R. Z. Guzman, D. F. Keller, J. L. Torres, P. K.
Kilpatrick,
and R. G. Carbonell
Biospecific Interactions of Affinity-Labelled Surfactants with Proteins
We are currently investigating the possibility of separating biological
molecules from dilute aqueous solutions using affinity-modified surface-active agents. A ligand that binds strongly and specifically to a desired biomolecule is covalently coupled to a surfactant. Several uses of the affinity-modified surfactant may be possible. For example,
the chemically-modified surfactant may be used to stabilize a
foam and thus provide an interface to separate the desired biomolecule. Alternatively, other types of interfaces may
be employed including an oil-water interface in an emulsion and a liposome-solution
interface.
The first step in developing these processes involves the synthesis of the ligand-modified surfactants. The surfactant Sandopan JA-36, an ethylene oxide-based surfactant that
contains carboxylic acid head groups, has been reacted with specific inhibitors
of beta-galactosidase (EC 3.2.1.23) and trypsin (EC 3.4.21.4) using the EDC reaction (1,2). The
beta-galactosidase inhibitor used is p-aminophenylthiogalactopyranside (APGP) and the trypsin inhibitor used is p-aminobenzamidine
(PAB). The binding of the inhibitor-modified surfactants to the enzymes was
then studied by kinetic assays.
With the large hydrophobic protein beta-galactosidase, two effects were observed. At surfactant-inhibitor concentrations below the critical micelle concentration (2 x 10-4 M), the inhibitor-modified surfactant was a stronger competitive inhibitor than the pure APGP. Above the CMC, the level of inhibition was found to be independent of surfactant-inhibitor concentration. These two effects were attributed to the non-specific binding of surfactant-inhibitor monomer to the hydrophobic protein below the CMC, and scavenging of excess free monomer by micelles above the CMC. With the more hydrophilic protein trypsin, the binding of the surfactant-inhibitor to the protein followed Michaelis-Menten kinetics with the identical binding constant as the pure PAB. Since the binding constant of PAB (8 x 10-4 M) is smaller than the CMC (2 x 10-4 M), complete inhibition was observed below this value. No significant loss of activity of either enzyme was observed in the presence of the pure surfactant JA-36 below concentrations of 10-3 M.
Despite the specific binding of both enzymes to their surfactant-inhibitors, the complex formed is insufficiently surface-active to effect a foam-based separation. We are currently exploring similar syntheses with surfactants which exhibit greater surface activity, and we are studying the emulsification and liposome-forming properties of the new compounds.
(1) Khorana, H. C., Chem,
Rev., 53, 145 (1953).
(2) Hoare, D. G., and Koshland, D. E., J.
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Department of