Advanced sample making tools for electron paramagnetic resonance spectroscopy

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.

Rapid freeze-quenching of biochemical reaction mixtures is the only way to freeze samples for EPR spectroscopy that would yield accurate kinetic data for the transient intermediates of the reaction. We propose to make devices and tools for making freeze-quenched samples for the EPR spectroscopy, both for the X-band and high field (HF) types of spectrometers, that would not use isopentane as the cryomedium. We will use an Update Instrument rapid freeze-quench apparatus but, instead of ejecting the reaction mixture into a flask with cold isopentane, we will spray the mixture over a cold surface kept at the liquid nitrogen temperature. We will explore two designs of the cold surface: a rapidly rotating metal disk kept almost fully submersed in liquid nitrogen and a stationary metal surface / for small volume samples. The devices will be easy to operate and will produce more samples per working day than with employment of isopentane. The packing procedure of the samples in the EPR tubes will be standardised by using original packing tools, for the X-band as well as for W-band samples, which would exclude the possibility of the EPR tube blocking during the packing procedure. The suggested packing tools will be free from the static electricity problem typically associated with isopentane use, and will produce samples with much greater spectra reproducibility than sample packing in isopentane. We also propose to make a tool for mixing two liquids in the thin HF EPR sample tubes (0.58 mm ID) with minimal reaction time of 1-2 s. This tool will not involve rapid freeze-quenching and therefore could not be used for obtaining accurate kinetic data. However, the tool will be very useful in preliminary studies, when it will produce much higher transient species concentrations as compared to manual mixing in such tiny tubes, which typically produces much longer minimal reaction time (~30 s to our experience). The tools will be tested on a biochemical reaction system.