A modified stopped-flow apparatus for time-resolved protein phosphorescence

Abstract

A type of commercial apparatus was modified and integrated in order to implement the detection of time-resolved protein phosphorescence in the stopped-flow technique. Laser excitation, photomultiplier protection from the intense fluorescence pulse, fluorescence integration, and data acquisition are all synchronized by a trigger module that takes over standard computer control of the stopped-flow apparatus. A detailed protocol is given for effective deoxygenation of the sample and flow lines and for avoiding contamination of the solutions by quenching impurities. The performance of the apparatus was tested by comparing the phosphorescence decay kinetics of the protein horse liver alcohol dehydrogenase in the stopped-flow apparatus and in a standard phosphorimeter. The time resolution of phosphorescence detection in the stopped-flow apparatus is 10 ms and the sensitivity in terms of chromophores concentration is about 0.1 μM.