Affinity surfactants as reversibly bound ligands for high-performance affinity chromatography

Area of Science:

• Biochemistry
• Analytical Chemistry
• Chromatography

Background:

• Cholinesterase enzymes are crucial in biological systems.
• High-performance liquid chromatography (HPLC) is a key separation technique.
• Developing efficient affinity supports is vital for enzyme purification.

Purpose of the Study:

• To synthesize a novel affinity surfactant for cholinesterase purification.
• To evaluate the surfactant's binding capacity and performance in HPLC.
• To demonstrate its utility in purifying cholinesterase from biological samples.

Main Methods:

• Covalent coupling of pyridine to octaethylene glycol n-hexadecyl ether to create a modified surfactant.
• Irreversible binding of the surfactant to octadecyl-bounded silica particles for HPLC.
• Affinity chromatography using the modified silica packing for enzyme purification.

Main Results:

• The modified surfactant acts as a reversible, competitive inhibitor of cholinesterase.
• The packing achieved a high ligand binding capacity (550 μmol/ml), exceeding typical agarose supports.
• Significant purification of horse serum cholinesterase (280-fold) and human serum cholinesterase (79-fold) was achieved with >80% yield in 7 minutes.

Conclusions:

• The developed affinity surfactant enables highly efficient and rapid purification of cholinesterase.
• This technique offers a generally applicable method for creating biospecific supports for high-performance affinity chromatography.
• The high binding capacity and efficiency represent a substantial improvement over existing methods.