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 aqueou s 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 path length between the plates in an IR cuvette suitable for use with (H2O) -H-2 and the high viscosity of the concentrated protein solutions requ ired to generate measurable IR signals. An apparatus, based largely on conventional stopped-Bow technology, is described which achieves mixi ng 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. T his is probably at least in part because of the high back pressure whi ch develops when aqueous fluid is passed rapidly through the short pat hlength of the cuvette and which promotes turbulent Bow. Several examp les of measurements of the deacylation of acylchymotrypsins are provid ed which demonstrate the operation of the apparatus in conjunction wit h a spectrometer capable of scanning at four scans/s. For cinnamoyl-ch ymotrypsin, isotope-edited spectra have been obtained which show somew hat lower resolution than is achieved by conventional scanning methods , since some smoothing has to be applied to the spectra, Difference sp ectra 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 thi s apparatus is likely to find a variety of applications in the study o f enzyme-catalysed reactions, since the spectra are relatively rich in structural information, and isotope editing greatly enhances the inte rpretability of the spectra.