PROBING THE CONFORMATION OF PROTEIN (BFGF) PRECIPITATES BY FLUORESCENCE SPECTROSCOPY

Aggregation and precipitation are major events in the handling and agi ng of most protein pharmaceuticals. We demonstrate the utility of fluo rescence spectroscopy in determining protein conformation in precipita tes using basic fibroblast growth factor (bFGF) as an example. Convers ion of the native to the soluble denatured form by chaotropes was acco mpanied by an increase in tryptophan emission. The emission spectra of resuspended precipitates were as reproducible as the spectra of the s oluble form. The sum of emission spectra of native soluble bFGF and de natured precipitated bFGF was superimposable on the spectrum of the un fractionated suspension, suggesting that quantitative analysis of dena tured 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, salti ng out by ammonium sulphate increased the fluorescence index (indicati ve of denaturation) which was reversible upon dissolution. In addition , aging (35 degrees C) of bFGF in the presence of sulphated ligands pr oduced precipitates with native-like fluorescence index, in contrast t o denatured precipitates formed without ligands.