Vf. Wendisch et al., ACCURATE DETERMINATION OF C-13 ENRICHMENTS IN NONPROTONATED CARBON-ATOMS OF ISOTOPICALLY ENRICHED AMINO-ACIDS BY H-1 NUCLEAR-MAGNETIC-RESONANCE, Analytical biochemistry, 245(2), 1997, pp. 196-202
A method for the accurate determination of C-13 enrichments in nonprot
onated carbon atoms of organic compounds that makes use of unresolved
C-13 satellites of proton(s) bonded to the vicinal carbon atom was dev
eloped. Using glutamate as a model molecule, this H-1 nuclear magnetic
resonance (NMR) inverse spin-echo difference spectroscopy method was
calibrated for inversion efficiency and relaxation effects which were
then shown to cause only a minor loss of the measured C-13 Satellite a
mplitude (2% for glutamate C-l and 7% for glutamate C-5). The determin
ation of C-13 enrichments in nonprotonated glutamate carbon atoms by t
his method was shown to be more precise than C-13 MMR, As a first appl
ication, a [5-C-13]glucose labeling experiment with Corynebacterium gl
utamicum ASK1 was performed. The labeling patterns of glutamate and ar
ginine extracted from cellular protein were determined using the newly
developed method and standard H-1 NMR with and without broadband C-13
decoupling. Determination of the C-13 enrichment in C-5 of glutamate
and arginine, respectively, by the two methods showed good agreement.
From the deduced labeling pattern of a-oxoglutarate, an in vivo carbon
flux distribution within the central metabolism of C. glutamicum ASK1
was calculated. Thus, the relative flux toward oxaloacetate via the t
ricarboxylic acid cycle enzyme malate dehydrogenase was determined as
45%, whereas that via anaplerotic C-3 carboxylation was determined as
55%. (C) 1997 Academic Press.