Properties of spinach ferredoxin in anaerobic urea solution: a comparison with the native protein.

The preparation of spinach ferredoxin by a modification of the method of Keres&es-Nagy and Margoliash which is particularly convenient for large-scale operation is described. The optical, CD, and EPR spectra of this protein are studied in 5 M urea as a function of ionic strength. At high ionic strength the various spectra are very similar to the native protein; at low ionic strength all three spectral parameters are greatly changed. The reactivity of the protein with mercurial and iodoacetamide is compared. The very high reactivity of the former is rationalized by assuming an initial reaction with the sulfide moieties. Iodoacetamide reacts only with accompanying denaturation by an all-or-none mechanism. At most only one sulfhydryl is available for reaction, even in 5 M urea. This is consistent with the assumption that the mercaptides function as metal ligands. A very sensitive instrument for the amperometric titration of SH groups is described.