption....................................................................................................42
4.8 Experiment...............................................................................................................................43
4.8.1 Investigation of the KM constant for ethanol..................................................................43
4.8.2 Investigation of the substrate specificity of the ADH......................................................45
4.8.3 Investigation of the KI constant of trifluoroethanol.........................................................46
4.8.4 Investigation of the KI constant of 1-butanol..................................................................48
4.8.5 Investigation of the KI constant of salicylic acid (substrate ethanol)..............................49
4.8.6 Investigation of the KI constant of salicylic acid (co-substrate NAD+)..........................51
4.8.7 Investigation of the KI constant of acetylsalicylic acid and comparison with salicylic
acid............................................................................................................................................52
2
Time schedule
Time schedule
First week:
Monday
Tuesday
Wednesday
Thursday
Friday
- Safety briefing
- Preparation of the over
night culture (2)
- Preparation of the
MicroPlates (3)
- Harvesting of the
bacteria (2)
- Talk: Phenotype
MicroArrays
- Talk: OMICS
- Derivatisation (2)
- Talk: GC
- Talk: MS
- Talk: T-Test, ANOVA
- Evaluation (2 and 3)
- Talk: PCA, HCA
- Talk: Databases
- Evaluation (2 and 3)
Monday
Tuesday
Wednesday
Thursday
Friday
- Preparation of
solutions
- Preparation of the over
night culture (3)
- Evaluation (2 and 3)
- Talk: Enzymes
- Investigation KM ADH
and substrate specificity
(4)
- Cultivation with
different C sources (3)
- Evaluation (4)
- Talk: MichaelisMenten kinetic
- Investigation KI
trifluoroethanol and 1butanol (4)
- Cultivation with
different C sources (3)
- Evaluation (3 and 4)
- Talk: Enzyme
inhibition
- Investigation KI
salicylic acid (ethanol
and NAD, 4)
- Evaluation (3 and 4)
- Investigation KI
acetylsalicylic acid (4)
- Evaluation (4)
Second week:
3
1General informations for the practical course
1 General informations for the practical course
1.1 Seminar topics
There will be a seminar whose contents are the topics of the practical course. The talks have to be
maximum 15 minutes long and give an overview about the topic, with a short in-depth information
about the methods used in the practical course. Each group prepares one topic. The seminar takes
place in the practical course room, which is equipped with a beamer.
1. Phenotype MicroArrays
2. OMICS-fields with the focus on metabolomics and metabolome analysis
3. GC/MS: GC in metabolome analysis (functional principle, derivatisation, retention time /
index, ...)
4. GC/MS: MS in metabolome analysis (functional principle, ionisation, analyser, ...)
5. Statistical analysis: T-test, ANOVA
6. Statistical analysis: Principal component analysis (PCA), hierarchical cluster analysis
(HCA)
7. Databases: BRENDA, KEGG and MetaCyc
8. Enzymes: Classification, structure (primary till quaternary structure), function and
regulation
9. Michaelis-Menten kinetic: identification of KM and vmax with different diagrams
10. Enzyme inhibition: inhibition types, Dixon plot
1.2 Safety in the laboratory
Each practical course participant is bound to inform herself/himself about the correct handling of
the chemicals used in the practical course before application. This contains the intensive reading of
the safety data sheets, particularly the listed “possible risks” and the “first aid measures”. Keep in
mind:
Safety first!!
4
1General informations for the practical course
Take care of a failure-free working environment (that means: take care for enough working space
without constrain your fellow students). Safety glasses, lab coat and gloves are obligatory!
Disregard of this instruction causes exclusion of the participant from the practical course. You will
work with easily flammable chemicals. Acquaint yourself with the locality (places of the fire
extinguishers, fire blankets and emergency exits). Keep free the accesses to the fire extinguishers,
fire blankets and emergency exits during the whole practical course. Personal bags and jackets
are not allowed in the practical course room. They have to be stored in the provided lockers.
The experiment description must be read completely and be completely understood before the
practical course begins.
1.3 Structure of the protocols
The protocols should be kept short, but anyway all important experimental changes, results and
discussion as well as possible mistakes have to be included. In the following some details are listed:
Approved structure:
1. Introduction
2. Material and methods
3. Results
4. Discussion
5. Literature
6. Attachment
1. Introduction
A few sentences are enough. What was done? What goal had the experiment?
2. Material and methods
Refer to the script, e.g. experiment 3 was performed as described in the script. Declare only
changes to the script, e.g. experiment 1 was performed as described in the script page xyz, but the
following changes were applied: chapter xy: instead of … … was used.
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1General informations for the practical course
3. Results
In the results part only graphically or tabularly prepared raw data is shown as well as results
are described shortly. Consider carefully what information is presented to the reader. Keep in
mind that every figure or table in the results or in the discussion part must be described or
discussed.
The protocol is mainly a result protocol. Because of this a detailed presentation of the results is
necessary. That means:
•
Clearly represented measuring results in tabular form.
•
Subscribe the tables correctly (above the table), state the correct units, choose the numbers
of decimal places expediently. Retain as possible to one unit e.g. mg or µg.
•
Subscribe the figures also correctly (below the figure) and state the correct units. Titles
within the diagram are not necessary, legends for one graph in the diagram are not inserted.
Scale the axes expedient, choose the accurate size for the figures.
•
The reader has to understand the content of the figures and tables only by reading the titles
of them.
•
Describe the results concisely.
4. Discussion
•
Detailed discussion of the results
•
Possible sources of errors
•
Comparison with data from literature (if possible)
5. Literature
All citations and numerical values from literature have to be presented with the according reference.
6. Attachment
The raw data, e.g. the chart recorder printouts, must be filed into the attachment and marked
correctly and unmistakable.
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1General informations for the practical course
The protocols must be delivered at the latest two weeks after the practical course ends! The
protocols are corrected only once. The delivery of the rectified protocols must occur within
one week (together with the first protocol)!
1.4 Continuative literature
1. Rehm,
H.
Der
Experimentator:
Proteinbiochemie/Proteomics
(2002)
Spektrum
Molekularbiologie/Genomics
(2003)
Spektrum
Akademischer Verlag
2. Mülhardt,C.
Der
Experimentator:
Akademischer Verlag
3. Stephenson, F.H. Mathematik im Labor – Ein Arbeitsbuch für Molekularbiologie und
Biotechnologie (2005) Spektrum Akademischer Verlag
4. Bärlocher, F. Biostatistik – Praktische Einführung in Konzepte und Methoden (1999)
Thieme Verlag
5. Voet, D./Voet, J.G./Pratt, C.W. Lehrbuch der Biochemie (2002) Wiley-VCH
6. Berg/Tymoczko/Stryer Biochemie (2003) Spektrum Akademischer Verlag
7. Nelson/Cox Lehninger Biochemie (2001) Springer-Verlag GmbH & Co. KG
8. Koolman, J./Röhm, K.H. Taschenatlas der Biochemie (2003) Thieme-Verlag
9. Villas-Boas/Roessner/Hansen/Smedsgaard/Nielsen Metabolome Analysis. An Introduction
(2007) Wiley-VCH
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2Metabolome analysis via GC-MS
2 Metabolome analysis via GC-MS
Metabolome analysis of intracellular metabolites will be carried out using the gram negative
bacterium Corynebacterium glutamicum (wild type Res167 and one mutant).
For this, the bacteria will be cultivated and harvested at a specific time point, cells will be disrupted
and metabolites extracted for analysis in a gas chromatograph coupled to a mass spectrometer.
The obtained chromatograms will be analysed using the software Metabolite Detector and
metabolites of the central metabolism will be identified with a specific library. Afterwards the
results will be further analysed for significance using different statistical approaches. Figure 2.1
shows all metabolites of the central metabolism present in the library used for identification in the
software.
2.1 Metabolome analysis
2.1.1
Overview
The metabolome comprises all metabolites present in a cell at a defined time and under controlled
conditions. These mostly small molecules are products and substrates for enzymatic reactions and
therefore respond to environmental changes. The metabolome is a network of different metabolic
pathways interacting with each other through these metabolites. Changes in metabolite
concentration are the result of changes in the enzyme function.
Metabolome analysis aims to cover all or selected metabolites present in the cell at the defined time
point and is integrated into the field of systems biology. An overview is shown in Figure 2.2.
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2Metabolome analysis via GC-MS
Fig. 2.1. Central metabolism with defined metabolites present in the library.
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2Metabolome analysis via GC-MS
Fig. 2.2. Overview „OMICS“ and definition of Metabolomics.
