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At NIH, Thressa and Earl have worked in the same
building, sharing common facilities and instruments.
Yet each has maintained her or his laboratory
space separately as an independent researcher.
A good example of a facility used in common is
the anaerobic laboratory in which they have conducted
various experiments in oxygen-free conditions.
Standard pieces of equipment for biochemical research,
such as the centrifuge and the Warburg apparatus,
are found in each laboratory. Here is a selected
list of facilities and instruments in their laboratories
over time.
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Anaerobic
Laboratory-An NIH First
In 1967, the first anaerobic laboratory for biomedical
research was built in Building 3 on the NIH's Bethesda
campus. This quarter of a million dollar facility demonstrated
the NIH's strong commitment to the research programs
led by Thressa and Earl Stadtman. Thressa's research
on methane biosynthesis and selenium biochemistry particularly
benefited from this unique facility. Until the closing
of Building 3's in 2002, the anaerobic laboratory served
numerous researchers at NIH as well as visiting scientists
from around the world.The anaerobic laboratory was furnished
with the usual laboratory equipment, but it was filled
with a mixture of nitrogen and hydrogen, instead of
ordinary air. An "oxygen-free" atmosphere is crucial
in dealing with bacteria that are killed in the presence
of oxygen or with biological compounds that are inactivated
when exposed to air. This unique facility allowed researchers
to conduct multi-step experiments with various instruments,
including manipulations that were extremely difficult
in conventional anaerobic "glove boxes." Because of
the danger of working in an anaerobic atmosphere, a
researcher must wear a special mask fitted with an air-delivery
tube while an observer outside monitors a two-way communication
system.
Video: Michael Poston on
the anaerobic laboratory.
Running time: 3:00 minutes
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Floor plan
of the anaerobic laboratory. |
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Inside the
anaerobic laboratory. |
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Respirator
mask used iside the anaerobic laboratory. |
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Fermenter
Room
The fermenter room in Building 3 had facilities for
growing and harvesting a large quantity of bacterial
or yeast cells from which various enzymes could be extracted.
Earl's research on metabolic regulation was particularly
dependent upon the mass-production of glutamine synthetase
in this room. The cell-growing procedure starts with
a small culture of cells. This culture is scaled up
to 10 or 20 liters in flasks, and then is introduced
into the large fermenter which has a maximum volume
of 500 liters. Typically, a volume of 350 liters is
used for cells growing in an aerobic condition. The
cells in the fermenter are then moved to a continuous
flow centrifuge. As the centrifuge bowl spins at high
speed, the cells are collected on the bowl's inside.
The supernatant liquid overlying centrifuged cells flows
out of the top and goes to a holding tank. This liquid
is discarded after being chemically treated. The cells
are scraped off, frozen in liquid nitrogen, and stored
for enzyme purification. The frozen cells are suspended
in a buffer solution and passed through a homogenizer,
which uses high pressure to break the cells open and
release the proteins. Finally, the enzyme is purified
from cell-free extracts, according to various precipitation
protocols. The 500-liter fermenter, installed around
1986, had an automatically-controlled valve system for
sterilization, temperature control, agitation, and gas
or air sparging . The fermenter prior to this model
had been manually operated with about fifty different
valves to open and close at various times. Earl and
his assistants usually grew two fermenter-loads of E.
coli a week. They then harvested 1200-1500 grams of
cells, from which one gram of glutamine synthetase was
purified. Now, the development of genetic engineering
makes a large-scale fermenter almost obsolete. Only
about 10 liters of the genetically modified organism
that specifically overproduces glutamine synthetase
are needed to harvest 30-50 grams of cells from which
one gram of pure enzyme is obtained.
Video: Barbara Berlett on
500-liter fermenter.
