A pitfall in the use of double-reciprocal plots to estimate the intrinsic molar fluorescence of ligands bound to albumin.

Double-reciprocal plots of fluorescence intensity versus protein concentration are often used to obtain the intrinsic molar fluorescence (Fb) of ligands bound to acceptor molecules such as albumin. In this paper we show that these plots develop upward concave curvature as the concentration of albumin increases. Thus linear extrapolation of such plots cannot be employed to provide accurate values of Fb. It is suggested that a direct plot of fluorescence intensity versus log protein concentration should be employed to obtain Fb.