The methods used in metabolome analysis comprise different sample preparation steps. These are
optimised for the used type of analysis. Typically, analyses are carried out on a gas chromatography
or a liquid chromatography coupled to a mass spectrometer (GC- or LC-MS). Furthermore,
capillary electrophoresis coupled to mass spectrometry (CE-MS) or nuclear magnetic resonance
spectroscopy (NMR) can be applied. The electrophoretic and chromatographic methods are used for
separation of the substances and enhance the resolution. The mass spectrometer is the analyser with
integrated detector. Further detector systems are e.g. flame ionisation detectors, thermal
conductivity detectors or UV-detectors.
2.1.2 Gas chromatography-Mass spectrometry
The coupling of gas chromatography and mass spectrometry is suitable for the analysis of volatile,
polar and lipophilic substances with low molecular mass. The method is fast, sensitive,
measurements are highly reproducible and analytes can be well identified and quantified.
During gas chromatographic separation a mixture of substances with different characteristics is
carried with the mobile phase (carrier gas) over the stationary phase on the column. The eluting
compounds are detected and is plotted in a chromatogram against the time. The mobile phase is the
10
2Metabolome analysis via GC-MS
inert gas helium, that does not interact with the stationary phase.
The separation on the column is caused by the retention of the analytes on the stationary phase.
Substances that are carried with the mobile phase can interact with the stationary phase and attach
to it before they detach. This causes a longer retention time of the substance in the column. The
retention is characteristic for each substance.
During gas chromatography, the temperature is increased continuously after injection of the sample.
Substances that are highly volatile vaporise and are carried over the column by the mobile phase.
Substances with higher boiling points vaporise at a later moment and are therefore carried over the
column later. Taken together, the separation is due to the substance specific affinity to the stationary
phase and to the vapour pressure.
Each substance that eluates from the column enters the ion source of the mass spectrometer, in
which the substances are ionised by electron impact. For this, electrons are emitted from a filament
into the electric field with a potential difference of 70 eV. The electrons are accelerated and hit the
molecules of the sample that enter the ion source. The voltage is high enough to cause
fragmentation of the molecules. The ionised substances are accelerated in the electric field and enter
the mass spectrometer in which they are separated by their mass-to-charge (m/z) ratio.
In the practical course a quadrupol mass spectrometer is used for the analysis. This is a mass filter
in which only ions with a defined m/z ratio can be detected at a time. Four parallel electrodes are
coordinated in the quadrupol. The combination of the alternating and direct current of the electrodes
causes that only ions with a defined m/z ratio can pass the quadrupol in a stable oscillating channel.
All ions with a differing m/z ratio are distracted, clash with the electrodes and stop. During one scan
of the mass range, ions with different m/z ratios can pass through the stable electric field of the
quadrupol and can be detected. The detection of the ions occurs at a secondary electron multiplier
and the signals are transferred to a computer with a software.
The results of the GC-MS analysis are displayed in a multidimensional chromatogram, in which the
x-axis represents the time and the y-axis the sum of all signal intensities of the detected ions (TIC =
total ion current). Each peak in the chromatogram also includes the detected fragments with a
defined m/z ratio at that time point.
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2Metabolome analysis via GC-MS
2.1.3
Derivatisation of substances in biological samples
The different substance classes like amino acids, sugars, steroids etc., that are present in biological
samples, are rarely or only volatile at high temperatures because of the intramolecular interactions
between the hydroxyl- and amine-residues of the molecules. To make these substances suitable for
GC-MS analysis and to increase their thermal stability, the polar functional groups are derivatised
with N-Methyl-N-(trimethylsilyl)-trifluoracetamid (MSTFA). During this reaction, the active
hydrogen atoms of hydroxyl- and amine-residues are nucleophilicly substituted through
trimethylsilyl-derivates (TMS-derivates). During derivatisation, more than one derivate of one
substance can be produced depending on the amount of functional groups in the molecule.
Many sugars are often present in biological samples. These can be present in the cyclic as well as in
the linear form. The derivatisation of all isomers would overload the chromatogram of a GC-MS
analysis and the identification would be difficult. Therefore, the samples are treated with
methoxyamine before MSTFA, to change the carbonyl-residue of the sugar into oximes. The
formation into an oxime fixes the linear form of the sugar molecules.
2.1.4 Identification of the metabolites
For identification of the metabolites in the biological samples it is important to apply a specific
retention index to each substance present in the chromatogram. The retention index is used for
identification of the components, because it is, in contrast to the retention time, stable against
technical routine work at the GC-MS machine (e.g. shortening of the column). To perform a
calibration of each measurement, an alkane mix is analysed each day as a time standard (Tab. 2.5).
Each alkane has a specific retention index, which corresponds to the number of the carbon atoms
present in the molecule multiplied with 100. Afterwards, the retention index of all other substances
can be calculated with the formula after Kováts (equation 2.1).
RI T =100[ y− x
t i −t x
 x]
t y −t x
Equation 2.1
with:
RI
retention index
T
in the temperature gradient
x
number of carbon atoms in the alkane, that eluates before the unknown
12
2Metabolome analysis via GC-MS
substance
y
number of carbon atoms in the alkane, that eluates after the unknown
substance
ti
retention time of the unknown substance
tx
retention time of the alkane, that eluates before the unknown substance
ty
retention time of the alkane, that eluates after the unknown substance
Additional to the retention index the mass spectrum of each substance is used for identification.
Each substance decomposes into a characteristic fragment pattern during ionisation in the ion
source. The display of all fragments is the mass spectrum.
With help of a substance library in which mass spectra and retention indices of different standard
substances is stored, a comparison of mass spectrum and retention index is done to identify the
unknown substance.
The analysis of the measurements (identification of the substances) is done as shown in Fig. 2.3.
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2Metabolome analysis via GC-MS
Fig. 2.3: Schedule for analysis of the GC-MS data.
Different programs are used for analysis of the data in the practical course. The software Metabolite
Detector first detects all peaks present in the chromatograms. Afterwards the peaks are identified
with the substance library that includes mass spectra and retention indices of all metabolites present
in Fig. 2.1. Afterwards, statistical analyses will be done. Details to the individual steps, you will
find later.
2.2 Introduction - Corynebacterium glutamicum
The bacterium used in the practical course is the gram-positive, aerobic, non-pathogen soil
bacterium Corynebacterium glutamicum (Fig. 2.4). It is a fast growing organism often used in
industry for the production of amino acids.
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2Metabolome analysis via GC-MS
Fig. 2.4: Electron microscopic picture of C. glutamicum Res 167.
The metabolic profile of the C. glutamicum wild type Res167 and a transposon mutant is analysed
in the practical course. The mutant originates from a transposon mutant bank constructed in the
group of Prof. Schomburg.
A transposon mutant is generated via random integration of the mobile transposon element into the
genome, that inhibits the function of the corresponding gene via interruption of the
OpenReadingFrame(ORF). For construction of the mutant bank the plasmid pAT6100 with the
transposon element IS6100 was used. The plasmid carries a kanamycin resistance as selection
marker.
For generation of a mutant bank, the transposon is transferred into C. glutamicum via
electroporation or conjugation with an E. coli helper strain and integrated into the genome via
homologue recombination. A positive integration of the transposon results in the kanamycin
resistance of the mutant. During cultivation of the mutant kanamycin has to be added to the medium
to prevent loss of the transposon.
Several methods exist to identify the integration point of the transposon in the genome. One method
uses amplification of the adjacent genomic regions with subsequent sequencing of the amplicon.
With BLAST analyses, the exact position of the transposon in the genome can be determined.
2.3 Schedule for metabolome analysis
For analysis of the intracellular metabolites, the bacterium is cultivated in buffled shaking flasks
until the desired growth phase is reached (e.g. exponential growth phase). Afterwards the cells are
harvested, washed to eliminate medium components and lysed. Polar metabolites are extracted and
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2Metabolome analysis via GC-MS
dried to eliminate water, that inhibits the following derivatisation reaction. During derivatisation the
volatility and thermal stability of the components is increased to make them suitable for GC-MS
analysis. The measurements are performed on a Trace GC-MS (Thermo). A principal overview for
preparation of the samples is shown in Fig 2.5.
Fig. 2.5: Preparation of samples for metabolome analysis.
2.3.1 Destruction of the cells and extraction of metabolites
For extraction of the intracellular metabolites, cells have to be destroyed. In contrast to protein
extraction, in metabolome analysis it is important to stop all enzymatic activity at the time of
harvesting to prevent ongoing metabolism of the cells that would change the metabolite
concentration.
