Steve Macko attending his handmade nitrogen isotope line, 1982 |
In September 1982, my first
postdoctoral fellow, Steve Macko, arrived from my old alma mater, the
University of Texas Marine Science Institute. Macko, as he is affectionately
called, sported a thick mustache and a shock of brown hair along with
coke-bottle lens glasses. His preferred outfit was a blue and red zippered sweatshirt
with a pair of slightly rumpled jeans. He is known the world over for his
thrifty ways—with food, his clothing, his hairstyles, and his lab’s gear. Almost
40 years later, Macko has held on to a perpetually youthful appearance.
Steve (right) and Sue Ziegler, Carnegie, 2016 |
We shared an office during his time as
a postdoc. Steve brought with him to the Geophysical Lab, not only boundless
enthusiasm and a plethora of ideas, but the technical wherewithal to measure
nitrogen isotopes in organic matter. He came with a host of marine science
projects, collaborators from around the globe, as well as a zeal to learn as
much as he could.
Often, after reading a new paper at his
desk, he would turn to me and say, “Ya’ wanna know something interesting?” He’d
have a devilish little smile on his face and follow this with a soft chuckle,
then follow that question up with “I have an idea!”
At the Geophysical Lab, Macko quickly
set up the methods he’d worked out as a PhD student for analyzing isotopes in
organic matter. He prided himself on his glassblowing talents. Tom Hoering had
been the Geophysical Lab’s only glass blower. The two of them had a bit of a
rivalry in terms of prowess at making connections and curves with the glass
tubing used to build vacuum extraction lines. Eventually they reached a détente
and managed to support each other’s skills.
Steve’s nitrogen isotope technique became
what is popularly known as the Sealed Tube method in which 5 milligrams of a sample—plant
or animal tissue-- was placed into a quartz tube with copper reagents and
sealed off after it had been evacuated on our homemade vacuum extraction line. We
used an oxygen-natural gas torch (at 2770°C!) to carefully melt the quartz and seal
the tubes shut. That was a white-knuckle operation that took patience and care
to do properly.
The next step required heating the
whole tube, with the sample sealed inside, in a furnace at 950°Ç, cooling it
carefully back to room temperature. We then cracked open the tube within a finicky
metal device that usually worked, but failed about 10% of the time. Once
cracked open, the gases from the combustion of the sample—carbon dioxide,
nitrogen, and water vapor—were distilled into a glass trap using liquid nitrogen
(-196°C) to freeze out the gases.
After several more steps, nitrogen gas
was isolated in a sample bulb, which was then taken to the isotope ratio mass
spectrometer for isotope analysis. Macko was a whiz at this and could process
15-16 samples in a day. I was slower, maxing out at around a dozen. It was not
unusual to have one or two of these samples be contaminated with air that had
somehow leaked into the vacuum extraction line. I used Steve’s method from 1982
until 1999, when I installed a new automated instrument used by all labs today.
During those 17 years, I trained multiple students and postdocs how to do this
procedure.
Nitrogen isotope line, Carnegie 1991 |
Macko’s methods spread to many other
laboratories like wildfire. During my career, I published 120+ papers using
nitrogen isotope measurements to study everything from humans to
extraterrestrial organic matter. Nitrogen isotope measurements now form the
backbone of stable isotope ecology research along with carbon isotopes. Who is
eating whom can be figured out with nitrogen isotopes. One of my first projects
with nitrogen isotopes was the study of the hot springs microbes, figuring out
how they were surviving in nutrient poor environments. In a subsequent chapter,
I describe how nitrogen isotopes allowed Noreen Tuross and me to figure out how long women nursed their babies during prehistoric times. I used
nitrogen isotopes to study pollution in the Delaware Estuary, the Atlantic
Ocean, and coastal coral reefs. Ocean scientists use nitrogen isotopes to learn
about ocean circulation and primary production.
Univ. of South Carolina's lab early 1980s with lots of glass 'lines', David Mucciarone (left) |
Macko’s main project was to culture
microorganisms, then separate and purify amino acids from them for isotopic
analysis, similar to the original work that Phil Abelson and Tom Hoering
published in 1961. We were interested in learning about the isotope patterns
originating from biochemical synthesis. Analyses of this type are used for
understanding unique biochemical pathways, sources of food for animal diets,
and the trophic position (e.g., primary consumer vs. top carnivore) for animals
in which their diets are unknown.
Our
work in 1982 involved all three Geophysical Lab staff members in biogeochemistry,
as well as Macko: me for culturing the organisms and interpreting the
biochemistry learned from the isotope patterns; Ed Hare for separation of the
amino acids; and Tom Hoering for IRMS and other isotope support. To carry out these experiments, I needed to
ramp up my culturing ability in order to grow the substantial amounts of
material needed for amino acid isotope analyses. Ed Hare needed to produce
grams of the special ion exchange resin required to separate milligram
quantities of amino acids. Hare and Macko worked hard in order to have the
amino acid analyzers going full time to assay compounds as they eluted from the
separation column. We used two IRMS instruments for the isotope measurements: a
newer Nuclide 6” IRMS for carbon and for nitrogen a much older IRMS constructed
at the Geophysical Laboratory by Hoering and others [the one previously
sandblasted…].
It took several weeks to grow up the microbial
cultures. We analyzed an aliquot of the microbes using the Sealed Tube method,
then hydrolyzed the remaining sample to produce hydrochloride salts of amino acids. Individual amino acids in the hydrolysate,
the solution after acid heating, were separated using column chromatography
that took 24 to 48 hours to complete.
