of Work: The development of electrophoretic methods was pursued in application to four technologies: i) capillary zone electrophoresis (CZE), ii) automated gel electrophoresis (HPGE), iii) miniaturized gel electrophoresis (PhastSystem) and iv) free- flow electrophoresis (FFE). i) The type, size and concentration of polymers in solution was correlated with retardation of migration and resolution by CZE of proteins and polystyrene size standards of up to 800 nm in diameter. In the size range of macromolecules and their complexes, up to 30 nm diameter, particles are retarded in their migration in polymer solutions in proportion to their size and independently of polymer length and field strength and in a polymer- type-specific fashion. Theory has been developed to predict particle size on the basis a known polymer size or vice versa. Larger particles in the size range of 30 to 250 nm diameter are retarded in their migration in inverse relation to particle size. Hypothetical mechanisms of retardation were proposed but no predictive theory of retardation exists as yet for that particle size range. An increase in resolution by a factor of 3 was demonstrated for particles with smooth surfaces. Particles with uneven surfaces (carboxyl modified, hairy) in the size range 110 to 430 nm diameter exhibit a resolution optimum, with several fold increase in resolution, at a polymer concentration at or near the entanglement threshold concentration, c*, at an optimal field strength in CZE of about 200 V/cm and using a relatively short polymer length. Particles with similar surface net charge density in the size range of 200 -800 nm in diameter were found to separate in a size dependent fashion in CZE within a medium range of ionic strength, in accordance with theoretical prediction. ii) HPGE provides a high yield, labor-saving preparative method for protein characterization by mass spectrometry, but is limited by its restriction to fluorescently labeled particles. That limitation was overcome in application to SDS-proteins, detectable in HPGE when non-covalently bound to Cascade Blue which can be separated prior to mass spectrometry. The sample applicator of the HPGE apparatus was modified by insertion of an agarose saturated filter paper seal, thereby solving the problem of hardware unavailability for that apparatus since the closing of its manufacturing firm in 1999. A method for transferring native protein into mass spectrometry was also elaborated. The protein zone is detected by a bromophenolphthalein ester at one spot along the band and electroeluted into a capillary tube close in width to the bandwidth and filled with buffer. With a protein recovery of up to 60%, 20 microgram protein loads suffice for MALDI-TOF analysis of the electroeluate. iii) The development of FFE into a preparative method for particles of subcellular and cellular size was initiated by demonstrating the proportionality of fraction number and mobility, the reproducibility of fraction numbers and the identification of fractions by mobility relative to that of a dye (Rf). Size separations in FFE at low ionic strength in the absence of polymers were demonstrated for polystyrene particles with similar surface charge density of 73 to 762 nm diameter, in accordance with the results of CZE (i). iv) Membrane complexes of proteins putatively involved in the docking and fusion of sea urchin egg cortical membranes were extracted in Triton-X-114. These complexes occur at too low concentrations to be readily detectable by Western blotting using the available antibodies directed against syntaxin, SNAP-25 and VAMP whereas recombinant forms of those proteins, available in larger amounts, were detected. To be able to detect at a 100-fold increase in sample load, a 0.5 ml volume sample applicator module for the PhastSystem was constructed. Its application to sample concentration by a moving boundary electrophoresis buffer system (stacking) is in the process of being elaborated. v) The previously postulated mechanism of electrophoretic peak spreading and peak asymmetry by interaction with the walls of the vesselwas tested by CZE in capillaries varying in diameter from 50 to 150 micron. Independently of capillary diameter, peak spreading was found to be protein-specific, to decrease with sample concentration of one protein out of three, and to be independent of field strength. Peak asymmetry was found to be correlated with peak spreading in the narrowest capillary, independent of peak spreading in the capillary of 100 micron diameter in application to one protein. Another protein failed to exhibit peak asymmetry altogether, and a third protein at high sample concentration showed an anomalous transition from negative to positive asymmetry as peak spreading increased. vi) Beta amyloid peptide (1-42), a component of neuritic plaques in Alzheimers disease, was shown to exhibit a size and surface net charge similar to those of the non-toxic (1-40) peptide rather than - as hypothesized in the literature - its toxic form. vii) CZE and PAGE conducted under nondenaturing conditions reveal 4 charge isomeric species of commercial recombinant Green Fluorescent Protein (rGFP) while rGFP isolated from eukaryotic cells in the laboratory as well as the SDS-derivative of commercial rGFP appear homogeneous. To the degree that the charge isomeric state of rGFP may impact on its use as a fluorescent tag, the demonstrated heterogeneity of the commercial native protein may be of importance. viii) A program for simulation of electrophoretic separations was created and is being validated by capillary electrophoresis.
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