Factors Affecting Protein Transfer into Surfactant-Isooctane Solution: A Case Study of Extraction Behavior of Chemically Modified Cytochrome c

Abstract

The extraction mechanism of proteins by surfactant molecules in an organic solvent has been investigated using a chemically modified protein. We conducted guanidylation on lysine residues of cytochrome c by replacing their amino groups with homoarginine to enhance the protein-surfactant interaction. Results have shown that guanidylated cytochrome c readily forms a hydrophobic complex with dioleyl phosphoric acid (DOLPA) through hydrogen bonding between the phosphate moiety and the guanidinium groups. Although improved protein-surfactant interaction activated the formation of a hydrophobic complex at the interface, it could not improve the protein transfer in isooctane. It has been established that the protein extraction mechanism using surfactant molecules is mainly governed by two processes: formation of an interfacial complex at the oil-water interface and the subsequent solubilization of the complex into the organic phase. In addition, a kinetic study demonstrated that guanidylation of lysine accelerated the initial extraction rate of cytochrome c. This fact implies that the protein transferability from aqueous phase into organic phase depends on the protein-surfactant interaction which can be modified by protein surface engineering.

Complex Formation of Cytochrome c with a Calixarene Carboxylic Acid Derivative: A Novel Solubilization Method for Biomolecules in Organic Media

“…Therefore, the extraction selectivity by t Oct[6]CH 2 COOH implies which cationic residue contributes the interaction with t Oct[6]CH 2 COOH. On the basis of the same strategy, guanidylated Cyt-c (G-Cyt-c) was prepared by chemical modification of native Cyt-c and the extraction behavior was also examined . The guanidylation with O -methylisourea altered the amino groups of the Lys residues to guanidinium groups.…”

“…Guanidylation of Lysine Residues of Cytochrome c. A guanidylated Cyt-c (abbreviated as G-Cyt-c) was prepared by modification of native Cyt-c with o -methylisourea providing homoarginine groups on the protein surface as shown in Scheme . The procedure was described in a previous paper . The number of modified amino groups was determined by a free amino group titration method using 2,4,6-trinitrobenzenesulfonic acid (TNBS) …”

“…13 The procedure was described in a previous paper. 14 The number of modified amino groups was determined by a free amino group titration method using 2,4,6trinitrobenzenesulfonic acid (TNBS). 15 Extraction of Cytochrome c with Calixarenes in Organic Solvents.…”

“…On the basis of the same strategy, guanidylated Cyt-c (G-Cyt-c) was prepared by chemical modification of native Cyt-c and the extraction behavior was also examined. 14 The guanidylation with O-methylisourea altered the amino groups of the Lys residues to guanidinium groups. Titration for free amino groups using 2,4,6-trinitrobenzenesulfonic acid (TNBS) indicated that 15.9 residues of Cyt-c were converted to the homoarginine moiety (Table 2…”

Extractive Solubilization, Structural Change, and Functional Conversion of Cytochrome c in Ionic Liquids via Crown Ether Complexation

“…Guanidylation of Lysine Residues of Cyt- c . To introduce arginine residues to the surface of Cyt- c , guanidylated Cyt- c (G-Cyt- c ) was prepared through modification of native Cyt- c with o -methylisourea according to methods described elsewhere (Scheme ). , Native Cyt- c was added to a phosphate buffer solution (pH 10.5) containing o -methylisourea hydrochloride (200 mol equiv) and triethylamine (200 mol equiv). The solution was stirred for 72 h under cooling.…”