A MODIFIED STOPPED-FLOW APPARATUS FOR TIME-RESOLVED PROTEIN PHOSPHORESCENCE

A type of commercial apparatus was modified and integrated in order to implement the detection of time-resolved protein phosphorescence in t he stopped-flow technique. Laser excitation, photomultiplier protectio n from the intense fluorescence pulse, fluorescence integration, and d ata acquisition are all synchronized by a trigger module that takes ov er 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 imp urities. The performance of the apparatus was tested by comparing the phosphorescence decay kinetics of the protein horse liver alcohol dehy drogenase in the stopped-flow apparatus and in a standard phosphorimet er. The time resolution of phosphorescence detection in the stopped-fl ow apparatus is 10 ms and the sensitivity in terms of chromophores con centration is about 0.1 mu M. (C) 1997 American Institute of Physics.