alpha-chymotrypsin and 1-butyl-3-methylimidazolium-chloride

Recently, studies have provided evidence for the negative effects of ionic liquids (ILs), "greener solvents," on proteins [Phys. Chem. Chem. Phys., 2014, 16, 5514]. The search to offset the negative effects of ILs on proteins has come into limelight as the maintenance of a "green solvent medium" is a great challenge for chemists and biochemists. As the first step in this search, 1-butyl-3-methylimidazolium bromide ([Bmim][Br]) has been applied to offset the deleterious action of 1-butyl-3-methylimidazolium iodide ([Bmim][I]) on α-chymotrypsin (CT). Fluorescence and circular dichroism (CD) experiments results have indicated that [Bmim][Br] significantly offsets the deleterious action of [Bmim][I] at lower concentrations (0.025 M). Surprisingly, the stabilizing action of [Bmim][Br] turns into a deleterious action for CT at higher concentrations (>0.1 M). On the other hand, [Bmim][I] acted as a destabilizer for CT at all investigated concentrations (0.025-0.6 M). The results obtained from this study lead to valuable insights into the selection of suitable ILs to attenuate the deleterious action of another IL without disturbing the protein structure.

Topics: Animals; Cattle; Chymotrypsin; Imidazoles; Iodides; Models, Molecular; Protective Agents; Protein Stability

We have recently developed a general approach to improve the utility of enzymes in ionic liquids (ILs) via tuning of the ratio of enzyme-containing positive to negative surface charges. In this work, the impact of enzyme surface charge ratio on the biophysical interaction of 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]) with chymotrypsin and lipase was investigated to understand this approach at the molecular level. Results of fluorescence quenching assays indicated that the extent of binding of the [BMIM] cation decreased (7- and 3.5-fold for chymotrypsin and lipase, respectively) as a function of increasing ratio of positive to negative surface charges. Conformational stability assays further showed a close correlation between thermodynamic stabilization and enzyme surface charge ratio as well as [BMIM] binding. As evidence of this correlation, succinylation and acetylation resulted in the stabilization of chymotrypsin in 10% (v/v) [BMIM][Cl] by 17.0 and 6.6 kJ/mol, respectively, while cationization destabilized chymotrypsin by 3.6 kJ/mol. Combined, these results indicate that altering the surface charge ratio mediates the organization of IL molecules, namely, [BMIM] and [Cl], around the enzymes. Preferential exclusion of [Cl], in particular, via lowering of the ratio of positive to negative surface charges, correlated with increased enzyme stability. Accordingly, these results more broadly provide insight into the mechanism of stabilization in ILs via charge modification.

Topics: Animals; Cattle; Chymotrypsin; Enzyme Stability; Fungal Proteins; Half-Life; Hydrogen-Ion Concentration; Imidazoles; Lipase; Protein Binding; Protein Denaturation; Spectrometry, Fluorescence; Static Electricity; Surface Properties; Thermodynamics