title: Water-soluble nonionic surfactants for affinity bioseparations.
abstract: Reversible competitive inhibitors of the three enzymes beta-galactosidase, trypsin, and serum cholinesterase have been covalently attached to nonionic ethoxylated surfactants. The binding of the resulting affinity-derivatized surfactants to the respective enzymes has been quantified by measuring Michaelis-Menten inhibition constants with kinetic assays. The surfactant-inhibitor of serum cholinesterase, octaethylene glycol monohexadecy ether pyridinium (C(16)E(8)-PYR), was adsorbed in aqueous solution to an octadecyl-bonded reverse-phase silica packing in a 2 x 0.2 cm stainless steel test column. The ability of the test column to function as a high-performance affinity chromatography (HPAC) column was determined by applying a mixture of bovine serum albumin and cholinesterase (4:1 w/w). Virtually all of the cholinesterase bound and was eluted by applying a gradient in ionic strength. The applied cholinfesterase was recovered with a yield of over 90% and an 11-fold purification. An aliquot of raw horse serum was then purified in the same fashion with a yield of 84% and a 280-fold purification. The surfactant-inhibitor was easily removed from the column with an alcohol wash for sterilization, cleaning, or application of a different affinity ligand. Moreover, the ligand density on the column can be easily manipulated by adsorbing mixtures of derivatized and underivatized surfactants. Leakage of ligands from the support seems to be minimal since the cholinesterase affinity column was operated efficiently after being exposed to 24,000 column volumes of buffer. The application of this technique to high-capacity, high-throughput reversible affinity purifications is limited only by the ability to identify suitable ligands.
authors.affiliation: Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina 27695, USA.
authors.forename: R
authors.initials: R
authors.lastname: Guzman
authors.forename: J L
authors.initials: JL
authors.lastname: Torres
authors.forename: R G
authors.initials: RG
authors.lastname: Carbonell
authors.forename: P K
authors.initials: PK
authors.lastname: Kilpatrick