For this, the cells are resuspended in a alcoholic solution and put in the ultra sonic bath for
destruction of the cells. This is a mechanical procedure. An overview about cell disruption methods
is shown in Fig. 2.6.
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2Metabolome analysis via GC-MS
Fig. 2.6: Cell disruption methods
For extraction of metabolites also different protocols exist. Because of the high amount of different
chemical characteristics of the metabolites (e.g. reactivity, polarity, thermal stability, etc.) a
complete extraction of all metabolites present in the cell at a time is not possible. The extraction
used in the practical course uses ethanol, water and chloroform to extract polar metabolites, that are
stable at 70°C.
2.3.2 Necessary preparations
All groups prepare in total 6 cultures of the wild type and 6 cultures of the mutant of C.
glutamicum. All prearrangements are done together.
1. Cultivation of the bacteria:
6 precultures per strain in full medium (prepared by the advisor)
•
5 mL full medium in a test tube are inoculated with bacteria from the agar plate
(mutant with 5 µL kanamycin solution)
6 over night cultures per strain in minimal medium
•
20 mL minimal medium in 100 mL flasks are inoculated with 500 µL preculture
•
add 20 µL of kanamycin solution to the medium of the mutant
6 main cultures per strain in minimal medium (prepared by the advisor )
•
determine the optical density of the over night culture (OD600nm)
•
inoculate 100 mL minimal medium in a 500 mL flask with the over night culture to a
optical density of 1 (see equation 2.2)
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2Metabolome analysis via GC-MS
Tab. 2.1 Pipetting schedule minimal medium
Component
Volume for 100 mL
Volume for 20 mL
Base solution 1
98,8 mL
19,76 mL
Base solution 2
1,1 mL
0,22 mL
Biotin solution
0,1 mL
0,02 mL
V inoculation =
V main
OD overnight
Equation 2.2
with:
Vinoculation = inoculation volume, i.e. needed volume of the over night culture
Vmain = volume of the main culture
ODovernight = optical density of the over night culture measured at a wavelength of 600 nm
2. Preparation of the solutions:
solutions prepared by the advisor :
•
1500 mL 0,9% NaCl solution (store at 4°C)
at the day of the experiment:
•
40 mL ethanol-ribitol-solution (4% (v/v) ribitol base solution)
•
ATTENTION: Always work under the flue!
1 mL methoxyamine-pyridine-solution (2% (w/v) methoxyamine in pyridine)
3. Preparation of the equipment:
•
put the rotor in the centrifuge and cool down to 4°C
•
preheat the ultrasonic bath to 70 °C
•
label (24) polypropylene test tubes, (24) 2 mL Eppendorf tubes and (24) glass vials
•
ice bath
IMPORTANT: Please ask the advisor for correct calculation of the solutions before preparation!
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2Metabolome analysis via GC-MS
2.3.3
Experimental procedure
The procedure is shown in Fig. 2.5. The steps have to be worked off direct successively. The
cultures/samples have to be kept on ice.
For the comparison of the different bacterial strains it is necessary to take a constant cell amount.
Therefore the optical density of the culture has to be determined before the harvesting. The needed
sampling volume (Vsampling) can be calculated with the equation 2.3.
V sampling =
50
OD main
Equation 2.3
•
From each culture two samples are taken. Each sample is put in a polypropylene test tube.
•
Centrifugation for 5 min at 4600 x g and 4°C. Decant the supernatants carefully. Resuspend
the pellet in 20 mL cold 0.9% NaCl solution.
•
Centrifugation for 5 min at 4600 x g and 4°C. Decant the supernatants carefully. Resuspend
the pellet in 20 mL cold 0.9% NaCl solution.
•
Centrifugation for 5 min at 4600 x g and 4°C. Decant the supernatant carefully.
•
Resuspend the pellets in 1.5 mL ethanol-ribitol-solution.
•
Destruction of the cells in the ultra sonic bath for 15 min at 70°C.
•
Cooling for 2 min in the ice bath.
•
Add 1.5 mL water to each sample and vortex for 1 min.
•
Add 1 mL chloroform to each sample (under the flue) and vortex for 1 min.
•
Centrifugation of all samples for 5 min at 14000 x g for separation of the polar and apolar
phase.
•
Take 1 mL of the upper polar phase and transfer it to a 2 mL Eppendorf tube under the flue.
Take care not to transfer parts of the interphase and the lower phase. If you have problems
contact the advisor!
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2Metabolome analysis via GC-MS
•
Dry the samples in the SpeedVac: at first for one hour with rotation under vacuum to avoid
boiling retardation of the ethanol. After this dry the samples completely over night without
rotation.
The non-volatile metabolites in the dried sample can not be used for GC-MS analysis. For this they
must be derivatised which occurs in two steps:
1. Methoxymation: add 40 µL methoxyamine-pyridine-solution to each sample and vortex for
1 min. Afterwards incubate the samples 90 min at 30°C and 600 rpm in a thermomixer.
2. Silylation: add 70 µL MSTFA to the sample and incubate it 30 min at 37°C and further
120 min at 25°C and 600 rpm.
The derivatised samples are centrifuged for 5 min at 13000 x g. The supernatants are transferred
into glass vials under the flue.
Prepare an additional sample with the alkane mix (8 different alkanes, time standard, see.
Tab. 2.5). Mix 6 µL alkane solution with 48 µL cyclohexane.
Put all samples into the autosampler of the GC-MS and start the measurement with the advisor.
Overview over the setting of the GC-MS
Injector (Programmed Temperature Vaporizer):
Tab. 2.2 PTV - parameter
Sample feeding
Start temperature
70 °C
Temperature ramp
14 K/s
End temperature
280 °C
Sample volume
1 µL
Modus
Split
Split ratio
25 : 1
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2Metabolome analysis via GC-MS
Gas chromatograph:
Tab. 2.3 Settings and parameters of the TRACE GC.
TRACE GC
Column
Agilent (DB-5MS)
Flow rate
1 mL/min
Start temperature
70 °C
1. hold time
1 min
1. temperature ramp
1 K/min
Intermediate temperature
76 °C
2. temperature ramp
6 K/min
End temperature
325 °C
2. hold time
10 min
Carrier gas
Helium
Mass spectrometer:
Tab. 2.4 Settings and parameter of the TRACE MS.
TRACE MS
Modus
Electron Ionisation
Ionisation voltage
70 V
Emission power
150 µA
Solvent dwell time
4.5 min
Measurement rate
2.5 scans/sec
Mass area
40 ... 460 m/z
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2Metabolome analysis via GC-MS
Alkane mix (time standard):
Tab. 2.5 Alkane mix parameter
Alkane
~Retention time [min]
Retention index
Decane
7
1000
Dodecane
13
1200
Pentadecane
21
1500
Nonadecane
28
1900
Docosane
33
2200
Octacosane
41
2800
Dotriacontane
46
3200
Hexatriacontane
49
3600
2.4 Evaluation
For the protocol you have to mention details of the cultivation of the bacterial strains. This includes
the name of the mutant, the optical density of the over night and the main culture, the inoculation
volume of the main culture and the sampling volume for the metabolome analysis.
2.4.1
Analysis of the raw data
With the raw data obtained from the GC-MS analysis the following analysis will be done:
At first load the chromatograms into the program Metabolite Detector.
With the help of the alkane mix a RI calibration of the samples is done. Thereby first of all the
peaks in the chromatogram are detected, the RIs are calculated and the peaks are identified by RI
and mass spectrum comparison. You get the needed settings for the software from the advisor.
In the Batch Analyse the loaded samples are compared and a result table is created. The
components in the table have to be controlled manually. For this purpose have a look on the mass
spectrum of the identified substance and compare it with the mass spectrum of the proposed
substance from the substance library. Sometimes there are shifts in the samples that have to be
corrected manually.
Before the export of the result table normalise all values with the internal standard ribitol. The table
can be exported as a .csv file and be loaded into the program i-tool. This program summarises all
derivatives of one substance.
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2Metabolome analysis via GC-MS
2.4.2
Statistical analysis and interpretation of the results
The statistic analysis will be performed with the program TigrMEV. With this program a t-test, a
principal component analysis (PCA), a hierarchical cluster analysis (HCA), heat maps etc. can be
performed. You get detailed descriptions of the different analyses from the advisor.
Illustration of the results:
1. Perform the statistical analysis with TigrMEV in order to show the significance of the
results. Thereby compare the wild type and the mutant. Choose different figures for the
illustration in the protocol.
2. Draw a bar diagram with all values that have a significance < 0.05 (one bar per metabolite).
3. Plot the relative metabolite concentrations of the mutant and the wild type double
logarithmic in a scatterplot. Show the calculated errors also in the same diagram. Label the
significant data points.
