8-anilino-1-naphthalene sulfonic acid (ANS) induces folding of acid unfolded cytochrome c to molten globule state as a result of electrostatic interactions

Hydrophobic interaction of 8-anilino-1-naphthalene sulfonic acid (ANS) with proteins is one of the widely used methods for characterizing/detecting pa rtially folded states of proteins. We have carried out a systematic investi gation on the effect of ANS, a charged hydrophobic fluorescent dye, on stru ctural properties of acid-unfolded horse heart cytochrome c at pH 2.0 by a combination of optical methods and electrospray ionization mass spectroscop y (ESI MS). ANS was found to induce, a secondary structure similar to nativ e protein and quenching of fluorescence of tryptophan residue, in the acid- unfolded protein. However, the tertiary structure was found to be disrupted thus indicating that ANS stabilizes a molten globule state in acid-unfolde d protein. To understand the mechanism of ANS-induced folding of acid-unfol ded cytochrome c, comparative ESI MS, soret absorption, and tryptophan fluo rescence studies using nile red, a neutral hydrophobic dye, and ANS were ca rried out. These studies suggested that, at low pH, electrostatic interacti ons between negatively charged ANS molecules and positively charged amino a cid residues present in acid-unfolded cytochrome c are probably responsible for ANS-induced folding of acid-unfolded protein to partially folded compa ct state or molten globule state. This is the first experimental demonstrat ion of ANS induced folding of unfolded protein and puts to question the use fulness of ANS for characterization/determination of partially folded inter mediates of proteins observed under low pH conditions.