Spectrofluorometric analysis of the dissociation of oligomeric macromolecules: correction for the absorption of exciting and emitted light in a side-bottom type fluorometer
Batke, J.
Analytical Biochemistry 121(1): 123-128
1982
ISSN/ISBN: 0003-2697 PMID: 7091672 Document Number: 189224
For the quantitative analysis of the association-dissociation of oligomeric macromolecules (proteins) the measurement of their concentration-dependent fluorescence (intrinsic or of a fluorescent dye attached covalently to the macromolecule) is proposed. The prerequisite of the application is that the different associated forms have distinct fluorescence quantum yields. A method, more extended than the simple inner filter correction, is presented for the calculation of the fluorescence concentration quenching. The calculation accounts for both absorption of exciting light and reabsorption of emitted light. Experiments with noninteracting (not dissociable or associable) fluorophoric compounds (fuschsin, aldolase, lactic dehydrogenase and bovine serum albumin) corroborate the applicability of the proposed correction for concentration quenching. By the aid of the above correction the dissociation of the tetrameric is characterized.