Describe the results with regard to the changes in the central metabolism (Fig. 2.1) between wild
type and mutant. Discuss the results in view of the present mutant.
Questions:
What is the metabolome?
How does metabolome analysis work?
Why can substances like e.g. sugars that are not volatile be separated by gas
chromatography?
What informations can be achieved from a chromatogram?
How does the identification of a metabolite occur in a GC-MS analysis?
23
3Growth phenotype of C. glutamicum: carbon metabolism
3 Growth phenotype of C. glutamicum: carbon metabolism
3.1 Growth phenotype
The phenotype is the sum of all properties of a cell, so that it does not only define the morphology
of a bacterium but also the molecular characteristic like e.g. the mRNA level. The growth
phenotype defines the growth behaviour of a bacterium. Does the bacterium grow and if yes, how
fast does it grow? Bacteria need essential nutrients in their environment for growth like carbon,
nitrogen, phosphor, sulphur, oxygen, hydrogen, etc. But there are also other properties of the
environment that influence the growth of a bacterium like toxic chemicals, temperature, pressure,
electromagnetic radiation, availability of water, etc.
The growth phenotype is from big importance for systems biology, that aims to understand the
whole organism and mimic it e.g. in a computer model to predict its behaviour.
3.2 Phenotype MicroArrays
The Phenotype MicroArrays is a method for a global detection of phenotypes. For this, a set of
nearly 2000 particular assays is used . The different assays are presented in 96-well plates. There are
about 200 assays for the investigation of the carbon metabolism, 400 assays for nitrogen
metabolism, 100 assays for phosphor and sulphur metabolism, 100 assays for biosynthetic
pathways, ion effects and pH effects and 1000 assays for chemical sensitivity (Fig. 3.1).
Fig. 3.1: The set of assays in the Phenotype MicroArrays (figure taken from B.R. Bochner, FEMS Micobiol Rev 33, 2009)
24
3Growth phenotype of C. glutamicum: carbon metabolism
The different assays consist of two components. The first component is a cell suspension, that
contains cells that were pregrown on agar plates containing a standard full medium for the
particular bacterium. The cell suspension has a standardized cell density which is set with a
turbidimeter. Additionally the cell suspension contains some salts and a tetrazolium redox dye. This
cell suspension is pipetted into the well of the 96-well plates, which are the second component.
These 96-well plates have the other nutrients needed for the assay dried down on the bottom. For
the investigation of the carbon metabolism the wells of the two different 96-well plates (PM1 and
PM2) contain a universal culture medium with all needed ingredients and a unique carbon source
(190 in total, Fig. 3.3). If the bacterium is able to catabolise the particular carbon source in the well
NADH is produced. The electron transport pathway in the bacterium will than transfer electrons
from NADH to the tetrazolium dye, which than gets a purple colour (Fig. 3.2a). With this method
respiration is measured instead of growth, which has the advantage of a higher sensitivity. Carbon
sources that are metabolised very good by the bacterium cause a dark purple colour and carbon
sources that are metabolised badly effect a light purple colour (Fig. 3.1).
a)
b)
Fig. 3.2: a) Linkage of carbon metabolism and tetrazolium (TV) reduction. b) Data collected using the Omnilog instrument and
software. The incubation time (x-axis) is plotted against the amount of formed purple colour (y-axis). Data from the
different bacteria strains are shown in red and green. Overlaps are shown in yellow (figures taken from B.R. Bochner,
FEMS Micobiol Rev 33, 2009).
25
3Growth phenotype of C. glutamicum: carbon metabolism
The amount of formed colour is monitored and recorded by the Omnilog instrument, which is an
incubator with a video camera and a linked computer. The outputs of the Omnilog instrument are
divers coloured graphs, so that two different strains can be compared (Fig. 3.2b).
PM1:
PM2:
Fig. 3.3: MicroPlate PM1 and 2: Carbon sources (source Biolog, Inc.)
26
3Growth phenotype of C. glutamicum: carbon metabolism
3.3 Goals and principle of the experiment
In the following experiment the growth phenotype of C. glutamicum wild type (WT) and a ptsG
transposon mutant (ptsG::tnIS6100) is investigated using different carbon sources with Phenotype
MicroAssays and shake flask cultivations. The gene ptsG encodes for the glucose-specific enzyme II
BC component of the phosphotransferase system (PTS, Fig. 3.4). The PTS is an active transport
system for mostly hexoses in microorganisms. During the transport, a phosphate group is
transferred from phosphoenolpyruvate via several protein kinases to the sugar. For Phenotype
MicroAssays the MircoPlates PM1 and PM2 (Fig. 3.3) are used. To verify the results from the
Phenotype MicroAssays shake flask cultivations with selected carbon sources are necessary, because
Phenotype MicroAssays detect only respiration and not growth. The bacterial growth is determined
by measuring the optical density at 600 nm.
Fig. 3.4: Phosphotransferase system using the example of glucose. PEP phosphoenolpyruvate, P phosphateee group, E enzyme, Hpr
histidine protein or heat-stable protein (figure taken from http://de.wikipedia.org/wiki/Phosphotransferase-System).
3.4 Experiment
3.4.1
Investigation of the carbon metabolism of C. glutamicum WT and ptsG::tnIS6100 by
Phenotype MicroAssays
Bacterial strains:
C. glutamicum RES167
C. glutamicum RES167 ptsG::tnIS6100
27
3Growth phenotype of C. glutamicum: carbon metabolism
Media:
Brain Heart Infusion (BHI) Medium: 37 g/L Brain Heart Infusion; for agar plates: 15 g/L agar agar
Solutions:
1.2x IF-0a fluid
1x IF-0a fluid: 5 mL 1.2x IF-0a fluid, 1 mL dH2O
100x Redox Dye Mix F
1000x biotin: 200 g/L, sterilised by filtration
1000x trace elements solution MM1: FeSO 4 · 7H2O 28.5 g/L, MnSO4 · H2O 16.5 g/L, ZnSO4 · 7H2O
6.4 g/L, CuSO4 · 5H2O 0.764 g/L, CoCl2 · 6H2O 0.128 g/L, NiCl2 · 6H2O 0.044 g/L,
Na2MoO4 · 2H2O 0.064 g/L, H3BO3 0.048 g/L, KAl(SO4)2 · 12H2O 0.028 g/L, adjust the pH
with 3 M sulphuric acid at 1 and sterilised by filtration
12x additive: 12 µL 1000x biotin, 12 µL 1000x trace elements solution, 976 µL dH2O
Inoculation fluid: 10 mL 1.2x IF-0a fluid, 0.12 mL 100x Redox Dye Mix F, 1 mL 12x additive
Material:
Phenotype MicroArrays MicroPlate PM1 and 2
Experimental procedure (Fig: 3.5):
•
Two groups work with one MicroPlate.
•
Grow C. glutamicum WT and ptsG::tnIS6100 on BHI agar plates overnight at 30°C.
•
Prepare a test tube containing 6 mL of 1x IF-0a fluid.
•
Remove cells from the agar plate using a sterile swab and transfer into the 6 mL of 1x IF-0a
fluid. Stir the cell suspension with the swab to obtain a uniform suspension. Check the
turbidity of the suspension with the turbidimeter. Add cells to achieve 81% T
(transmittance).
28
3Growth phenotype of C. glutamicum: carbon metabolism
•
Add 0.88 mL of the 81% T cell suspension to 11.12 mL inoculation fluid.
•
Inoculate the MicroPlate with this inoculation cell suspension, 100 µL per well.
•
Incubate the inoculated MicroPlate in the Omnilog instrument for 48 h at 30°C.
Fig.3.5: Experimental procedure for Phenotype MicroArrays MicroPlate PM1 and 2 (source Biolog, Inc.).
Evaluation:
Evaluate the data collected using the Omnilog instrument with the Omnilog software. Use the
results from all groups. What carbon sources does C. glutamicum metabolise? What are the
differences between the WT and the mutant ptsG::tnIS6100? How can this differences be
explained?
3.4.2
Investigation of the carbon metabolism of C. glutamicum WT and ptsG::tnIS6100 by
shake flask cultivations
For the investigation of the carbon metabolism by shake flask cultivations C. glutamicum WT and
ptsG::tnIS6100 are cultivated with different carbon sources (interesting carbon sources from the
Phenotype MicroArrays experiment). During the cultivation the cell growth is analysed by
measuring the optical density of the culture at 600 nm (OD600).