The fact that amino acids do not separate at regular or specific time
intervals was a problem. The trick was to use a separate liquid chromatography
system to determine when each amino acid started and finished eluting.
With this information, we could isolate
and capture each amino acid in its entirety but separate it from other amino
acids in the sample. Fractions were collected from the eluant every few
minutes. After analysis via liquid chromatography, fractions were pooled to
capture the entire amino acid eluting from the column. This resulted in 13-15 different amino acid
samples, each containing the hydrochloride salts of a single amino acid. The
samples were then dried by evaporation in the fume hood. The amino acids were then
analyzed by the Sealed Tube method described above.
This was an elaborate and slow process
where each sample took a total of about 6 weeks from start to finish, depending
on whether everything was working in tip-top order (Macko et al., 1987). We
were the only research group in the world doing these detailed, complicated
analyses and in the early 1980s were twenty years ahead of our time. Today,
experiments of this sort can be accomplished with much less material, and by
automated analysis, where five to ten samples can be measured in a week. The framework we established in the 1980s through this laborious work
was important for subsequent
measurements of nitrogen isotopes in amino acids from animals, a critical
method for studying the mechanisms that are now used by laboratories globally
to study ecological trophic levels.
Another pivotal
postdoctoral fellow from this time was Michael Engel. Engel and Macko became
fast friends and colleagues, a relationship that has lasted over 30 years. Engel came to work with Ed Hare to learn as much as he could about
amino acid analytical techniques. Engel is the opposite of Macko in many ways.
He’s a flashy dresser, loves to eat at the finest restaurants, spends money
without guilt, and sometimes seems a bit down in the dumps.
Dr. Engel and Macko, Hoeringfest, 1995 |
He’d come into my lab sometimes and
shake his head, “I don’t know…I can’t figure something out.” Then, he’d proceed
to argue with himself, while I listened, and figure things out for him self. I
call him “Dr. Engel”, rarely Mike, and today when we see each other, we laugh
and giggle about the Olden Days like a bunch of teenagers.
Engel and his major professor Bart Nagy at
the University of Arizona studied the organic chemistry of one of the most
famous meteorites—the Murchison, which fell in Australia several decades ago
(Engel and Nagy, 1982). This particular meteorite contains abundant organic
matter that has been studied by scientists around the world. Dr. Engel
developed detailed procedures to extract and identify amino acids found in the
meteorite. Many of these amino acids have similar structures to those found in
the proteins of living organisms on Earth.
All
living things use proteins to do the metabolic work of a cell. These proteins
are composed of building blocks known as amino acids. There are about 20
different amino acids that are found in proteins. Anywhere from 50 to several
hundred amino acids are linked together to form a long chain-like protein.
Almost every amino acid comes in two forms, levorotary (left-L) and
dextrorotary (right-D). These forms of amino acids are mirror images of each
other but cannot be overlaid onto each other, just like right and left hands.
The words come from the fact that if you shine a light on left-handed
molecules; they bend light to the left. The same is true for the right-handed
variety. However, most living things only use the L amino acids. When a
creature dies the L amino acids begin to convert to D amino acids through a
process called amino acid racemization in which one mirror image molecule turns
into another.
Other geochemists had found amino acids in
the Murchison meteorite before, but they did not report the ratio of L- to D-
amino acids. Engel and Nagy found a slight enrichment in the L-form of the
amino acids and argued that this enrichment might have been important in
influencing the ultimate form (L-) that was “chosen” by living organisms. Other
geochemists denounced their findings as contamination from minute quantities of
terrestrial amino acids, all of which would have had just the L- form.
This plagued Engel, then
a postdoc looking for a permanent faculty position, for many years. He was subsequently
proved right by other researchers and by developing a new stable isotope method
that I’ve used for nearly thirty years. Collaborating with Steve Macko, they published their first paper in 1991 using continuous flow
methods in which amino acids are separated by gas chromatography then swept
one-by-one into the mass spectrometer without all the messy chemistry. Engel
and Macko published multiple papers in the coming decade on isotopic
compositions of amino acids from meteorites, which confirmed Engel’s earlier
assertion that the enantiomeric excess he measured and published with Nagy in
1982 was in fact correct.
While Engel and Macko
were at the Geophysical Laboratory, we conducted experiments measuring nitrogen
isotopic fractionation by the enzyme that adds the amino (NH2) group
to acid carbon compounds, thereby forming amino acids. This enzyme, glutamate-aspartate
transaminase, was the second enzyme system that I used to measure isotope
fractionation. It was much easier to do these experiments than working with
Rubisco during my PhD. First, we didn’t have to grow organisms, could purchase
the enzyme directly from Sigma Chemical Company rather than purify it ourselves.
The chemicals needed to carry out the reaction (i.e., reactants--aspartate,
glutamate, α-ketoglutarate and oxaloacetate) were all inexpensive and readily
obtainable. We determined the nitrogen isotope fractionation for the reaction going
in one direction, then the other. This is one of the most basic and important enzymatic
reactions for producing amino acids in all organisms. The paper has been cited
hundreds of times but never repeated.
Having the capability
for measuring nitrogen, carbon, and hydrogen isotopes in my laboratory opened
up the entire world of isotope biogeochemistry for me. Every experiment that we
could think of was new, and the outcomes unknown. At that time, the literature
on stable isotopes was so small that you knew each publication that came out,
memorized its contents, and thought about it long and hard. The work on amino
acids quickly progressed to investigating fossil collagen samples and lab grown
animals. The amino acid isotope field plodded long incrementally for a decade before
resurging as advances in instrumentation permitted reliable automated measurements.
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