Overview
The amino acid Trp-214 within HSA (human serum albumin) acts as a FRET donor and the ligand 3-hydroxyflavone acts as the acceptor.
Acceptor ligand is successively added and the decreasing donor fluorescence and increasing acceptor fluorescence due to FRET-complexes is recorded.
The binding affinity is investigated from both the donor-quenching and analysis of the average FRET-efficiency in a binding isotherm. The latter also gives information about the D-A distance.