th  - 1988 - 51st ENC Conference

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NATURAL ABUNDANCE 13 C and 14 N NMR OF BACTERIAL OSMOLYTES IN VlVO. B.A.
Lewis,~'S.C.Cayley, S Pedmanabhan, and M.T. Record, Jr. Dept. of Chemistry,
University of Wisconsin, Madison Wl 53706.
Bacteria such as E. Coli and _5. Typhimurium are capable of growing under conditions of moderately
high osmotic stress, up to about 0.7 molar salt. To adapt to such high-osmolarity environments, the bacterial
cell accumulates potassium ions and also synthesizes or accumulates one or more small organic molecules.
These include the anion glutamate and the neutral or zwitterionic molecules praline, glycine betaine ( N,N,N-
trimethyl glycine) and/or trehalose, a glucose dimer. Because these small molecules are~ccumulated to
intracollular concentrations on the order of 0.5 molal, they are readily observed in dense cell slurries by
natural abundance 13 c NMR on our Bruker AM360 with a I 0 mm broadband probe. 14 N NMR is also useful to
observe glycine betaine, which has a relatively narrow 14 N spectrum due to the symmetric environment of
the nitrogen and its lack of exchangeable protons.
We are able to measure the relative and absolute amounts of the various organic osmolytes accumulated
by the bacteria in viva under a variety of environmental conditions. In minimal medium with 0.5 M NaCl,
trehalose and glutamate are the only small organic molecules present in high amounts. If I mM praline is added
to the medium, it is accumulated to nearly 0.4 M intracellularly, with some diminution of the trehalose and
glutamate levels. 61ycine heroine, however, also supplied at I mM, is accumulated to about 0.5 M, and
trehalose is completely eliminated.
Under these high salt conditions, significant amounts of rf power are absorbed by the sample,
particularly at the high frequencies of 90 MHz for 13 c and 360 for I H. Thus for the i 3 C experiments we
employ gated proton dacoupling to minimize sample heating. In addition, the pulse lengths must be calibrated
for each sample, and internal standards must be used for quantitative measurement.
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CHARACTERIZATION OF HUMAN BLOOD PLASMA USING VERY HIGH FIELD
DIFFEERENCE SPECTROSCOPY.
Dadok, J.*, Bothner-By, A. A., Mishra, P.K., Carnegie Mellon
15213
Wilkinson, D. A., Giles, R. H., Acevedo, H. F., Shrivastava,
Allegheny-Singer Research Institute, Pittsburgh, PA 15213
RESOLUTION ENHANCED PMR
Univ., Pittsburgh, PA
P.N., Jarmillo, B.,
Blood plasma from cancerous patients was compared with plasma from healthy males and
females using 620 MHz PMR spectra with various degrees of resolution enhancement. The
variations in the content of VLDL, LDL and HDL as well as of other plasma components
was evaluated with the use of difference spectroscopy. Preliminary results indicate
that this technique may provide useful information on the physiological state of the
blood plasma. We could see also systematic and substantial differences in plasma of
healthy males and females and we feel that comparison should be made within well de-
fined groups of the same sex.
References:
I. Fossel,
2. Bell, D.
Letters,
E.T., Carr, J.M. and McDonagh, J., N. Engl. J. Med.,315 1369-1376 (1986).
J., Sandler, P. J., Macleod, A. F., Turner, P. R., LaVille, A., FEB
219, 239-273 (1987).
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