Measurements of Absolute Values in Biochemical Fluorescence Spectroscopy

Overview

Journal J Res Natl Bur Stand A Phys Chem

Specialty Chemistry

Date 2021 Sep 27

PMID 34565885

Citations 8

Authors

Raymond F Chen

Affiliations

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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 (), 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 for tryptophan has been variously reported as 0.13 and 0.20, and 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 commerically available instruments in absolute measurements, as well as the special complications arising in complex biochemical systems where the fluorescence is heterogeneous.

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