Running time: 2:10 minutes
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Warburg
Apparatus
The Warburg apparatus is an analytical instrument for
measuring the pressure of gases and vapors from biochemical
reactions. It was named after its inventor, the German
biochemist Otto Heinrich
Warburg (1883-1970). Warburg
pioneered research on cell respiration and tumor metabolism,
work for which he won the Nobel Prize in Physiology
or Medicine in 1931. Originally, the Warburg apparatus
was used to study "respiration" or the uptake of gaseous
oxygen and the production of carbon dioxide by various
cells or tissues. Later, it also proved useful in examining
"fermentation," a process of breaking down organic compounds
in the absence of oxygen. It was an ideal instrument
for both aerobic and anaerobic enzyme studies until
it was replaced by various spectrometers.
The Warburg apparatus is based on the principle that,
at constant temperature and constant gas volume, any
changes in the amount of a gas can be measured by changes
in its pressure. As a closed system, it consists of
a detachable flask equipped with one or more sidearms
for additions of chemicals and a manometer (pressure
gauge) containing a liquid of known density. The sample
of interest is placed in the main chamber and the flask
is immersed in a constant temperature water bath. It
is then shaken to facilitate rapid gas exchange between
the fluid and the gas phase. At equilibrium a starting
reference point is read on the manometer, and then the
reaction is started. The volume of gas produced or absorbed
is determined at specific time intervals.
Earl used this apparatus for his early research on
fatty acid metabolism and also for his research on
aging in the 1980s. It was also an important tool
for Thressa in studying the role of vitamin B12 -dependent
enzymes in methane biosynthesis and selenium biochemistry.
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Earl using
the Warburg apparatus, 1952. |
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The Warburg apparatus. |
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Micro-Combustion
Furnace
This micro-combustion furnace, manufactured around 1942
by the Fisher Scientific and Eimer & Amend company,
was used for Thressa's research on methane fermentation
in the 1950s and 1960s. Radioactive methane was oxidized
to radioactive carbon dioxide by a catalyst in a tube
heated in the furnace. In this way, a given amount of
radioactive methane could be determined quantitatively.
Later on other direct methods of measuring methane using
special chromatographic equipment became available.
The furnace uses an electrical current to heat substances
placed in a tube. The heating coils are controlled
by a variable transformer, which converts the electrical
current into heat inside the furnace so that no heat
is lost. The electrical current input-and therefore
the temperature-is controlled by the dial on the base
of the instrument. The furnace can produce temperatures
ranging from 200o -1,700oF.
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Fisher Scientific
and Eimer and Amend Micro-Combustion Furnace. |
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Amino Acid/Peptide
Analyzer
The amino acid/peptide analyzer was sold as a kit by
the Dionex Corporation. In 1980, Thressa's research
assistant, Joe Davis, and her postdoctoral fellow, Greg
Dilworth, assembled it, like an erector set, by attaching
equipment to a frame of connected rods on a work cart
in the laboratory. They used the analyzer for identifying
amino acids in selenium-containing proteins. Earl's
group also took advantage of it for analyzing amino
acids in oxidized proteins. These days, high pressure
liquid chromatography (HPLC) systems are available from
many manufacturers, and any of them can be configured
to do the job of analyzing amino acids and peptides.
The instrument consists of several parts. First, it
is controlled by a Dionex Chromatograph Programmer,
a controlling computer, which programs the times at
which different solvents are pumped and the temperatures
at which they are pumped. Second, solvents are kept
in glass bottles on top of the rack, and their temperature
is usually kept at 40o to
65oF by the Dionex
Column Heater Control. Third, connected to the mixing
column is the filtering and detecting device, the
Gilson Spectra/flo Fluorometer. Finally, all the
information-the pH level of solvents and the program
of solvents/temperatures/time is recorded in the
Shimadzu Chromatopac C-R3A Recorder. This particular
recorder was a later addition to the instrument.
The operation of the amino acid/peptide analyzer is
largely automated but requires some experience. A mixture
of amino acids is injected into the system and pumped
into the mixing column. Next, each amino acid is then
eluted or washed out from the column in a specific order.
This is accomplished by pumping in solvents of increasing
pH and by varying the column temperature. The eluted
amino acid in buffer is then mixed with the detecting
materials at the bottom of the column. Subsequently,
the mixture goes into the detecting device, in which
the filter controls the specificity of the fluorometer.
