Unit

UNIT 7.7 Determining the Fluorescence Spectrum of a Protein

  1. Roger H. Pain

Published Online: 1 JAN 2005

DOI: 10.1002/0471140864.ps0707s38

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Pain, R. H. 2005. Determining the Fluorescence Spectrum of a Protein. Current Protocols in Protein Science. 38:7.7:7.7.1–7.7.20.

Author Information

  1. Jozef Stefan Institute, Ljubljana, Slovenia

Publication History

  1. Published Online: 1 JAN 2005
  2. Published Print: NOV 2004

Abstract

Fluorescence spectra of proteins are determined chiefly by the polarity of the environment of the tryptophan and tyrosine residues and by their specific interactions. A thorough consideration of fluorescence spectrometers and their calibration is provided along with important information regarding spectrometer cells, buffers and clarification of samples. Protocols are provided for recording fluorescence spectra and for measuring fluorescence quenching to probe the accessibility of tryptophan residues to small molecules (to yield information about the structural environment of the tryptophan). The technique involves quantifying the decrease in protein fluorescence intensity in the presence of increasing concentrations of quencher, followed by analysis of the data to give details of the interaction of the quencher with the tryptophan residue. Finally, a Support Protocol gives details on how to interpret fluorescence spectra.