Analysis of protein folding using polarity-sensitive fluorescent probes

Polarity-sensitive fluorescent probes like 8-anilino-1-naphthalenesulphonat e (ANS) and 1,1'-bis(4-anilino5-naphthalenesulphonic acid (bis-ANS) have be en frequently used to detect equilibrium folding intermediates like the mol ten globule state, as the formation of the latter involves increase in hydr ophobic exposure. The ability of these fluorescent probes to bind to the in termediate state thus provides a convenient method for detection of folding intermediates in a multiple-state folding transition. However, there is no convenient method available to quantitatively analyse the fluorescent chan ges detected as a function of denaturant concentration. Here we describe a new method for quantitative analysis that permits one to estimate free ener gy of folding and determine the relative population of the various states o f protein in the solution.