Date of Award:

5-1982

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Chemistry and Biochemistry

Advisor/Chair:

Thomas F. Emery

Abstract

The isolation, purification, and partial characterization of a novel iron-containing protein from the sulfate-reducing anaerobic bacterium, Desulfovibrio gigas, is described. The highly insoluble protein was isolated from the cell debris following osmotic shock of the bacteria. The insoluble fraction consistently contained about 90% of the cell-associated iron. This protein was treated with acid, chelating agents, detergents and proteases in order to study their effect on the iron solubility. The results of elemental analysis of a crude protein preparation were 5.3% iron, 2.9% sulfur and 11 .9% nitrogen. An independent colorimetric iron analysis showed 6.4% iron. The iron could be dissociated from the protein by treatment with 5% sodium dodecyl sulfate. The iron-free protein was purified by a combination of organic extraction and DEAE-cellulose chromatography. The purified protein showed only one major band, MW 14,000, by SDS polyacrylamide gel electrophoresis. The protein could be reconstituted upon treatment with an appropriate mixture of FeS and B-mercaptoethanol. The reconstituted protein had the same physical and chemical properties as the native protein. The amino acid composition was not unusual except for the high isoleucine content. The amino acid composition was similar to that of a number of membrane proteins.

Growth and iron assimilation in D. gigas were studied and the form of iron present in the medium were also examined. The intracellular distribution of iron and sulfur assimilated from the medium was determined. The presence of an iron chelating compound was also briefly examined.

Some interesting observations and experiments dealing with colored iron complexes and their characteristics are included. An unusual basic component tentatively identified as cadaverine is also discussed briefly.

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