White A J, Drabble K, Wharton C W
School of Biochemistry, University of Birmingham Edgbaston, U.K.
Biochem J. 1995 Mar 15;306 ( Pt 3)(Pt 3):843-9. doi: 10.1042/bj3060843.
IR spectroscopy has been widely applied in the study of photo-activated biological processes such as photosynthesis, but has not been applied to the study of reacting systems which require rapid mixing of aqueous solutions. This has been due in part to the high pressure needed to make an aqueous solution flow rapidly through the 50 microns optical pathlength between the plates in an IR cuvette suitable for use with 2H2O and the high viscosity of the concentrated protein solutions required to generate measurable IR signals. An apparatus, based largely on conventional stopped-flow technology, is described which achieves mixing well within the time-resolved performance (approximately 40 ms) of modern Fourier-transform IR (FTIR) spectrometers, since the dead time of the mixing device is approximately 15 ms. It has proved possible to achieve efficient mixing by using a simple six-jet mixing device. This is probably at least in part because of the high back pressure which develops when aqueous fluid is passed rapidly through the short pathlength of the cuvette and which promotes turbulent flow. Several examples of measurements of the deacylation of acylchymotrypsins are provided which demonstrate the operation of the apparatus in conjunction with a spectrometer capable of scanning at four scans/s. For cinnamoyl-chymotrypsin, isotope-edited spectra have been obtained which show somewhat lower resolution than is achieved by conventional scanning methods, since some smoothing has to be applied to the spectra. Difference spectra of the acylation of chymotrypsin by glycylglycine p-nitrophenyl ester have been obtained by averaging ten stopped-flow shots and show good signal-to-noise ratio without smoothing. It is predicted that this apparatus is likely to find a variety of applications in the study of enzyme-catalysed reactions, since the spectra are relatively rich in structural information, and isotope editing greatly enhances the interpretability of the spectra.
红外光谱已广泛应用于光合作用等光激活生物过程的研究，但尚未应用于需要快速混合水溶液的反应体系的研究。部分原因在于，要使水溶液快速流过适用于重水的红外比色皿中板间50微米的光程，需要较高压力，而且产生可测量红外信号所需的浓缩蛋白质溶液粘度很高。本文描述了一种主要基于传统停流技术的仪器，由于混合装置的死时间约为15毫秒，因此该仪器能在现代傅里叶变换红外（FTIR）光谱仪的时间分辨性能（约40毫秒）内实现良好混合。事实证明，使用简单的六射流混合装置就可以实现高效混合。这可能至少部分是因为当水性流体快速通过比色皿的短光程时会产生高背压，从而促进了湍流。文中提供了几个酰基胰凝乳蛋白酶脱酰化测量的例子，展示了该仪器与能够以每秒四次扫描进行扫描的光谱仪配合使用时的运行情况。对于肉桂酰基胰凝乳蛋白酶，已获得同位素编辑光谱，但其分辨率略低于传统扫描方法，因为必须对光谱进行一些平滑处理。通过平均十次停流测量获得了甘氨酰甘氨酸对硝基苯酯酰化胰凝乳蛋白酶的差示光谱，且未经平滑处理就显示出良好的信噪比。预计该仪器可能会在酶催化反应研究中找到多种应用，因为光谱中结构信息相对丰富，而且同位素编辑大大增强了光谱的可解释性。
