An investigation is proposed to evaluate the feasibility of utilizing the unique critical phenomena of neutral (i.e. nonionic and zwitterionic) surfactant micellar systems as a new general type of extraction and concentration step for the purification of biomolecules. The technique is based upon the fact that aqueous solutions of such surfactants, upon temperature alteration, exhibit clouding in which the surfactant-rich phase separates from the bulk aqueous phase. Thus, any biomolecules or other species which partition to the micellar aggregate will be enriched and concentrated in the surfactant-rich phase as well as separated from those substances present in the aqueous phase that do not bind to the micellar pseudophase. Previously, this approach has been utilized to separate and concentrate different metal ions. Preliminary work in our labs demonstrate that the same principle can be used for the separation and extraction organic and bioorganic molecules. In our feasibility study, the main focus will concentrate on identifying the factors that can be manipulated in order to achieve the selective partitioning required in such separation and purification technique. This will be done by determining the interaction and partition coefficient between different neutral surfactants and several series of model biomolecules (amino acids, peptides, proteins, enantiometric drugs, and medicinals) as a function of variation of experimental parameters, such as pH, pI, ionic strength, concentration of surfactant and biomaterial initially present. In order to improve the specificity, appropriate alkyl affinity ligands, which will serv as a co- surfactant, will be synthesized that are specific for certain classes of biomolecules and their effect upon the extraction evaluated. The other main, key studies to be conducted include: (i) determination, where applicable, of the effect of the surfactant and cloud point extraction technique upon the biomolecules' activity or function; (ii) an evaluation of the compatibility of this extraction step with other prior or subsequent steps (chromatographic) that may be required for ultimate purification; (iii) examination of the ability to easily scale up the extraction from the submicrogram to kilogram range of the biomolecule originally present; and (iv) evaluation of the different possible means for removing the surfactant from the concentrated, extracted biomolecular material. In addition, the technique will be tested on some real biomaterial samples and the results compared to that obtained using conventional purification schemes. The successful development of a new extraction scheme to the health-related sciences should be obvious.
| Saitoh, T; Hinze, W L (1991) Concentration of hydrophobic organic compounds and extraction of protein using alkylammoniosulfate zwitterionic surfactant mediated phase separations (cloud point extractions). Anal Chem 63:2520-5 |
