A Spectroscopic Insight on Ethanol Induced Aggregation of Papain.

In this contribution, the structural and dynamical changes occurring to papain molecules in ethanol-water binary solvent mixture have been investigated and compared with its denatured state induced by guanidine hydrochloride. Steady-state fluorescence, solvation dynamics, time-resolved rotational anisotropy, circular dichroism and single molecular level fluorescence correlation spectroscopic studies were carried out for this purpose. In ethanol-water mixture with XEtOH = 0.6, DACIA-tagged papain was found to undergo a blue shift of 12 nm, while in presence of 5 M GnHCl, a red shift of 5 nm was observed. Solvation dynamics of the system was also found to be different in presence of these external agents. In ethanol-water mixture the average solvation time was found to increase almost 2 fold as compared to that in water, while in presence of GnHCl only a marginal increase could be observed. These changes of DACIA-tagged papain in ethanol-water mixture is attributed to the aggregation of the protein in the presence of ethanol. The residual anisotropy was found to increase 14 fold and the rotational time component corresponding to the rotation of the probe molecule was found to increase by 4 fold in the ethanol-water mixture also gives a notion of the papain aggregation. Atomic force microscopy (AFM) confirms this aggregate formation, which is also quantified by fluorescence correlation spectroscopic (FCS) study. The hydrodynamic radius of the protein aggregates in ethanol-water mixture was calculated to be ~155 Å as compared to the corresponding value of 18.4 Å in the case of native monomer papain. Also, it confirmed that the aggregate formation takes place even in nanomolar concentration of papain. Analysis of circular dichroism spectra of papain showed that an increase in the β-sheet content of papain at the expense of α-helix and the random coil with an increase of ethanol mole fraction may be responsible for this aggregation process.