Probing the conformation of protein (bFGF) precipitates by fluorescence spectroscopy

Aggregation and precipitation are major events in the handling and aging of most protein pharmaceuticals. We demonstrate the utility of fluorescence spectroscopy in determining protein conformation in precipitates using basic fibroblast growth factor (bFGF) as an example. Conversion of the native to the soluble denatured from by chaotropes was accompanied by an increase in tryptophan emission. The emission spectra of resuspended precipitates were as reproducible as the spectra of the soluble form. The sum of emission spectra of native soluble bFGF and denatured precipitated bFGF was superimposable on the spectrum of the unfractionated suspension, suggesting that quantitative analysis of denatured aggregates in turbid protein formulations is possible. The ratio of tryptophan to tyrosine emissions increased with increasing extent of denaturation both in solution and in suspension. For example, salting out by ammonium sulphate increased the fluorescence index (indicative of denaturation) which was reversible upon dissolution. In addition, aging (35 degrees C) of bFGF in the presence of sulphated ligands produced precipitates with native-like fluorescence index, in contrast to denatured precipitates formed without ligands.