Open AccessAnalysis of protein self‐association at constant concentration by fluorescence‐energy transfer
Author(s) -
Hassiepen Ulrich,
Federwisch Matthias,
Mülders Thomas,
Lenz Volker J.,
Gattner HansGregor,
Krüger Peter,
Wollmer Axel
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2550580.x
Subject(s) - chemistry , fluorescence , dimer , förster resonance energy transfer , acceptor , intermolecular force , equilibrium constant , dissociation (chemistry) , dissociation constant , dilution , crystallography , molecule , biochemistry , organic chemistry , thermodynamics , physics , receptor , quantum mechanics , condensed matter physics
Fluorescence‐resonance‐energy transfer from subunits labelled with a fluorescence donor group to subunits labelled with a fluorescence acceptor group can be used for quantitative analysis of protein self‐association. The present approach evaluates fluorescence measurements on mixtures of equimolar solutions of donor‐labelled and acceptor‐labelled protein composed by systematic variation of the volume ratio. Its attractive feature is that it allows the determination of equilibrium constants at fixed total concentration. Problems encountered by most other methods, which require the equilibria to be followed to high dilution, are avoided. Conditions to be fulfilled are that a reactive site is available on the protein for specific introduction of the labels and that labelling neither affects the conformation nor interferes with the intermolecular interactions. It is desirable that the Förster distance of the donor/acceptor pair complies with its separation. While dimerisation constants can be determined exclusively by fluorescence measurements, the analysis of more complex cases of self‐association depends on additional independent information. This communication reports on an application of the approach to the association/dissociation equilibrium between insulin monomers and dimers. Labelling of insulin at the ε‐amino group of LysB29 does not disturb the conformation nor does it affect dimerisation. 2‐Aminobenzoyl and 3‐nitrotyrosyl residues served as the donor/acceptor pairs. Because they are less bulky than most other fluorescence labels and are of balanced polarity they do not alter the chemical nature of the protein. Their Förster distance of 29 Å matches their 32‐Å separation in the insulin dimer. Energy transfer was measured as a function of the molar fractions of donor‐insulin and acceptor‐insulin at constant total concentration. Evaluation of this dependence resulted in a dimerisation constant, K 12 , of 0.72×10 5 M −1 . Its agreement with values obtained with other methods demonstrates that the present approach is a reliable alternative.