A STOPPED-FLOW APPARATUS FOR INFRARED-SPECTROSCOPY OF AQUEOUS-SOLUTIONS

IR spectroscopy has been widely applied in the study of photoactivated 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 mu m optical pathlength between the plates in an IR cuvette suitable for use with (H2O)-H-2 and the high viscosity of the concentrated protein solutions required to generate measurable IR signals. An apparatus, based largely on conventional stopped-Bow technology, is described which achieves mixing well within the time-resolved performance (approximate to 40 ms) of modern Fourier-transform IR (FTIR) spectrometers, since the dead time of the mixing device is approximate to 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 Bow. 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.