Isoelectric Focusing in Space
- PDF / 1,206,981 Bytes
- 6 Pages / 414.72 x 648 pts Page_size
- 103 Downloads / 173 Views
261
ISOELECTRIC FOCUSING IN SPACE
M. BIER, N. B. EGEN, R. A. MOSHER AND G. E. TWITTY Biophysics Technology Laboratory, University of Arizona,
Tucson,
Arizona, USA
ABSTRACT The potential of space electrophoresis is conditioned by the fact that all electrophoretic techniques require the suppression of gravity-caused convection. Isoelectric focusing (IEF) is a powerful variant of electrophoresis, in which amphoteric substances are separated in a pH gradient according to their isoelectric points. A new apparatus for large scale IEF, utilizing a recycling principle, has been developed. In the ground-based prototype, laminar flow is provided by a series of parallel filter elements. The operation of the apparatus is monitored by an automated array of pH and ultraviolet absorption sensors under control of a desk-top computer. The apparatus has proven to be useful for the purification of a variety of enzymes, snake venom proteins, peptide hormones, and other biologicals, including interferon produced by genetic engineering techniques. In planning for a possible space apparatus, a crucial question regarding electroosmosis needs to be addressed. To solve this problem, simple focusing test modules are planned for inclusion in an early Shuttle flight.
INTRODUCTION Electrophoresis of biological materials without the interference of gravity may become an interesting and possibly significant use of orbiting spacecraft (1). Electrophoresis is defined as the transport of electrically charged molecular species under the influence of a direct current electrical field. Because of its singular usefulness for the analysis and separation of complex protein mixtures a number of electrophoretic techniques have been developed (2). An essential feature of these is the necessity to stabilize the liquid medium against gravity-driven convective flows. The microgravity environment of orbiting spacecraft provides an alternate means for avoidance of convection. This has been verified in pilot experiments conducted aboard the Skylab, Apollo 16, and Apollo-Soyuz space missions (3-7). Isoelectric focusing (IEFY is a particualrly powerful variant of electrophoresis, in which the electrophoretic separation is carried out in a pH gradient. The proteins migrate, i.e., focus, to the pH region corresponding to their isoelectric point, where they become immobilized due to zero net charge. The isoelectric point of a protein is a rather characteristic parameter, and analytical IEF can resolve proteins differing by only 0.01 pH units. The pH gradient is established 'naturally', through the focusing of appropriate buffer mixtures, usually commercially available mixtures of carrier ampholytes, marketed under a variety of tradenames, such as Ampholine. The objective of our project was the development of new technology for large scale purification of peptide hormones, proteins and other biologicals, with particular emphasis on the possible usefulness of the microgravity environment. The basis of this technology was the development of a novel type of focusing
Data Loading...