29
3Growth phenotype of C. glutamicum: carbon metabolism
Bacterial strains:
C. glutamicum RES167
C. glutamicum RES167 ptsG::tnIS6100
Media:
Brain heart infusion (BHI) medium: 37 g/L Brain heart infusion; for agar plates: 15 g/L agar agar
MM1 medium (111 mM carbon source): base solution 1: 98.8 mL, base solution 2: 1.1 mL, 1000x
biotin 0.1 mL
Solutions:
Base solution 1: MM1 base solution: 94.7 mL, 25x glucose or an other carbon source: 4 mL, 1000x
calcium chloride: 0.1 mL
Base solution 2: 100x magnesium sulphate: 1 mL, 1000x trace elements solution: 0.1 mL
1000x biotin: 200 g/L, sterilised by filtration
MM1 base solution: ammonium sulphate 5 g, urea 5 g, di-potassium hydrogen phosphate 1.53 g,
potassium dihydrogen phosphate 2g, dissolve the substances in 0.85 mL dH2O and adjust the
pH with 5 M KOH at 7, fill up with dH2O to 0.947 mL and autoclave the solution
25x glucose solution (2.78 M): 550 g/L Glucose · H2O, autoclave the solution
1000x calcium chloride solution: 10g/L alcium chloride· 2H2O, autoclave the solution
100x magnesium sulphate solution: 25 g/L magnesium sulphate · 7H2O, autoclave the solution
1000x trace elements solution MM1: FeSO 4 · 7H2O 28.5 g/L, MnSO4 · H2O 16.5 g/L, ZnSO4 · 7H2O
6.4 g/L, CuSO4 · 5H2O 0.764 g/L, CoCl2 · 6H2O 0.128 g/L, NiCl2 · 6H2O 0.044 g/L,
Na2MoO4 · 2H2O 0.064 g/L, H3BO3 0.048 g/L, KAl(SO4)2 · 12H2O 0.028 g/L, adjust the pH
with 3 M sulphuric acid at 1 and sterilised by filtration
1000x kanamycin solution: 25 mg/mL, sterilised by filtration
30
3Growth phenotype of C. glutamicum: carbon metabolism
Experimental procedure :
•
Grow C. glutamicum WT and ptsG::tnIS6100 on BHI agar plates respectively BHI
kanamycin (25 µg/mL) agar plates overnight at 30°C.
•
Inoculate 5 mL BHI medium respectively BHI kanamycin (25 µg/mL) medium with bacteria
from the agar plate with an inoculation loop and incubate at 30°C and 180 rpm for 6 - 7
hours in a incubator.
•
Inoculate 20 mL MM1 medium respectively MM1 kanamycin (25 µg/mL) with 500 µL of
the preculture and incubate at 30°C and 180 rpm over night in an incubator.
•
Inoculate 100 mL MM1 medium at a OD 600 = 1 with the over night culture and incubate at
30°C and 180 rpm.
•
Measure the OD600 every half an hour.
Evaluation:
Draw growth curves (Fig. 3.6) of the different cultivations and compare them. Are the results from
the Phenotype MicroArrays experiment confirmed?
Fig. 3.6: Growth curve of a bacterial culture (source: http://science.jrank.org/pages/12415/bacterial-growth-curve.html).
31
4Enzyme kinetics
4 Enzyme kinetics
4.1 Enzymes
Enzymes are biocatalysts that accelerate biochemical reactions in cells. The substance whose
transformation is catalysed through an enzyme is called the substrate of this enzyme. Enzymes
recognize their substrates with a high specificity and accelerate the reaction up to 10 9 fold and more.
This reaction acceleration is caused by the stabilization of the transition state through the enzyme
and the thereby released binding energy that decreases the activation energy of the reaction. The
binding energy is realised by the following factors:
Catalysis mechanisms:
1. Acid-base catalysis
2. Covalent catalysis
3. Metal ion catalysis
4. Electrostatic catalysis
5. Catalysis of the neighbour groups and orientation effects
6. Catalysis by stabilization of the transition state
The substrate is mostly dehydrated in the active centre i.e. the contacts between enzyme and
substrate replace contacts between water and substrate. Normally one or multiple active side chains
in the active centre are directly involved in the catalysis, e.g. SH or OH groups of particular amino
acids. Many enzymes contain an additional prosthetic group in the active centre. Prosthetic groups
(“non-protein groups”) are normally covalently bound metal ions (Cu 2+, Zn2+, Fe2+) or heme metal
complexes, co-substrates are not covalently bound organic compounds (e.g. NAD+ or biotin). The
prosthetic groups have chemical reactivities, which the functional groups of the amino acids do not
have.
32
4Enzyme kinetics
4.2 Enzyme kinetics
The most simplest chemical reaction is the reaction of first order:
S
→
P
(S substrate; P product)
The reaction rate v in the absence of an enzyme is:
v=
−d [ S ] d [P ]
=
=k⋅[S ]
dt
dt
Equation 4.1
(v reaction rate; S substrate; t time; P product; k rate constant)
The reaction rate is here only dependent on the concentration of the reaction participant S. The
relationship between velocity and concentration of S is in this case linear. The proportionality factor
k is called rate constant. This constant is dependent on the external reaction conditions as pH-value,
temperature etc.
In the presence of an enzyme the reaction rate and the concentration are not longer proportional to
each other. With very little substrate concentrations [S] the reaction rate v rises at first nearly linear
with the substrate concentration; with increasing [S] the increasing of v is slowed down till a
constant value, the maximal reaction rate v max is reached at very high [S]. A kinetic of first order (v
is proportional to [S]) is here becoming continuously a kinetic of zero order (v is independent from
[S]). The relationship between velocity and substrate concentration has a shape of a right-angled
hyperbola.
For the description of this curve the Michaelis constant K M was established. KM equates the
substrate concentration at which the half maximal reaction rate (v max/2) is reached. It is an
approximate dimension for the affinity (strength of the enzyme-substrate-binding) of an enzyme:
the higher the affinity the steeper the curve and the smaller KM (Fig. 4.1).
33
4Enzyme kinetics
Fig. 4.1: Michaelis-Menten plot
The Michaelis-Menten kinetic is described by the following formula:
v=
v max⋅[ S ]
K M [S ]
Equation 4.2
(v reaction rate; vmax theoretical maximal reaction rate at infinite high [S]; [S] substrate
concentration; KM Michaelis constant; is [S] = KM the reaction rate becomes v =vmax/2)
The unit for the catalytic activity of an enzyme is katal (kat). It defines the enzyme yield which
catalyses the transformation of 1 mol substrate per second at 30°C (1 kat = 1 mol substrate / sec)
With the Michaelis-Menten diagram (reaction rate v plotted against substrate concentration [S], see
figure 4.1) vmax and KM can only be approximated.
For an easier identification of vmax and KM the Michaelis-Menten equation can be linearised. One
method is the direct linear plot by Eisenthal and Cornish-Bowden. In this plot each line represents
one measurement. The negative value of the substrate concentration (x-axis) is joined with a
straight line with the measured reaction rate v (y-axis). The intersection point of the lines from all
measurements yields vmax and KM (Fig. 4.2). The Eisenthal-Cornish-Bowden plot is described by the
following formula:
34
4Enzyme kinetics
v max=v
v
⋅K
[S ] M
Equation 4.3
(v reaction rate; vmax maximal reaction rate; [S] substrate concentration; KM Michaelis constant)
Fig. 4.2: Eisenthal-Cornish-Bowden plot
Another linearisation method is the Lineweaver-Burk plot. In this double reciprocal diagram the
reciprocal substrate concentration 1/[S] (x-axis) is plotted against the reciprocal reaction rate 1/v (yaxis). With the help of the intersection points of the straight line with the x- and the y-axis v max and
KM can be calculated (Fig. 4.3). The Lineweaver-Burk plot is described by the following formula:
1 KM 1
1
=
⋅ 
v v max [ S ] v max
Equation 4.4
(v reaction rate; vmax maximal reaction rate; [S] substrate concentration; KM Michaelis constant)
35
4Enzyme kinetics
Fig. 4.3: Lineweaver-Burk plot
In the Eadie-Hofstee plot the reaction rate v divided by the substrate concentration [S] (x-axis) is
plotted against the reaction rate v. The slope of the straight line is equal to -K M and the intersection
point of the line with the y-axis is vmax (Fig. 4.4). The Eadie-Hofstee plot is described by the
following formula:
v
v=−K M⋅ v max
[S]
Equation 4.5
(v reaction rate; vmax maximal reaction rate; [S] substrate concentration; KM Michaelis constant)
Fig. 4.4: Eadie-Hofstee plot
36
4Enzyme kinetics
In the also linearising Hanes-Woolf plot the substrate concentration [S] (x-axis) is plotted against
the substrate concentration [S] divided by the reaction rate v (y-axis). The intersection point of the
straight line with the x-axis is -K M and the slope of the line is 1/v max (Fig. 4.5). This plot is described
by the formula:
K
[S ]
1
=
⋅[S ] M
v v max
v max
Equation 4.6
(v reaction rate; vmax maximal reaction rate; [S] substrate concentration; KM Michaelis constant)
Fig. 4.5: Hanes-Woolf plot
4.3 Enzyme inhibition
Enzyme activity is not always desirable. For all organisms it is very important to control its enzyme
activity e.g. by inhibition. In nature many inhibitors with different inhibition mechanisms are
known. Reversible inhibitors have no covalent binding to the enzyme and the inhibition is
reversible. There are three different mechanisms of reversible enzyme inhibition:
1. A competitive inhibitor competes against the substrate for the active centre of the enzyme.
Both can not bind at the same time to the same enzyme. The inhibitor in contrast to the
substrate can not be transformed by the enzyme (Fig. 4.6). In the presence of a competitive
inhibitor a higher substrate concentration must be used to reach the half maximal reaction
rate. In this case KM gets a higher value but the maximal reaction rate stays stable (Fig. 4.7).
