Initial purification study of the cytochrome b561 of bean hypocotyl plasma membrane

The plasma membrane (PM) of higher plants contains a major ascorbate-reducible, high-potential b-type cytochrome, named cytochrome b(561) (cyt b(561)). In this paper a rapid purification protocol for the cyt b(561) of bean hypocotyls PM is described. An almost 200-fold increase of cyt b(561) specific concentration was achieved with respect to the PM fraction, which contained about 0.2 nmol of ascorbate-reducible heme per mg protein. The procedure can be performed in one day starting from purified PMs obtained by the phase-partitioning procedure. However, cyt b(561) proved to be unstable during chromatographic purification and the amount of protein finally recovered was low. Purified cpt b(561) eluted as a 130,000 Da protein-detergent complex from gel-filtration columns. It was completely reduced by ascorbate and reduced-minus-oxidized spectra showed alpha-, beta- and gamma-bands at 561, 530, and 429 nm respectively, not unlike the spectra of whole PMs. This work represents an initial approach to the biochemical characterization of the cyt b(561) of higher plants, formerly suggested to be related to cyt b(561) of animal chromaffin granules.