Hyperpolarization of nuclear spins is the most powerful method of
signal enhancement in NMR, in those cases where we can find ways to
apply it. In hyperpolarization, a physical means is used to repopulate
the Zeeman energy levels of the nuclear spin system before an NMR
experiment. Because the NMR sensitivity is proportional to the
population, this enhances the detectable signal by orders of magnitude.
Dynamic nuclear polarization (DNP) is a newly available technology (J.H. Ardenkjaer-Larsen et al., Proc. Natl. Acad. Sci. USA 2003, 100, 10158-10163) for
providing hyperpolarization of various classes of molecules. It makes
use of the high polarizability of electron spins. Even at a moderate
magnetic field of 1T, and at a temperature of 1K, electron spins are
almost completely polarized. At the same temperature, the polarization
of nuclear spins is still small even at the highest magnetic fields
available. In DNP, polarization is transferred from the electron spins
of free radicals to nuclear spins by means of microwave irradiation.
Using hyperpolarization, in a single scan we routinely achieve a signal gain of three to four orders of magnitude, when compared to conventional NMR. The availability of such a large signal in one instant permits the observation of rapid, transient processes in real-time
, that would otherwise be too fast for NMR spectroscopy.
We are using this capability of hyperpolarization enhanced NMR for the study of the mechanisms and kinetics of biochemical reacthions, in particular enzymes. The above figure demonstrates the time-resolved observation of a reaction on the example of trypsin catalyzed hydrolysis of benzoyl arginine ethyl ester. After rapid injection of the hyperpolarized substrate, the reaction was followed by 13
C NMR in time intervals of 0.5 seconds. From the measured data points, the reaction rate can readily be determined. In addition to the measurement of enzynme kinetics of non-chromogenic substrates, or of multi-step reactions, this technique holds particular promise for the characterization of transient reaction intermediates. Identification of intermediates can readily be achieved through observation of chemical shift on the sub-second time scale, and has the potential of leading to a detailed understanding of the reaction mechanism.