期刊
MAGNETIC RESONANCE IN CHEMISTRY
卷 47, 期 -, 页码 S138-S146出版社
WILEY
DOI: 10.1002/mrc.2420
关键词
NMR; H-1; C-13; water suppression; metabolites; fermentation; cyanobacteria; Arthrospira (Spirulina); Synechococcus
资金
- Air Force Office of Scientific Research (MURI) [FA9550-05-1-0365]
We highlight a range of cryoprobe-assisted NMR methods for studying metabolite production by cyanobacteria, which should be valuable for a wide range of biological applications requiring ultra sensitivity and precise concentration determination over a large dynamic range. Cyroprobe-assisted H-1 and C-13 NMR have been applied to precise determination of metabolic products excreted during autofermentation in two cyanobacterial species: filamentous Arthrospira (Spirulina) maxima CS-328 and unicellular Synechococcus sp. PCC 7002. Several fermentative end products were identified and quantified in concentrations ranging from 50 to 3000 mu M in cell-free media (a direct measurement of native-like samples) with less than 5.5% relative error in under 10 min of acquisition per sample with the assistance of an efficient water-suppression protocol. Relaxation times (T1) of these metabolites in aqueous ((H2O)-H-1) solution were measured and found to vary by nearly threefold, necessitating generation of individual calibration curves for each species for highest precision. However, using a 4.5 x longer overall recycle delay between scans, the metabolite concentrations can be predicted within 25% error by calibrating only to a single calibration standard (succinate); other metabolites are then calculated on the basis of their signal integrals and known proton degeneracies. Precise ratios of concentrations of C-13-labeled versus unlabeled metabolites were determined from integral ratios of H-1 peaks that exhibit C-13-H-1 J-couplings and independently confirmed by direct measurement of areas of corresponding C-13 resonances. C-13 NMR was used to identify and quantify production of osmolytes, trehalose, and glucosylglycerol by A. maxima. Copyright (C) 2009 John Wiley & Sons, Ltd.
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