A double-beam rapid-scanning stopped-flow spectrophotometer

Abstract

A double-beam rapid-wavelength-scanning stopped-flow spectrophotometer system based on the Norcon model 501 spectrometer was construced, which enables u.v.-or visible absorbance spectra to be recorded at the rate of 800/s after the rapid mixing (within 3ms) of two reactant solutions. Each spectrum spans about 200nm in 1ms. It is possible to record difference spectra during reactions with half-lives less than 10ms involving absorbance changes of less than 0.1 absorbance unit. Analogue circuitry is used to produce spectra of absorbance against wavelength. Up to 32 such spectra can be recorded at pre-selected times during a reaction and stored in an 8K×8-bit-word hard-wired data-capture system to be subsequently displaned individually or simultaneously. Time-courses at different wavelengths can also be displayed. By averaging up to 216 spectra it is possible to record spectra under conditions of low signal-to-noise ratios. Examples are given of the application of the system in recording: (a) the spectrum of NADH (2mol/mol of enzyme) produced in the pre-steady-state phase of the horse liver alcohol dehydrogenase-catalysed oxidation of ethanol; (b) the spectrum of NADH (4mol/mol of enzyme) produced in a single turnover in the glyceraldehyde 3-phosphate dehydrogenase reaction; (c) spectral changes during the reaction of aquocobalamin and dithionite; (d) spectral changes during thereaction ofcyanmethaemoglobin and dithionite; (e) the release of thionitrobenzoate and production of NADH when thiol-modified glyceraldehyde 3-phosphate dehydrogenase is mixed with dithiothreitol and substrates.