Ultracentrifuge electrophoresis apparatus

A combination ultracentrifuge-electrophoresis apparatus has been proposed for macromolecular separations by placing electrodes at the top and bottom of the centrifuge cell. Such a device takes advantage not only of difference in mass but also charge. 

The electrophoresis apparatus has an advantage over the ultracentrifuge in the ease of detecting extraneous DNA which may result from degradation of either the host cell or the virus. Kozloff and Putnam (161) used the electrophoretic separation cell to remove rapidly migrating DNA present as an impurity in bacteriophage T6. In a later study of T5 it was found that DNAase effectively removed all electrophoretically detectable DNA so that an apparently homogeneous preparation was obtained. 

Painstaking attempts to identify the size of the infectious unit have in the past been necessitated for plant viruses, and also for animal viruses for which hitherto no method of assay for single virus particles has materialized. These include chemical fractionation, physical fractionation by means of separation cells in the ultracentrifuge and electrophoresis apparatus, and inactivation of the virus (Lauffer, 181,182,184,299). Because of electron microscopy and the excellent linearity of the plaque count, these methods have not proved necessary with the bacteriophages. 

Another purified enzyme preparation which produces laminaripentaose from insoluble laminarin and from heat-treated pachyman is produced by a strain of Arthrobacter luteus (100,101,102) when grown on yeast cells or / -(1 —>3)-glucan. The enzyme, which was named Zymolase (also referred to as Zymolyase) appeared to be homogeneous by electrophoresis in a Tiselius apparatus and by ultracentrifugation. The molecular weight of the enzyme was estimated from ultracentrifugation to be ca. 20,500. The optimum pH for lysis of viable yeast cells was 7.5. The optimum temperature was 35°C. The optimum pH for heat-treated pachyman hydrolysis was 6.5, and the optimum temperature was 45°C. A Lineweaver-Burk plot with heat-treated pachyman yielded a Km value of 0.04% when the solubilized carbohydrate was assayed by the phenol-sulfuric acid method. Zymolase lost all its activity after incubation at 60°C for 5 min. 

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