Purification of cytochrome b-561 : An integral heme protein of the adrenal chromaffin granule membrane

Abstract

1. 1. Cytochrome b-561, an integral heme protein of the adrenal chromaffin granule membrane, has been solubilized and isolated in highly purified and water soluble form by an non-ionic detergent procedure. 2. 2. The prosthetic group of cytochrome b-561 is protoheme IX. 3. 3. Gel electrophoresis in sodium dodecylsulfate of the isolated cytochrome revealed a single band of a low molecular weight polypeptide, and on the basis of amino acid analyses, a minimum molecular weight of 4380 was computed for the apoprotein. 4. 4. The cytochrome is a typical integral membrane protein with a high percentage of hydrophobic amino acids (i.e. 36%) and a high average hydrophobicity (i.e. 1173 cal/residue). A considerable amount of Triton X-100, firmly bound to the apoprotein, is responsible for its solubility in aqueous solution. 5. 5. Molecular-sieve chromatography indicated that the cytochrome exists in an aggregated form in aqueous solution. 6. 6. The absorption spectra revealed peaks at 360, 415 and 531 nm in the oxidized form, and at 427, 530 and 561 nm in the reduced form. The millimolar absorptivity (ε) at the α-band (reduced) was 28.3 mM −1·cm −1 at 561 nm.