Advanced sample making tools for electron paramagnetic resonance spectroscopy

包括许多由酶催化的生物化学过程，随着瞬时中间体物质的形成而进行，所述瞬时中间体物质可以通过其特定的吸光度及时监测，并且如果所述物质是顺磁性的，则通过其EPR光谱（电子顺磁共振）监测。光谱获得的这些物种的时间依赖性可用于计算机模拟反应的动力学机制，使研究人员能够检查关于反应机制的不同假设。虽然停流二极管阵列分光光度法可用于在两种反应物混合后从短至约2 ms的真实的时间内监测光谱老化过程，但EPR光谱法在真实的时间内使用的灵敏度要低得多。因此，在大多数应用中，EPR动力学数据是在反应开始后不同时间冷冻的样品上获得的。重要的是，EPR样品的冷冻速率至关重要。将具有反应混合物的石英EPR管浸入到低温介质中，在世界范围内的实验室中广泛使用，其特征在于秒的冷冻时间，这太长而不能产生与液相中的光谱学获得的数据一致的足够的动力学数据。另一种方法是冷冻淬火样品的EPR光谱。这通常通过将反应混合物强制注入冷（~140 K）异戊烷中来完成。以这种方式制备的样品产生与光学获得的动力学数据一致的动力学数据。然而，在异戊烷中冷冻淬灭是一个复杂的过程，每个工作日只能制备几个样品。更重要的是，在异戊烷中的冷冻淬灭产生具有低光谱再现性的样品。可以理解的是，这种冷冻猝灭并不经常用于获得生化反应顺磁中间体的高质量动力学数据。我们建议制造工具和设备，使冷冻淬火样品的EPR光谱不使用异戊烷。这不仅使冷冻淬灭更容易，而且还将导致EPR谱的更高再现性。此外，新方法将允许每个工作日制作更多样品，这意味着获得的动力学数据的统计显著性更高。我们建议制作的工具将使用样品冷冻在一块金属表面上，用液氮（77 K）热平衡。将有可能使用这些工具为EPR光谱的不同波段和光谱通常覆盖的整个反应时间范围制作样品-从2 ms开始，没有上限。将使用Update Instrument快速冷冻骤冷装置，但是，我们将在冷金属表面上喷洒混合物，而不是将反应混合物喷射到具有冷异戊烷的烧瓶中。一旦样品在表面冻结，样品的薄片和外壳必须转移到EPR管中进行测量。因此，我们还建议制造用于在EPR管中容易且可重复地包装冷冻样品的工具，同时将管和样品保存在液氮中。一些EPR光谱仪使用非常薄的样品管（OD<1 mm）。如上所述，通过将管直接浸入低温介质中来冷冻这些管中的反应混合物将不会产生一组适合于精确动力学研究的样品。然而，这仍然是一种常见的做法，对于不针对动力学问题的试点研究是有用的。当使用皮下注射针时，用液体填充这种细管需要在填充过程中连续地从管中取出针。这是相当难以快速完成的，因此当手动操作注射器时，实际上可实现的最小反应时间约为30秒。我们建议制作一个工具，使这个时间减少到1-2秒。我们将利用这些新的仪器和工具来研究马甲脒与H_2O_2反应的中间体。

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.