Design and Performance of a Differential Spectrofluorometer

A differential spectrofluorometer has been designed and built. This instrument is capable of measuring and recording the difference fluorescence spectrum between two samples which share a common light source, monochromator, and photomultiplier. The two fluorescent signals are synchronously demodulated, filtered, and subtracted in a differential voltmeter. The model compounds, indole and N -acetyl- l -tryptophanamide, as well as lysozyme and an inhibitor have been studied. Difference fluorescence spectroscopy offers a sensitive method to detect protein conformational changes involving the aromatic residues and to reveal small emission differences in aromatic molecules. Used concurrently with solvent perturbation it can be used to probe the topology of the surface to evaluate the accessibility of aromatic residues.