As it passes through the filter, the emission of fluorescence
is detected.
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The instrument
set up in Thressa's laboratory. |
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Drawing
of the amino acid/peptide analyzer. |
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Electron Paramagnetic
Resonance (EPR)
Electron Paramagnetic Resonance (EPR) is an instrument
that detects substances with a particular magnetic
property called "paramagnetism." Paramagnetism can
be found in such metal atoms as aluminum and platinum,
or such metal ions as Fe3+ and
Cu2+, which become
magnetized in a magnetic field but lose their magnetic
power when the field is removed. This property is partly
due to the "spin" movement
of electrons. The electron, one of the fundamental
particles that constitute an atom, orbits around the
atom's nucleus. But it also spins by itself, like
a spinning top. Only two directions of spin, opposite
to each other, are allowed. When two electrons of
different spin properties are paired, the effect of
spinning-mathematically represented by the angular
momentum-is cancelled out. However, when there is
an unpaired electron in the substance, the spin effect
can be detected. Hence EPR is also called Electron
Spin Resonance (ESR). Since it is one of the most
sensitive instruments that can identify substances
having unpaired electrons, EPR has been successfully
applied in various fields of study, such as detecting
impurities in semi-conductors. In biochemistry, it
has become a powerful tool for finding
"free radicals" in cells. For this reason, Earl and
his co-workers began to use it in 1988. After updating
the first model installed at that time (the Bruker
ESP 300) several times, the laboratory has purchased
two more advanced models, Elexsys E580 and EMX, which
are now installed in Building 50. Recently, Thressa's
group also has taken advantage of this instrument
in research on enzymes that contain selenium as well
as molybdenum (a heavy metal).
The most distinctive feature of EPR is the two big
magnets. The sample is placed in the microwave resonator
located in the gap between the magnets. When a strong
magnetic field is applied to the sample, a small magnetic
moment arising from the spin of an unpaired electron
is oriented in a direction either parallel or anti-parallel
to the applied field. This creates a unique energy level
for the sample in the given magnetic field. Then, EPR's
microwave generator sends a wave of a specific energy
(measured in frequency) to the microwave resonator where
the sample is placed. When the microwave's energy (i.e.,
its frequency) matches exactly with the energy level,
it is absorbed by the sample. This "energy-absorption"
or "frequency-matching" phenomenon is called "resonance."
For technical reasons, EPR detects resonance at a fixed
microwave frequency by changing or scanning the strength
of the magnetic field. With the positions of resonance
lines and their amplitudes, it is possible to determine
what kind of free radicals are in the sample and how
much of them there is. In addition, since EPR's resonance
lines are split further according to the overall environment
for electrons-in particular, the presence of nuclei
nearby-the analysis of this splitting can also provide
useful information on the structural identity of the
sample.
Video: Moon Bin Yim on electron
paramagnetic resonance.
Running time: 1:17 minutes
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Drawing
of EPR. |
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Spectrophotometer
and High Pressure Liquid Chromatography (HPLC)
Since the early 1980s, Earl and his co-workers have
used various analytical methods to measure the level
of oxidized, and thus inactivated, enzymes for their
research on protein degradation and aging. For example,
the spectrophotometer, which measures the relative intensities
of light in different parts of the spectrum, can detect
changes in the level of oxidized proteins.
In separating and identifying oxidized segments of
proteins (i.e., oxidized peptides or amino acids), various
chromatographic techniques are useful. Among them is
the high pressure liquid chromatography commonly known
as HPLC. Its operational principle is simple: the sample
of interest is injected into HPLC's column, and as the
mobile phase passes through the column, the different
products are separated and detected. In Earl's laboratory,
a variety of columns are used to separate and identify
compounds, depending on their molecular properties,
such as size, ionic charge, and affinity to specific
molecules. In Thressa's research, HPLC separation of
selenium containing proteins, peptides and amino acids
is important.
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Spectrophotometer
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High Pressure
Liquid Chromatograph. |
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