Rhodamine B-labeled thyroid hormone forms high molecular weight aggregates in solution: a possible source of artifacts in binding experiments

The fluorescence intensity of rhodamine B-labeled thyroid hormone is a nonlinear function of probe concentration. The concentration dependence was analyzed in terms of a model with continuous aggregation of the probe molecules to give very high molecular weight species. The apparent association constant was 3.65 microM-1 at 25 degrees C, pH 7.2. Studies at different temperatures showed apparent delta H = 6.8 kcal/mol and delta S = -7.3 cal/mol/degree for association. Based on their absorbance spectra, these aggregates seem to be linear, with the rhodamine chromophores slanted with respect to the long axis.