ANALYSIS OF PROTEIN SELF-ASSOCIATION AT CONSTANT CONCENTRATION BY FLUORESCENCE-ENERGY TRANSFER

Fluorescence-resonance-energy transfer from subunits labelled with a f luorescence donor group to subunits labelled with a fluorescence accep tor group can be used for quantitative analysis of protein self-associ ation. The present approach evaluates fluorescence measurements on mix tures of equimolar solutions of donor-labelled and acceptor-labelled p rotein composed by systematic variation of the volume ratio. Its attra ctive feature is that it allows the determination of equilibrium const ants 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 a vailable on the protein for specific introduction of the labels and th at labelling neither affects the conformation nor interferes with the intermolecular interactions. It is desirable that the Forster distance of the donor/acceptor pair complies with its separation. While dimeri sation constants can be determined exclusively by fluorescence measure ments, the analysis of more complex cases of self-association depends on additional independent information. This communication reports on a n application of the approach to the association/dissociation equilibr ium between insulin monomers and dimers. Labelling of insulin at the E -amino group of LysB29 does not disturb the conformation nor does it a ffect dimerisation. 2-Aminobenzoyl and 3-nitrotyrosyl residues served as the donor/acceptor pairs. Because they are less bulky than most oth er fluorescence labels and are of balanced polarity they do not alter the chemical nature of the protein. Their Forster distance of 29 Angst rom matches their 32-Angstrom separation in the insulin dimer. Energy transfer was measured as a function of the molar fractions of donor-in sulin and acceptor-insulin at constant total concentration. Evaluation of this dependence resulted in a dimerisation constant, K-12, of 0.72 X10(5) M-1. Its agreement with values obtained with other methods demo nstrates that the present approach is a reliable alternative.