Purification of cytochrome b6 A tightly bound protein in chloroplast membranes

Purification of cytochrome b₆ was pursued to further develop rational technology for purification, proof of purity, and study of properties of membrane proteins. Cytochrome b₆ was purified—the first time from any source—from spinach chloroplast membranes; yield of pure cytochrome b₆ was 30% of that found in ethanol-extracted particles. The three-step procedure (pH 8) employed: (I) extraction in Triton X-100-4 M (optionally 2 M) urea, (II) chromatography in a Bio-Gel A-1.5m Column (Triton X-100-4 M urea). Without this step, subsequent electrophoresis failed. (III) Preparative disc gel electrophoresis.

Properties of cytochrome b₆: Cytochrome b₆ migrated in undenatured form as a single band in disc electrophoresis (pH 8, 7 or 8.9). None of the limited, accepted properties of the cytochrome in particles was altered by the purification procedure: Reduced b₆ has absorption maxima (22 °C) at 434, 536, and 563 nm; at ?199 °C the a absorption region shows two peaks of equal intensity at 561 and 557 nm. Cytochrome b₆ is reduced by dithionite (not by ascorbate) and is autoxidizable. The prosthetic group of b₆ is protohaemin and is fully extractable by acid-acetone. No non-haem iron is present. The millimolar extinction coefficient of reduced b₆ (563–600nm) per mole of haem is 21. The protein equivalent weight is 40000 g per mole of haem. Cytochrome b₄ is an intrinsically aggregatable molecule. The reduced cytochrome does not react with CO except when Triton X-100 is present.