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Sub-minute kinetics of human red cell fumarase: 1H spin-echo NMR spectroscopy and 13C rapid-dissolution dynamic nuclear polarization

Sub-minute kinetics of human red cell fumarase: 1H spin-echo NMR spectroscopy and 13C rapid-dissolution dynamic nuclear polarization
Sub-minute kinetics of human red cell fumarase: 1H spin-echo NMR spectroscopy and 13C rapid-dissolution dynamic nuclear polarization
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.
fumarase kinetics, human red blood cell, hyperpolarization, malate, Michaelis-Menten model
1099-1492
1-13
Shishmarev, Dmitry
11bac3f9-42c3-42ba-b3d1-e5fc06365580
Wright, Alan J.
fa8b30ea-13ba-4c19-b5bc-f92d330fd2c5
Rodrigues, Tiago B.
81d395c1-c552-4e99-bb8d-6c7872b387a9
Pileio, Giuseppe
13f78e66-0707-4438-b9c9-6dbd3eb7d4e8
Stevanato, Gabriele
13f2e0a2-2ce7-4938-a2ac-274a81196a72
Brindle, Kevin M.
316bceba-bd08-4979-89b3-28d35adf882a
Kuchel, Philip W.
62f434b1-d370-4552-85f4-05b76c625945
Shishmarev, Dmitry
11bac3f9-42c3-42ba-b3d1-e5fc06365580
Wright, Alan J.
fa8b30ea-13ba-4c19-b5bc-f92d330fd2c5
Rodrigues, Tiago B.
81d395c1-c552-4e99-bb8d-6c7872b387a9
Pileio, Giuseppe
13f78e66-0707-4438-b9c9-6dbd3eb7d4e8
Stevanato, Gabriele
13f2e0a2-2ce7-4938-a2ac-274a81196a72
Brindle, Kevin M.
316bceba-bd08-4979-89b3-28d35adf882a
Kuchel, Philip W.
62f434b1-d370-4552-85f4-05b76c625945

Shishmarev, Dmitry, Wright, Alan J., Rodrigues, Tiago B., Pileio, Giuseppe, Stevanato, Gabriele, Brindle, Kevin M. and Kuchel, Philip W. (2018) Sub-minute kinetics of human red cell fumarase: 1H spin-echo NMR spectroscopy and 13C rapid-dissolution dynamic nuclear polarization. NMR in Biomedicine, 31 (3), 1-13. (doi:10.1002/nbm.3870).

Record type: Article

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–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.

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More information

Accepted/In Press date: 30 October 2017
e-pub ahead of print date: 8 January 2018
Keywords: fumarase kinetics, human red blood cell, hyperpolarization, malate, Michaelis-Menten model

Identifiers

Local EPrints ID: 417516
URI: https://eprints.soton.ac.uk/id/eprint/417516
ISSN: 1099-1492
PURE UUID: 574c3c0e-e9da-45b9-94cc-614bf158d7f0
ORCID for Giuseppe Pileio: ORCID iD orcid.org/0000-0001-9223-3896

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Date deposited: 01 Feb 2018 17:30
Last modified: 03 Dec 2019 05:37

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