Analysis of protein folding using polarity-sensitive fluorescent probes

Polarity-sensitive fluorescent probes like 8-anilino-1naphthalenesulphonate (ANS) and 1,1′-bis(4-anilino5-naphthalenesulphonic acid (bis-ANS) have been frequently used to detect equilibrium folding intermediates like the molten globule state, as the formation of the latter involves increase in hydrophobic exposure. The ability of these fluorescent probes to bind to the intermediate 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 changes detected as a function of denaturant concentration. Here we describe a new method for quantitative analysis that permits one to estimate free energy of folding and determine the relative population of the various states of protein in the solution.