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In Agrobacterium tumefaciens the main part of c-type cytochromes is tightly bound to the bacterial cell envelope structures. Several techniques were attempted to solubilize these cytochromes. The highest yield of cytochromes released is obtained by treatment of particle suspensions with 5% Triton X-100. Further purification confirms that the proteins are not really solubilized, but still aggregated in small heterogeneous complexes. Chromatography on a CM-cellulose column demonstrates that at least three different c-type cytochromes are present: cyt c-550, cyt c-552 and cyt c-556.

Cytochrome c-556 from Agrobacterium mefaciens B2a was isolated in a pure, homoneous state. The best purification procedure volved ammonium sulphate fractionation, delting on Sephadex G-25, column chromatographic fractionation on DEAE- and CM-cellulose, and gel filtration on Sephadex G-75 superfine. Substitution of the CM-cellulose step by isoelectric focusing was successful. The purity of the final preparation is warranted by the purity index value, the electrophoretic patterns in the absence and presence of sodium dodecylsulphate, the sedimentation profile and the N-terminal amino acid analysis (alanine). The absorption spectrum of reduced cytochrome c-556 has maxima at 318, 419, 526 and 555.5 nm. The molar extinction coefficient for the alpha-band is 20 200M-1cm-1. The isoelectric point, determined both by preparative and analytical isoelectric focusing, is 5.55 +/- 0.10. The molecular weight of cytochrome c-556 was determined by gel filtration as 12000 and by dodecylsulphate gel electrophoresis as 11 500.