Initial purification study of the cytochromeb 561 of bean hypocotyl plasma membrane

Summary The plasma membrane (PM) of higher plants contains a major ascorbate-reducible, high-potentialb-type cytochrome, named cytochromeb₅₆₁ (cytb₅₆₁). In this paper a rapid purification protocol for the cytb₅₆₁ of bean hypocotyls PM is described. An almost 200-fold increase of cytb₅₆₁ 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, cytb₅₆₁ proved to be unstable during chromatographic purification and the amount of protein finally recovered was low. Purified cytb₅₆₁ 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 -, - and -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 cytb₅₆₁ of higher plants, formerly suggested to be related to cytb₅₆₁ of animal chromaffin granules.Abbreviations cytb₅₆₁ cytochromeb₅₆₁ - PM plasma membrane - UPV upper-phase vesicles - GSII glucan synthase II - CCR NADH-dependent cytochromec reductase - CCO cytochromec oxidase - TX-100R reduced Triton X-100