Measurements of absolute values in biochemical fluorescence spectroscopy

Abstract

Fluorescence spectroscopy is an important tool of the biochemist studying the structure and interactions of proteins and nucleic acids. The four basic quantities to measure accurately are: 1. spectra (corrected excitation and emission), 2. quantum yields (Q), 3. fluorescence decay characteristics, and 4. polarization. Commercially available instruments, with little modification, can be used to obtain these measurements, but the biochemist in this field is very dependent on the accuracy of measurements of substances he uses as standards. Confusion arises from disagreement between reported values for standards which may be used to calibrate a detector system to obtain quantum yields, or to set up lifetime and polarization photometers. For instance, the protein chemist is fond of using tryptophan and quinine as quantum yield standards, but Q for tryptophan has been variously reported as 0.13 and 0.20, and Q values for quinine bisulfate range from 0.4 to 0.7. The biochemist should also be aware of the problems inherent in the use of commercially available instruments in absolute measurements, as well as the special complications arising in complex biochemical systems where the fluorescence is heterogeneous.