Clostridium pasteurianum rubredoxin and its recombinant counterpart purified from Escherichia coli have been analysed by electrospray ionization m.s. (e.s.i.m.s.). Whereas the N-terminal methionine of the native protein is formylated, the recombinant one has a free N-terminal methionine. E. coli cells also produce a colourless protein from the cloned gene. This protein is absent from C. pasteurianum and was shown to be zinc-substituted rubredoxin. The molecular forms of rubredoxin detected by e.s.i.m.s. depended on the experimental conditions used. Significant conversion into apo-rubredoxin occurred when the proteins were ionized at acidic pH and detected in the positive-ion mode. This conversion was quantitative in the case of Zn-rubredoxin. In contrast, when the proteins were analysed at neutral pH in the negative-ion mode, only the holoproteins, i.e. the species initially present in the solutions, were detected in the spectra. The e.s.i.m.s. experimental conditions set up here may prove useful for the analysis of other acidic metalloproteins with weakly bound metals.

Petillot, Y., Forest, E., Mathieu, I., Meyer, J., & Moulis, J. M. (1993). Analysis, by electrospray ionization mass spectrometry, of several forms of Clostridium pasteurianum rubredoxin. Biochemical Journal, 296(3), 657–661.