37
4Enzyme kinetics
Fig. 4.6: Model for competitive inhibition. E enzyme; S substrate; I inhibitor; P product; ES enzyme-substrate-complex; EI enzymeinhibitor-complex; KI inhibition constant (Figure taken from: Nelson & Cox, Lehninger Biochemie, Springer Lehrbuch)
Fig. 4.7: Lineweaver-Burk plot of a competitive inhibited enzyme
The inhibition constant KI is defined as the equilibrium constant for the dissociation
reaction of the enzyme-inhibitor-complex. The smaller KI the stronger the binding of the
inhibitor to the enzyme and with it the inhibition effect of the inhibitor.
2.
A uncompetitive inhibitor does not bind to the free enzyme. It only binds to the enzymesubstrate-complex (Fig. 4.8). In contrast to a competitive inhibition here K M and vmax are
decreased (Fig. 4.9).
38
4Enzyme kinetics
Fig. 4.8: Model for uncompetitive inhibition. E enzyme; S substrate; I inhibitor; P product; ES enzyme-substrate-complex; EI
enzyme-inhibitor-complex; ESI enzyme-substrate-inhibitor-complex; K I inhibition constant.(Figure taken from: Nelson &
Cox, Lehninger Biochemie, Springer Lehrbuch)
Fig. 4.9: Lineweaver-Burk plot of a uncompetitive inhibited enzyme
3. A non-competitive or mixed inhibitor binds to the free enzyme as well as to the enzyme
-substrate-complex (Fig. 4.10), so that vmax is decreased and KM stays stable (noncompetitive inhibition, Fig. 4.11 a), increases (mixed inhibition, Fig. 4.11 b).
Fig. 4.10:Model for non-competitive or mixed inhibition. E enzyme; S substrate; I inhibitor; P product; ES enzyme-substratecomplex; EI enzyme-inhibitor-complex; ESI enzyme-substrate-inhibitor-complex; K I inhibition constant. (Figure taken
from: Nelson & Cox, Lehninger Biochemie, Springer Lehrbuch)
39
4Enzyme kinetics
a)
b)
Fig. 4.11: Lineweaver-Burk plot of a non-competive inhibited enzyme. a) non-competitive inhibition, K M stays stable; b) mixed
inhibition, KM increases.
The inhibitor constant KI can be determined with the help of a dixon plot. Here the inhibitor
concentration (x-axis) is plotted against 1/v. The intersection point of all lines of the measurement
(different substrate concentrations) corresponds to the constant KI (Fig. 4.12).
Fig. 4.12: Dixon plot
4.4 Photometric activity tests
Photometry names measurement methods in the area of the visible light. The absorption photometry
measures indirectly the light absorption (“attenuation of light”) and concludes out of it the
concentration of an analyte. When light radiographes a cuvette that is filled with a transparent
solution one part of the light is absorbed by photometric active particles in the solution, one part
passes and a small part is scattered. Therefore by the measurement of the light absorption the
concentration of the light absorbing particles can be calculated.
The base for the measurement is the “Lambert-Beer law”. This law says that the photometric
40
4Enzyme kinetics
absorption A is proportional to the distance the light travels through the measurement solution d and
the concentration of the solute substance c. The proportionality factor ε (“ molar absorption
coefficient”) is amongst others dependent on the substance and the used wave length. The enzyme
activity EA is measured as the transformed particle amount of the substrate Δn per time t (Equation
4.10).
Lambert-Beer law:
A=⋅d⋅c
Equation 4.7
respectively
 A=⋅d⋅ c
Equation 4.8
 c=
n
V
Equation 4.9
EA=
n
t
Equation 4.10
The outcome of this is:
EA=
 A⋅V
t⋅⋅d
Equation 4.11
In our experiment:
EA=158.7
A
t
Equation 4.12
(EA enzyme activity [nkat]; t time [s]; ΔA absorption change; ε molar absorption coefficient e.g. for
NADH = 6300 L · mol-1 · cm-1; d distance the light travels through the solution [cm]; V volume [L])
4.5 Alcohol dehydrogenase from Saccharomyces cerivisiae
The yeast Saccharomyces cerivisiae ferments glucose under anaerobic conditions to ethanol and
CO2. In this process the alcohol dehydrogenase (ADH) reduces acetaldehyde to ethanol. NADH 2
provides the reduction equivalents.
ADH
Ethanol
+
NAD
+
↔
Acetaldehyde
+
NADH+H+
41
4Enzyme kinetics
4.6 Goals and principle of the experiment
In the following experiment the activity of the ADH from the yeast Saccharomyes cerivisiae will be
investigated with different inhibitors. The activity is measured optically by measuring the increasing
amount of the co-substrate NADH at a wavelength of 340 nm. NADH has no pyrimidine ring with
an aromatic character any more so that absorption behaviour changes (Fig. 4.13a. NADH has a
second absorption maximum at 340 nm (Fig. 4.13b). The increase of the absorption per time unit
for the transformation of NAD+ to NADH is a direct size for the reaction rate.
a)
b)
Fig. 4.13: Structure of NAD+ and NADH (a) and the absorption maxima of NAD+ and NADH (b)
4.7
Recording of the absorption
For the recording and visualisation of the absorption (Fig. 4.14) a photometer is used.
Fig. 4.14: Absorption-time-curve
42
4Enzyme kinetics
From the recorded absorption change the linear initial velocity is investigated graphically by
determine the slope of the linear area. The slope of the line is the absorption change per time unit
(ΔA/s). Out of this the reaction rate (in ΔA/s) and hence the corresponding activity (in nkat) can be
calculated.
4.8 Experiment
4.8.1
Investigation of the KM constant for ethanol
For the investigation of the KM constant different amounts of substrate will be used.
Solutions:
Glycine buffer: 0.1 M; pH 9.0
Ethanol solution: 0.5 M in glycine buffer pH 9.0
ADH solution: 5 mg/100 mL in glycine buffer pH 9.0
NAD solution: 24 mg/mL in glycine buffer pH 9.0
Experimental procedure:
•
Keep the NAD and ADH solutions on ice. All other solutions have to be at room
temperature.
•
Pipette the following samples in Eppendorf tubes:
Tab. 4.1: Pipetting schedule for the investigation of the K M constant for ethanol
Final
1.25 mM 2.5 mM 3.5 mM 5 mM
concentrati
on ethanol
6.5 mM 10 mM 12.5 mM 25 mM
50 mM
100 mM
µL NAD
solution
100
100
100
100
100
100
100
100
100
100
µL ethanol 2.5
solution
5
7
10
13
20
25
50
100
200
µL glycine
buffer
845
843
840
837
830
825
800
750
650
847.5
43
4Enzyme kinetics
•
Connect a USB stick with the photometer.
•
Press the key Test.
•
Select Kinetik with the arrow keys and confirm with Enter.
•
Either the test program with the name ADH is already loaded or press Gespeich. Tests in
the bottom of the display and choose Internen Test and then the test program ADH.
•
The following settings should be selected:
• Messmodus: Absorption
• Wellenlänge: 340 nm
• Ref.-Wellenlg.korrektur: Aus
• Delay-Zeit: 0:00
• Intervallzeit: 0:02
• Gesamtlaufzeit: 0:03:00
• Zeige Ergebnis: Ein
continuing with weitere Parameter
• Faktor: 1.000
• Einheit: C
• Linearitätswert: 0.005
• Probenposition: Einfachhalter
• ID#: 1
• Untere/Obere Grenze: -9999/9999
• Auto Speichern: Aus
•
Select Durchlauf Test in the bottom of the display.
