Water-soluble nonionic surfactants for affinity bioseparations.

Area of Science:

• Biochemistry
• Chromatography
• Biotechnology

Background:

• Enzyme inhibitors can be functionalized to create affinity ligands.
• Nonionic ethoxylated surfactants offer a versatile platform for ligand immobilization.
• High-performance affinity chromatography (HPAC) requires stable and efficient affinity supports.

Purpose of the Study:

• To synthesize and characterize reversible competitive inhibitors covalently attached to ethoxylated surfactants.
• To evaluate the performance of these affinity-derivatized surfactants as ligands in HPAC for enzyme purification.
• To demonstrate the high purification capacity and stability of the developed HPAC system.

Main Methods:

• Covalent attachment of enzyme inhibitors (beta-galactosidase, trypsin, serum cholinesterase) to nonionic ethoxylated surfactants.
• Quantification of surfactant-enzyme binding using Michaelis-Menten inhibition constants and kinetic assays.
• Adsorption of a surfactant-inhibitor (C(16)E(8)-PYR) onto an octadecyl-bonded silica column for HPAC.
• Purification of cholinesterase from bovine serum albumin mixture and raw horse serum using a gradient elution.

Main Results:

• Affinity-derivatized surfactants were successfully synthesized and characterized.
• The C(16)E(8)-PYR surfactant column demonstrated effective binding and elution of cholinesterase.
• Purification of cholinesterase from raw horse serum achieved an 84% yield and 280-fold purification.
• The affinity ligand showed minimal leakage and high stability over extended use (24,000 column volumes).

Conclusions:

• Reversible competitive inhibitors can be effectively immobilized onto surfactants for HPAC applications.
• This method provides a versatile and efficient approach for high-capacity, high-throughput enzyme purification.
• The developed HPAC system offers excellent stability, easy ligand removal, and tunable ligand density.