Computer-interfaced rapid scanning molecular absorption and emission spectrophotometer for stopped-flow studies of enzyme reactions.

Abstract

An improved stopped-flow system with a rapid-scanning spectrometer permits measurement of either light absorption or emission at scan speeds of up to 150 spectra per second. The entire flow system, including valves, syringes and quartz flow cell is contained in a thermostat bath and is leak-tight from 5 to 45 degrees C. Pneumatic valves control the flow through Teflon tubing. Quartz fiber-optic light guides are used to transmit light to and from the flow cell. Experimental data are given to demonstrate the absorbance and fluorescence modes. The growth and decay of the fluorescence spectrum of NADH was followed in a reaction catalyzed by lactate dehydrogenase (LDH). The kinetics of binding of 1-anilino-8-naphthalene sulfonate (ANS) to bovine serum albumin (BSA) was studied by both scanning and fixed-wavelength fluorescence emission. The fluorescence of BSA was completely quenched within two milliseconds accompanied by an abrupt increase in the fluorescence of ANS which was followed by a slower first-order growth.