Haem staining in gels, a useful tool in the study of bacterial c-type cytochromes

Abstract

c-Type cytochromes are the only cytochromes to retain quantitatively their haem during SDS gel electrophoresis and can be identified in complex mixtures by their haem peroxidase activity. Although weak staining bands may be due to residual haem attachment to b-type cytochromes or to migration of haem, these effects could be abolished by prior extraction with organic solvent. The colour yield of haem staining allowed an estimate of the relative amounts of a particular cytochrome, particularly if loadings were below 50 pmol. At greater loadings, a plateau of colour development was observed. Freshly made gels gave much poorer colour development. The haem-staining method was shown to be useful in three particular areas of study in bacterial respiration. Firstly, it allows assessment of the results of sphaeroplast formation in gram negative bacteria. Secondly, quantitation of the haem stain was useful in the investigation of the induction effects of growth conditions on c-type cytochromes. Thirdly, the interpretation of complex chromatographic profiles was greatly simplified by the use of haem-stained SDS electrophoretic gels.