•
Transfer the sample in a cuvette and place it in position B in the photometer.
•
Select Leerprobe einstellen in the bottom of the display to perform a zero compensation.
•
Pipette 50 µL of the ADH solution in the sample: Open the lid of the photometer; pipette
the enzyme in the solution in the cuvette; mix the sample with a small spattle; close the lid
of the photometer.
•
Immediately press Probe messen in the bottom of the display.
•
The photometer will now measure the absorption for 3 min every 2 sec. You can observe this
on the display.
•
After the measurement select Grafik bearbeiten in the bottom of the display.
•
Select Speichern Daten in the bottom of the display.
44
4Enzyme kinetics
•
Generate a file name (each measurement file must have another name!) and press Bez.
übernehmen in the bottom of the display.
•
The file will now be saved in csv format on the USB stick.
•
Repeat this measurement with the other samples.
Evaluation:
From the linear beginning region of the recorded curves the reaction rate is achieved. The slope of
the line is the absorption change per time unit (ΔA/s). With the help of the equation 4.12 now the
enzyme activity can be calculated. For this calculation use only data from the linear region of the
measurement.
Draw a Michaelis-Menten plot with the calculated values (v vs. [S]; v = enzyme activity) and
estimate vmax and KM. For a more precise determination of vmax and KM a Eisenthal-Cornish-Bowden,
a Lineweaver-Burk, a Eadie-Hofstee and a Hanes-Woolf plot are to be draw (in all cases v =
enzyme activity). Compare the results from the different plot types. What are the advantages and
disadvantages of the particular plots? Compare also the calculated values with the values from
literature (e.g. BRENDA Database: http://www.brenda-enzymes.org/).
4.8.2
Investigation of the substrate specificity of the ADH
For the investigation of the substrate specificity of the ADH different substrates are used.
Solutions:
Glycine buffer: 0.1 M; pH 9.0
Substrate solutions (in glycine buffer pH 9.0):0.5 M ethanol; 0.5 M 1-butanol, 0.5 M
trifluoroethanol, 0.5 M salicylic acid, 10 mM acetylsalicylic acid
ADH solution: 5 mg/100 mL in glycine buffer pH 9.0
NAD solution: 24 mg/mL in glycine buffer pH 9.0
45
4Enzyme kinetics
Experimental procedure:
•
Keep the NAD and ADH solutions on ice. All other solutions have to be at room
temperature.
•
Pipette the following samples in Eppendorf tubes:
Tab. 4.2: Pipetting schedule for the investigation of the substrate specificity of the ADH
Substrate
Ethanol
(50 mM)
1-butanol Trifluoroethanol
(50 mM) (50 mM)
Salicylic acid Acetylsalicylic
(50 mM)
acid (8.5 mM)
µL NAD
solution
100
100
100
100
100
µL
substrate
solution
100
100
100
100
850
µL glycine 750
buffer
750
750
750
/
•
Continue as in 4.8.1
Evaluation:
Draw a bar diagram with the calculated enzyme activity. Compare the calculated activities. Why are
the substrates transformed differently well by the ADH?
4.8.3
Investigation of the KI constant of trifluoroethanol
For the investigation of the KI constant of trifluoroethanol different substrate and inhibitor
concentrations are used.
Solutions:
Glycine buffer: 0.1 M; pH 9.0
Ethanol solution: 0.5 M in glycine buffer pH 9.0
Trifluoroethanol solution: 0.5 M in glycine buffer pH 9.0
ADH solution: 5 mg/100 mL in glycine buffer pH 9.0
NAD solution: 24 mg/mL in glycine buffer pH 9.0
46
4Enzyme kinetics
Experimental procedure:
•
Keep the NAD and ADH solutions on ice. All other solutions have to be at room
temperature.
•
Pipette the following samples in Eppendorf tubes:
Tab. 4.3: Pipetting schedule for the investigation of the K I constant of trifluoroethanol
Substrate
10 mM
concentration
10 mM
10 mM
10 mM
10 mM
20 mM
20 mM
20 mM
20 mM
20 mM
Inhibitor
0 mM
concentration
0.5 mM
1 mM
2 mM
4 mM
0 mM
0.5 mM
1 mM
2 mM
4 mM
µL NAD
solution
100
100
100
100
100
100
100
100
100
100
µL inhibitor
solution
/
1
2
4
8
/
1
2
4
8
µL substrate
solution
20
20
20
20
20
40
40
40
40
40
µL glycine
buffer
830
829
828
826
822
810
809
808
806
802
Substrate
30 mM
concentration
30 mM
30 mM
30 mM
30 mM
50 mM
50 mM
50 mM
50 mM
50 mM
Inhibitor
0 mM
concentration
0.5 mM
1 mM
2 mM
4 mM
0 mM
0.5 mM
1 mM
2 mM
4 mM
µL NAD
solution
100
100
100
100
100
100
100
100
100
100
µL inhibitor
solution
/
1
2
4
8
/
1
2
4
8
µL substrate
solution
60
60
60
60
60
100
100
100
100
100
µL glycine
buffer
790
789
788
786
782
750
749
748
746
742
•
Continue as in 4.8.1
Evaluation:
Draw a Michaelis-Menten plot with the calculated values (v vs. [S]; v = enzyme activity; all
inhibitor concentrations in one plot) and estimate vmax and KM. For a more precise determination of
vmax and KM draw a Lineweaver-Burk plot(also here v = enzyme activity and all inhibitor
concentrations in one plot). For the investigation of the K I constant draw a Dixon plot. How do v max
and KM change with increasing inhibitor concentration in the Michaelis-Menten and Lineweaver47
4Enzyme kinetics
Burk plot? What type of inhibition causes trifluoroethanol?
4.8.4
Investigation of the KI constant of 1-butanol
For the investigation of the KI constant of 1-butanol different substrate and inhibitor concentrations
are used.
Solutions:
Glycine buffer: 0.1 M; pH 9.0
Ethanol solution: 0.5 M in glycine buffer pH 9.0
1-butanol solution: 0.5 M in glycine buffer pH 9.0
ADH solution: 5 mg/100 mL in glycine buffer pH 9.0
NAD solution: 24 mg/mL in glycine buffer pH 9.0
Experimental procedure:
•
Keep the NAD and ADH solutions on ice. All other solutions have to be at room
temperature.
•
Pipette the following samples in Eppendorf tubes:
Tab. 4.4: Pipetting schedule for the investigation of the K I constant of 1-butanol
Substrate
10 mM
concentration
10 mM
10 mM
10 mM
10 mM
20 mM
20 mM
20 mM
20 mM
20 mM
Inhibitor
0 mM
concentration
10 mM
20 mM
50 mM
100 mM
0 mM
10 mM
20 mM
50 mM
100 mM
µL NAD
solution
100
100
100
100
100
100
100
100
100
100
µL inhibitor
solution
/
20
40
100
200
/
20
40
100
200
µL substrate
solution
20
20
20
20
20
40
40
40
40
40
µL glycine
buffer
830
810
790
730
630
810
790
770
710
610
48
4Enzyme kinetics
Substrate
30 mM
concentration
30 mM
30 mM
30 mM
30 mM
50 mM
50 mM
50 mM
50 mM
50 mM
Inhibitor
0 mM
concentration
10 mM
20 mM
50 mM
100 mM
0 mM
10 mM
20 mM
50 mM
100 mM
µL NAD
solution
100
100
100
100
100
100
100
100
100
100
µL inhibitor
solution
/
20
40
100
200
/
20
40
100
200
µL substrate
solution
60
60
60
60
60
100
100
100
100
100
µL glycine
buffer
790
770
750
690
590
750
730
710
650
550
•
Continue as in 4.8.1
Evaluation:
Draw a Michaelis-Menten plot with the calculated values (v vs. [S]; v = enzyme activity; all
inhibitor concentrations in one plot) and estimate vmax and KM. For a more precise determination of
vmax and KM draw a Lineweaver-Burk plot (also here v = enzyme activity and all inhibitor
concentrations in one plot). For the investigation of the K I constant draw a Dixon plot. How do v max
and KM change with increasing inhibitor concentration in the Michaelis-Menten and LineweaverBurk plot? What type of inhibition causes 1-butanol?
4.8.5
Investigation of the KI constant of salicylic acid (substrate ethanol)
For the investigation of the K I constant of salicylic acid different substrate and inhibitor
concentrations are used.
