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

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 H spin-echo and C-NMR spectra. H-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 C-NMR spectroscopy. The rapid-dissolution dynamic nuclear polarization (RD-DNP) technique was used to hyperpolarize [1,4- C]fumarate, which was injected into concentrated hemolysates. The kinetic data were analyzed using recently developed FmR analysis and modeling of the enzymatic reaction using Michaelis-Menten equations. In RD-DNP experiments, the decline in the C-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 C-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.

Enhanced Ca2+ influx in mechanically distorted erythrocytes measured with 19F nuclear magnetic resonance spectroscopy

“…This range is due to the sensitivity of the 19 F atom (attached to the two benzene rings in this case) to redistribution of electron density in the aromatic system. Because 19 F atoms are also hydrogen bond acceptors (e.g., 5,30,31 ), it is expected that in the presence of high concentrations of amino acid side chains that the 5FBAPTA 19 F NMR resonance would shift; the interaction is likely to be enhanced if two of the four carboxyl groups are neutralized by binding to Ca 2+ . This was born out by the appearance of the broad resonance at ~ 7 ppm in RBCs that had been loaded with 5FBAPTA and then made permeable to Ca 2+ either with Yoda1 or A23187 (Figs.…”

Rapid zero-trans kinetics of Cs+ exchange in human erythrocytes quantified by dissolution hyperpolarized 133Cs+ NMR spectroscopy

“…The equations are applicable in a situation where there is no evidence of substrate concentration-dependence of k 1 and k −1 that can occur with enzymes 26,27,33 and membrane transport systems that operate with hyperpolarized substrates when there are large extents of reaction. This means that substrate concentrations change during the reaction to such an extent that they span the apparent K m value(s) of the enzyme/carrier 11,25 .…”

Glyoxalase activity in human erythrocytes and mouse lymphoma, liver and brain probed with hyperpolarized 13C-methylglyoxal

“…For the RD-DNP experiments with cell suspensions or lysates, a Bruker Avance II 600 MHz spectrometer (150.9 MHz for 13 C) with a 14.1 T vertical magnet and 10-mm Bruker broadband probe were used to record the 13 C NMR spectra. The 13 C NMR RD-DNP spectra were acquired as reported recently; 23 viz., each was based on a single free induction decay (FID) of 32,768 complex data points acquired with a repetition time of 1 s. FID acquisition time was 0.806 s; spectral width was 269.35 ppm; the carrier frequency was set to 120 ppm; the duration of the radio-frequency pulse was 1 μs, which corresponded to a nutation (flip) angle of 4 o . The data were processed with one degree of zero filling and multiplication with an exponential line-broadening factor of 1 Hz before Fourier transformation.…”

“…The sample underwent rapid dissolution in 6 mL of a solution containing 10 mM Na phosphate, 137 mM NaCl and 2 mM KCl (pH 7.4 at 37 °C, at 10 bar and 180˚C) to give a final MeGx concentration of ~80 mM. Up to 2 mL of the hyperpolarized solution was injected via a heat-exchange apparatus 23 into samples of cells or lysates, which were pre-equilibrated at 37 °C in a 10-mm NMR tube. The time from sample ejection to injection into the lysate/cell-suspension was typically ~17 s. For the experiments on anaesthetized mice, after a similar delay, 100 μL of the hyperpolarized solution was injected via a caterer into the tail vein over ~10 s.…”