Phospholipid‐lysozyme coating for chiral separation in capillary electrophoresis

Abstract

A phospholipid coating with lysozyme as chiral recognition reagent permeated into the phospholipid membrane was developed for the chiral capillary electrophoretic (CE) separation of D- and L-tryptophan. As a kind of carriers, coated as phospholipid membranes onto the inner wall of a fused-silica capillary, liposomes are able to interact with basic proteins such as lysozyme, which may reside on the surface of the phospholipid membrane or permeate into the middle of the membrane. The interaction results in strong immobilization of lysozyme in the capillary. Coatings prepared with liposomes alone did not allow stable immobilization of lysozyme into the phospholipid membranes, as seen from the poor repeatability of the chiral separation. When 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide (M1C4) was applied as a first coating layer in the capillary, the electroosmotic flow (EOF) was effectively suppressed, the phospholipid coating was stabilized, and the lysozyme immobilization was much improved. The liposome composition, the running buffer, and the capillary inner diameter all affected the chiral separation of D- and L-tryptophan. Coating with 4 mM M1C4 and then 1 mM phosphatidylcholine (PC)/phosphatidylserine (PS) (80:20 mol%), with 20 mM (ionic strength) Tris at pH 7.4 as the running buffer, resulted in optimal chiral separation with good separation efficiency and resolution. Since lysozyme was strongly permeated into the membrane of the phospholipids on the capillary surface, the chiral separation of D- and L-tryptophan was achieved without lysozyme in the running buffer. The effects of different coating procedures and separation conditions on separation were evaluated, and the M1C4-liposome and liposome-lysozyme interactions were elucidated. The usefulness of protein immobilized into phospholipid membranes as a chiral selector in CE is demonstrated for the first time.

Cholesterol‐rich membrane coatings for interaction studies in capillary electrophoresis: Application to red blood cell lipid extracts

“…Liposomes are attracting considerable interest as a coating material in CE [1][2][3][4][5]. The simple and fast method of preparing the semipermanent phospholipid coating, and its versatility, make CE a promising technique for studies on phospholipid-analyte interactions.…”

Piperazine-based buffers for liposome coating of capillaries for electrophoresis

“…Among the buffer ions studied (i.e, HEPES, phosphate, Tricine, and Tris), successful separation of neutral model compounds was achieved only when HEPES was used as liposome solvent and as BGE solution. In addition, we recently demonstrated efficient phospholipid coating, followed by protein immobilization of capillaries with use of 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide, a quaternary monoammonium salt [13]. When 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide was applied as a first coating layer in the capillary, the EOF was effectively suppressed, the phospholipid coating was stabilized, and protein immobilization was much improved.…”

Immobilization of phospholipid‐avidin on fused‐silica capillaries for chiral separation in open‐tubular capillary electrochromatography

“…In our earlier phospholipid coating, on which lysozyme was immobilized, the stability of the coating was improved by suppressing the EOF with 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide [13]. Because avidin has a high pI value of 10 and a high molar mass of 60 kDa, it would be expected to yield strong electrostatic and hydrophobic interactions, and, owing to the strong immobilization on phospholipid membrane and silica wall, it should effectively reverse the EOF direction in CEC.…”

“…Development of new supporting matrixes for protein immobilization in capillaries, making the capillaries capable of high chiral separation efficiency, is of great interest. We recently introduced a phospholipid bilayer coating with lysozyme as chiral recognition reagent permeated into the phospholipid membrane for the chiral separation of D-and L-tryptophan by open-tubular CEC [13]. 1-(4-Iodobutyl)-1,4-dimethylpiperazin-1-ium iodide was applied as coating layer in the capillary to effectively suppress the EOF, and then the capillary was coated with phospholipids, onto which lysozyme was immobilized.…”