Initial purification study of the cytochrome b(561) of bean hypocotyl plasma membrane

The plasma membrane (PM) of higher plants contains a major ascorbate-reduci ble, 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 hy pocotyls PM is described. An almost 200-fold increase of cyt b(561) specifi c concentration was achieved with respect to the PM fraction, which contain ed 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 pha se-partitioning procedure. However, cyt b(561) proved to be unstable during chromatographic purification and the amount of protein finally recovered w as low. Purified cpt b(561) eluted as a 130,000 Da protein-detergent comple x from gel-filtration columns. It was completely reduced by ascorbate and r educed-minus-oxidized spectra showed alpha-, beta- and gamma-bands at 561, 530, and 429 nm respectively, not unlike the spectra of whole PMs. This wor k 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.