Solutions:
Glycine buffer: 0.1 M; pH 9.0
Ethanol solution: 0.5 M in glycine buffer pH 9.0
Salicylic acid solution: 0.5 M in glycine buffer pH 9.0
ADH solution: 5 mg/100 mL in glycine buffer pH 9.0
NAD solution: 24 mg/mL in glycine buffer pH 9.0
49
4Enzyme kinetics
Experimental procedure:
•
Keep the NAD and ADH solutions on ice. All other solutions have to be at room
temperature.
•
Pipette the following samples in Eppendorf tubes:
Tab. 4.5: Pipetting schedule for the investigation of the K I constant of salicylic acid
Substrate
5 mM
concentration
5 mM
5 mM
5 mM
5 mM
10 mM
10 mM
10 mM
10 mM
10 mM
Inhibitor
0 mM
concentration
2.5 mM
5 mM
10 mM
25 mM
0 mM
2.5 mM
5 mM
10 mM
25 mM
µL NAD
solution
100
100
100
100
100
100
100
100
100
100
µL inhibitor
solution
/
5
10
20
50
/
5
10
20
50
µL substrate
solution
10
10
10
10
10
20
20
20
20
20
µL glycine
buffer
840
835
830
820
790
830
825
820
810
780
Substrate
20 mM
concentration
20 mM
20 mM
20 mM
20 mM
50 mM
50 mM
50 mM
50 mM
50 mM
Inhibitor
0 mM
concentration
2.5 mM
5 mM
10 mM
25 mM
0 mM
2.5 mM
5 mM
10 mM
25 mM
µL NAD
solution
100
100
100
100
100
100
100
100
100
100
µL inhibitor
solution
/
5
10
20
50
/
5
10
20
50
µL substrate
solution
40
40
40
40
40
100
100
100
100
100
µL glycine
buffer
810
805
800
790
760
750
745
740
730
700
•
Continue as in 4.8.1
Evaluation:
Draw a Michaelis-Menten plot with the calculated values (v vs. [S]; v = enzyme activity; all
inhibitor concentrations in one plot) and estimate vmax and KM. For a more precise determination of
vmax and KM draw a Lineweaver-Burk plot (also here v = enzyme activity and all inhibitor
concentrations in one plot). For the investigation of the K I constant draw a Dixon plot. How do v max
and KM change with increasing inhibitor concentration in the Michaelis-Menten and Lineweaver50
4Enzyme kinetics
Burk plot? What type of inhibition causes salicylic acid?
4.8.6
Investigation of the KI constant of salicylic acid (co-substrate NAD+)
For the investigation of the KI constant of salicylic acid different co-substrate and inhibitor
concentrations are used.
Solutions:
Glycine buffer: 0.1 M; pH 9.0
Ethanol solution: 0.5 M in glycine buffer pH 9.0
Salicylic acid solution: 0.5 M in glycine buffer pH 9.0
ADH solution: 5 mg/100 mL in glycine buffer pH 9.0
NAD solution: 24 mg/mL (36 mM) in glycine buffer pH 9.0
Experimental procedure:
•
Keep the NAD and ADH solutions on ice. All other solutions have to be at room
temperature.
•
Pipette the following samples in Eppendorf tubes:
Tab. 4.6: Pipetting schedule for the investigation of the K I constant of salicylic acid
Co-substrate 0.5 mM
concentration
0.5 mM
0.5 mM
0.5mM
0.5 mM
1 mM
1 mM
1 mM
1 mM
1 mM
Inhibitor
0 mM
concentration
2.5 mM
5 mM
10 mM
25 mM
0 mM
2.5 mM
5 mM
10 mM
25 mM
µL NAD
solution
14
14
14
14
14
28
28
28
28
28
µL inhibitor
solution
/
5
10
20
50
/
5
10
20
50
µL substrate
solution
50
50
50
50
50
50
50
50
50
50
µL glycine
buffer
886
881
876
866
836
872
867
862
852
822
51
4Enzyme kinetics
Co-substrate 2 mM
concentration
2 mM
2 mM
2 mM
2 mM
4 mM
4 mM
4 mM
4 mM
4 mM
Inhibitor
0 mM
concentration
2.5 mM
5 mM
10 mM
25 mM
0 mM
2.5 mM
5 mM
10 mM
25 mM
µL NAD
solution
56
56
56
56
56
111
111
111
111
111
µL inhibitor
solution
/
5
10
20
50
/
5
10
20
50
µL substrate
solution
50
50
50
50
50
50
50
50
50
50
µL glycine
buffer
844
839
834
824
794
789
784
779
769
739
Co-substrate 8 mM
concentration
8 mM
8 mM
8 mM
8 mM
Inhibitor
0 mM
concentration
2.5 mM
5 mM
10 mM
25 mM
µL NAD
solution
222
222
222
222
222
µL inhibitor
solution
/
5
10
20
50
µL substrate
solution
50
50
50
50
50
µL glycine
buffer
678
673
668
658
628
•
Continue as in 4.8.1
Evaluation:
Draw a Michaelis-Menten plot with the calculated values (v vs. [CoS]; v = enzyme activity; all
inhibitor concentrations in one plot) and estimate vmax and KM. For a more precise determination of
vmax and KM draw a Lineweaver-Burk plot (also here v = enzyme activity and all inhibitor
concentrations in one plot). For the investigation of the K I constant draw a Dixon plot. How do v max
and KM change with increasing inhibitor concentration in the Michaelis-Menten and LineweaverBurk plot? What type of inhibition causes salicylic acid? What conclusions can be drawn from the
two experiments with ethanol and NAD+ as substrates?
4.8.7
Investigation of the KI constant of acetylsalicylic acid and comparison with salicylic
acid
For the investigation of the KI constant of acetylsalicylic acid and salicylic acid different substrate
and inhibitor concentrations are used.
52
4Enzyme kinetics
Solutions:
Glycine buffer: 0.1 M; pH 9.0
Ethanol solution: 0.5 M in glycine buffer pH 9.0
Acetylsalicylic acid solution: 10 mM in glycine buffer pH 9.0
Salicylic acid solution: 10 mM in glycine buffer pH 9.0
ADH solution: 5 mg/100 mL in glycine buffer pH 9.0
NAD solution: 24 mg/mL in glycine buffer pH 9.0
Experimental procedure:
•
Keep the NAD and ADH solutions on ice. All other solutions have to be at room
temperature.
•
Pipette the following samples in Eppendorf tubes:
Tab. 4.7: Pipetting schedule for the investigation of the K I constant of acetylsalicylic acid
Substrate
5 mM
concentration
5 mM
5 mM
5 mM
5 mM
10 mM
10 mM
10 mM
10 mM
10 mM
Inhibitor
0 mM
concentration
0.1 mM
0.3 mM
0.4 mM
0.5 mM
0 mM
0.1 mM
0.3 mM
0.4 mM
0.5 mM
µL NAD
solution
100
100
100
100
100
100
100
100
100
100
µL inhibitor
solution
/
10
30
40
50
/
10
30
40
50
µL substrate
solution
10
10
10
10
10
20
20
20
20
20
µL glycine
buffer
840
830
810
800
790
830
820
800
790
780
53
4Enzyme kinetics
Substrate
20 mM
concentration
20 mM
20 mM
20 mM
20 mM
50 mM
50 mM
50 mM
50 mM
50 mM
Inhibitor
0 mM
concentration
0.1 mM
0.3 mM
0.4 mM
0.5 mM
0 mM
0.1 mM
0.3 mM
0.4 mM
0.5 mM
µL NAD
solution
100
100
100
100
100
100
100
100
100
100
µL inhibitor
solution
/
10
30
40
50
/
10
30
40
50
µL substrate
solution
40
40
40
40
40
100
100
100
100
100
µL glycine
buffer
810
800
780
770
760
750
740
720
710
700
•
Continue as in 4.8.1.
Evaluation:
Draw a Michaelis-Menten plot (only for acetylsalicylic acid) with the calculated values (v vs. [S]; v
= enzyme activity; all inhibitor concentrations in one plot) and estimate vmax and KM. For a more
precise determination of vmax and KM draw a Lineweaver-Burk plot (one for every inhibitor, also
here v = enzyme activity and all inhibitor concentrations in one plot). For the investigation of the K I
constant draw a Dixon plot (one for every inhibitor). How do v max and KM change with increasing
inhibitor concentration in the Michaelis-Menten and Lineweaver-Burk plot? What type of inhibition
causes acetylsalicylic acid? Compare the results using the different inhibitors acetylsalicylic acid
and salicylic acid.
54