Thermodynamic insights into the binding of ANS with the salt induced molten globule states of cytochrome c

The molten globule (MG) state or the A-state of cytochrome c (cyt c) has been induced by addition of salts sodium perchlorate (NaClO4), sodium thiocyanate (NaSCN), and sodium sulphate (Na2SO4) at pH 2.0. Isothermal titration calorimetry (ITC) has been used for determining the energetics of binding of 8-anilino naphthalene sulphonate (ANS) to the salt induced A-state of cyt c, and the accompanying thermodynamic parameters have been analyzed to elucidate the nature of the interactions between ANS and the A-state of cyt c. Temperature dependent studies of the binding process reveal that the binding is not a two state process and there are more than a single type of interactions involved. Addition of a bulky salt tetraethylammonium bromide (TEAB) increases the stoichiometry of binding significantly, with a reduction in the binding affinity at a higher concentration. The results provide quantitative information on the binding of ANS to the salt induced molten globule states of cyt c. It is further inferred that the binding involves a combination of hydrophobic and electrostatic interactions.