Abstract

A spectroscopic and enzymatic investigation of the cytochromes of B. megaterium and B. subtilis was conducted. Attempts were made to isolate and characterize the individual cytochromes as well as to determine the locale of such activities as cytochrome c oxidase, DPNH oxidase, succinic dehydrogenase, cytochrome c reductase and diaphorse. Only two methods proved effective in liberating the cytochromes; disruption of the cells with sonic osillations and grinding tlhe cells with alumina. These procedures caused a fragmentation of the cell membrane. It was found that the entire cytochrome complement of these two bacteria was bound intimately to particles which most likely arise from the cell membrane. The spectrum of these cytochrome containing particles displayed the typical four banded spectrum found in mammalian tissues and yeast. From the various studies made, the presence of cytochromes of the a, b, and c type was confirmed. A concentration of the cytochrome bearing particles could be brought about by either an ammonium sulfate fractionation or by centrifugation at 144,000 x g. for three hours. When the particles were centrifuged, a concentration of these particles in an intense color layer at the bottom of the centrifuge tube resulted. The spectrum of this concentrate showed peaks which were several times more intense than were those shown by the original sonic extract. With these sharpened peaks it was easier to note changes in cytochrome composition of different batches of cells. A few batches of cells contained a high content of cytochrome c, while most batches of cells had only a low content of this particular cytochrome. The content of the a type cytochrome also varied markedly in the different batches of cells. The content of cytochrome b was more resistant to change, but even the amount of this cytochrome showed some variation. A procedure for obtaining a partially purified cytochrome c from the cytochrome concentrate is presented. Extraction was brought about by pre-treating the fraction with Triton X-100 before performing a digestion with the hydrolytic enzyme lipase. This treatment liberates a soluble cytochrome c which can be precipitated from a concentrated ammonium sulfate solution with trichloroacetic acid in the usual manner. The absorption maxima of the reduced spectrum of the partially purified cytochrome were at 550, 520, and 415 mμ. The Soret peak shifted to 408 mμ. when the pigment was oxidized. A short discussion of the similarities between this bacterial cytochrome c and a lipid soluble cytochrome c obtained from mammalian tissues is included. The enzymatic studies showed that cytochrome c oxidase, DPHN oxidase, and succinic dehydrogenase are associated witht he particle described above, while the diaphorase and cytochrome c reductase were found in the more soluble fractions. Inhibition studies with cytochrome c oxidase, using cyanide, carbon monoxide and azide revealed a similar inhibition pattern to that shown by mammalian cytochrom oxidase.

Degree

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

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