Solubilization of heme proteins in low polar solvents by chemical modification on a protein's surface

The use of nonaqueous solvents in biocatalytic reactions has gained much attention. Especially, biocatalysis in ionic liquids (ILs) has been investigated by many researchers recently. In this study, we tried to solubilize proteins in low polar solvents including water-immiscible ILs. The proteins cytochrome c (cyt. c) and cytochrome P450 (P450) were chosen as model proteins. We used covalent modification with longer chain poly(ethylene oxide) (PEO) or with both PEO and a hydrophobic modifier (n-hexylamine) to improve the solubility of proteins in low polar nonaqueous solvents. The chemically modified proteins were dissolved in chloroform and hydrophobic water-immiscible ILs. PEO150/C-6-cyt. c was slightly dissolved in chloroform. Alkyl amine modification of the carboxyl residues increased the solubility of PEO150-modified cyt. c in chloroform. Cyt. c modified with PEO5000 is soluble in chloroform and low polar ILs. PEO2000/C-6-modified P450st and PEO5000-modified P450st were dissolved in all low polar solvents used in this study. UV-Vis spectroscopy and resonance Raman spectroscopy revealed the structure of chemically modified heme proteins in the nonaqueous solvents. Copyright (C) 2008 John Wiley & Sons, Ltd.