Immunological Properties and Conformational Differences Detected by Tritium-Hydrogen Exchange of Clostridial Ferredoxins and Apoferredoxins

Abstract

Abstract Antisera were prepared against the ferredoxins from Clostridium acidi-urici and Clostridium pasteurianum and characterized by quantitative precipitin and microcomplement fixation methods. The clostridial ferredoxins are thus the smallest naturally occurring polypeptides that have been shown to produce precipitating antibodies. The ferredoxin antibody of the immune rabbit serum is contained in the IgG fraction. The antiserum prepared against C. pasteurianum ferredoxin does not react with C. acidi-urici ferredoxin. Reconstituted ferredoxin is antigenically indistinguishable from the native ferredoxin as tested by microcomplement fixation. The antisera react much more strongly with ferredoxin than with apoferredoxin. Interpretation of this observation with respect to showing the difference in the conformation of ferredoxin and apoferredoxin is limited because the possible role of the iron-sulfur chromophore of ferredoxin as an antigenic determinant is not known. However, experiments with the tritium-hydrogen exchange technique showed conformational differences between the polypeptide portions of apoferredoxin and ferredoxin. Differences between the conformations of the polypeptide chain of the oxidized and the reduced states of C. acidi-urici ferredoxin were also shown by the tritium-hydrogen exchange technique. The reduced state was found to have fewer sites for equilibration with tritium, and thus to have a conformation more compact than the oxidized state of the iron-sulfur protein. Ferredoxin reconstituted from the fully tritiated apoferredoxin was found to retain more replaceable hydrogen atoms than the native oxidized ferredoxin.