Advanced sample making tools for electron paramagnetic resonance spectroscopy

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Biochemical processes including many catalysed by enzymes, proceed with formation of transient intermediate species that can be monitored in time by their specific optical absorbance and, if the species are paramagnetic, by their EPR spectra (electron paramagnetic resonance). The spectroscopically obtained time dependencies of these species can be used in computer simulation of the kinetic mechanism of the reaction, allowing researchers to check different hypotheses about the reaction mechanism. Whilst stopped-flow diode-array spectrophotometry can be used to monitor the process of optical spectra aging in real time from as short as ~2 ms after two reactants are mixed, EPR spectroscopy is far less sensitive to be used in real time. Therefore in most applications the EPR kinetic data are obtained on the samples frozen variable time after the reaction starts. Importantly, the rate of the EPR sample freezing is crucial. Immersion of a quartz EPR tube with the reaction mixture to a cryogenic medium, extensively used in the laboratories worldwide, is characterised by the freezing time of seconds, which is too long to yield adequate kinetic data consistent with the data obtained by the optical spectroscopy in the liquid phase. The alternative is freeze-quenching of the samples for the EPR spectroscopy. This is usually done by forceful injection of a reaction mixture into cold (~140 K) isopentane. The samples made in this way yield kinetic data consistent with those obtained optically. However, freeze-quenching in isopentane is a complex procedure, allowing just a few samples to be made per working day. More importantly, freeze-quenching in isopentane yields samples with a low spectral reproducibility. Understandably, such freeze-quenching is not very often used for obtaining high quality kinetic data for the paramagnetic intermediates of biochemical reactions. We propose to manufacture tools and devices for making freeze-quenched samples for the EPR spectroscopy without use of isopentane. Not only this will make the freeze-quenching easier, it will also result in a higher reproducibility of the EPR spectra. In addition, the new method will allow making more samples per working day, meaning a higher statistical significance of the kinetic data obtained. The tools we propose to make will use sample freezing on the surface of a piece of metal thermally equilibrated with liquid nitrogen (77 K). It will be possible to use the tools for making samples for different bands of EPR spectroscopy and for the whole range of reaction time typically covered by optical spectroscopy - from 2 ms and with no upper limit. An Update Instrument rapid freeze-quench apparatus will be employed, but, instead of ejection the reaction mixture into a flask with cold isopentane, we will spray the mixture over a cold metal surface. Once a sample is frozen on the surface, the flakes and crust of the sample have to be transferred to an EPR tube for measuring. Therefore, we also propose to make tools for easy and reproducible packing of the frozen samples in the EPR tubes, whilst the tube and the sample are kept in liquid nitrogen. Some EPR spectrometers use very thin sample tubes (OD<1 mm). Freezing of a reaction mixture in such tubes by direct immersion of the tubes into a cryomedium will not, as indicated above, yield a set of samples suitable for accurate kinetic studies. However, this is still a common practice and is useful for pilot studies not aimed at the kinetic issues. Filling such thin tubes with a liquid when using a hypodermal needle requires continuous removal of the needle from the tube as the filling goes on. This is rather difficult to do fast, so the minimal reaction time practically achievable when handling a syringe manually is about 30 s. We propose to make a tool that would allow this time to be reduced down to 1-2 s. We will use the new devices and tools in studying the intermediates of the reaction of horse metMb with H2O2.