| 1. | Dmitry Shishmarev, Alan J Wright, Tiago B Rodrigues, Giuseppe Pileio, Gabriele Stevanato, Kevin M Brindle, Philip W Kuchel: Sub-minute kinetics of human red cell fumarase: 1H spin-echo NMR spectroscopy and 13C rapid-dissolution dynamic nuclear polarization. In: NMR in Biomedicine, vol. 31, no. 3, pp. e3870–n/a, 2018, ISSN: 1099-1492, (e3870 NBM-17-0125.R2). @article{NBM:NBM3870,
title = {Sub-minute kinetics of human red cell fumarase: 1H spin-echo NMR spectroscopy and 13C rapid-dissolution dynamic nuclear polarization},
author = {Dmitry Shishmarev and Alan J Wright and Tiago B Rodrigues and Giuseppe Pileio and Gabriele Stevanato and Kevin M Brindle and Philip W Kuchel},
url = {http://dx.doi.org/10.1002/nbm.3870},
doi = {10.1002/nbm.3870},
issn = {1099-1492},
year = {2018},
date = {2018-01-01},
journal = {NMR in Biomedicine},
volume = {31},
number = {3},
pages = {e3870--n/a},
abstract = {Fumarate is an important probe of metabolism in hyperpolarized magnetic resonance imaging and spectroscopy. It is used to detect the release of fumarase in cancer tissues, which is associated with necrosis and drug treatment. Nevertheless, there are limited reports describing the detailed kinetic studies of this enzyme in various cells and tissues. Thus, we aimed to evaluate the sub-minute kinetics of human red blood cell fumarase using nuclear magnetic resonance (NMR) spectroscopy, and to provide a quantitative description of the enzyme that is relevant to the use of fumarate as a probe of cell rupture. The fumarase reaction was studied using time courses of 1H spin-echo and 13C-NMR spectra. 1H-NMR experiments showed that the fumarase reaction in hemolysates is sufficiently rapid to make its kinetics amenable to study in a period of approximately 3 min, a timescale characteristic of hyperpolarized 13C-NMR spectroscopy. The rapid-dissolution dynamic nuclear polarization (RD-DNP) technique was used to hyperpolarize [1,4-13C]fumarate, which was injected into concentrated hemolysates. The kinetic data were analyzed using recently developed FmRα analysis and modeling of the enzymatic reaction using Michaelis\textendashMenten equations. In RD-DNP experiments, the decline in the 13C-NMR signal from fumarate, and the concurrent rise and fall of that from malate, were captured with high spectral resolution and signal-to-noise ratio, which allowed the robust quantification of fumarase kinetics. The kinetic parameters obtained indicate the potential contribution of hemolysis to the overall rate of the fumarase reaction when 13C-NMR RD-DNP is used to detect necrosis in animal models of implanted tumors. The analytical procedures developed will be applicable to studies of other rapid enzymatic reactions using conventional and hyperpolarized substrate NMR spectroscopy.},
note = {e3870 NBM-17-0125.R2},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Fumarate is an important probe of metabolism in hyperpolarized magnetic resonance imaging and spectroscopy. It is used to detect the release of fumarase in cancer tissues, which is associated with necrosis and drug treatment. Nevertheless, there are limited reports describing the detailed kinetic studies of this enzyme in various cells and tissues. Thus, we aimed to evaluate the sub-minute kinetics of human red blood cell fumarase using nuclear magnetic resonance (NMR) spectroscopy, and to provide a quantitative description of the enzyme that is relevant to the use of fumarate as a probe of cell rupture. The fumarase reaction was studied using time courses of 1H spin-echo and 13C-NMR spectra. 1H-NMR experiments showed that the fumarase reaction in hemolysates is sufficiently rapid to make its kinetics amenable to study in a period of approximately 3 min, a timescale characteristic of hyperpolarized 13C-NMR spectroscopy. The rapid-dissolution dynamic nuclear polarization (RD-DNP) technique was used to hyperpolarize [1,4-13C]fumarate, which was injected into concentrated hemolysates. The kinetic data were analyzed using recently developed FmRα analysis and modeling of the enzymatic reaction using Michaelis–Menten equations. In RD-DNP experiments, the decline in the 13C-NMR signal from fumarate, and the concurrent rise and fall of that from malate, were captured with high spectral resolution and signal-to-noise ratio, which allowed the robust quantification of fumarase kinetics. The kinetic parameters obtained indicate the potential contribution of hemolysis to the overall rate of the fumarase reaction when 13C-NMR RD-DNP is used to detect necrosis in animal models of implanted tumors. The analytical procedures developed will be applicable to studies of other rapid enzymatic reactions using conventional and hyperpolarized substrate NMR spectroscopy. |
