[27] Microfluorometry of product formation in single enzyme-sepharose beads using fluorogenic substrates

This chapter presents the method for the measurement of turnover characteristics in single beads of immobilized enzyme gels. It is based on applying microfluorometric techniques, originally developed for measuring intracellular enzymic reactions in single cells, using fluorogenic substrates. It is noted that microscope fluorometry not only produces an integral fluorescence signal (in analogy to conventional fluorometry) of a tiny specimen, but also yields its image or projection owing to the image forming function of the optical path of the microscope. This method thus permits a fluorometric signal to be correlated with the geometry of the specimen. Therefore, it is also possible to obtain information on concentration profiles of substrates and products within the catalytic particle. In addition to the experiments described in the chapter, some cell-physiological techniques, especially microspectrophotometry, open up new opportunities for research on immobilized enzymes.