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Construction and Performance of a Fluorescence Polarization Spectrophotometer

From the ¹ From the Biochemistry Division, Department of Chemistry and Chemical Engineering, University of Illinois, Urbana, Illinois 61803

The construction and performance of a fluorescence polarization spectrophotometer is described, the parts of which are mostly commercially available items. The instrument uses two photomultipliers to detect separately each polarized component of the fluorescent light. The ratio of the components is plotted continuously by means of a ratio recorder while the wave length of the exciting light is changed at uniform speed. A servomechanism keeps the slits to the minimum value compatible with the sensitivity of the instrument. We obtained maximum resolution on the excitation side by allowing the broadest possible band of fluorescence, selected by broad band or low pass optical filters, to reach the photomultipliers. The use of fluorescence polarization spectrophotometry in investigation of proteins is discussed.

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