To date, preparative electrophoretic methods for subcellular-sized particles and complexes have been in use which are responsive to net charge differences among such particles, but poorly responsive to differences in size and shape. To exploit size and shape differences among large particles, this laboratory has developed two techniques: An adaptation of preparative automated gel electrophoresis apparatus for use with concentrated polymer solutions in addition to use with gels, and a free-flow electrophoresis apparatus with capillary dimensions between the glass walls of the vessel through which the flow is conducted. During the past year, both of those techniques were successfully applied to preparative separations of subcellular-sized particles. The free-flow electrophoresis technique, using narrowly spaced glass plates, was applied to the separation of sea urchin egg vesicles. Vesicles derived from fused and non-fused sea urchin eggs, and, more importantly, fertilized and unfertilized eggs, were separated The capacity of the fractions to fuse in presence of Ca was maintained. The preparative mode of the automated gel apparatus was applied to rat liver microsomes. These had been show during the previous year to separate into three major electrophoretic components. Each of those was now isolated. The yield of the main component was near-quantitative after the first electroelution step, that of the two other components after no more than two steps. Each of the three fraction gave rise to identical SDS-peptide patterns, showing that their differences did not reside in the dominant elements of their protein composition. The major component exceeded the others in size, while these differed in surface net charge only. The isolated products of two of the components were not detectably impure, while a polymeric impurity in the third amounted to 10-15% by weight only. Thus, both types of preparative size separations tools have been found to be capable of yielding fractions capable of yielding functional information in future studies.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Intramural Research (Z01)
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