Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin

The non-linearity of the fluorescence emission on increasing the probe to protein ratio has long been regarded as problematic and has lead to the development of dyes to overcome this effect. One of the causes of this non-linear response can be ascribed to the overlap of the label's own absorption and emission spectra. At higher labelling ratios, this affords the possibility of a reasonably efficient energy migration pathway, thus reducing the observed quantum yield of the dye. In this work we study the photophysics of fluorescein isothiocyanate (FITC) when conjugated to bovine serum albumin (BSA) at different labelling ratios (in the range FITC : BSA 1 : 17-15 : 1) using both steady state and time-resolved fluorescence techniques where on going from under labelled to over labelled samples a decrease in the initial (and steady state) anisotropy is observed, accompanied by an increase in the complexity of the decay kinetics and a decrease in the average lifetime. The band structure, elucidated by synchronous scan fluorescence spectroscopy, is also found to change on increased labelling. These results can be applied to the study of protein conformation and were confirmed by the analysis of denaturing BSA using urea.