Helicorubin and cytochrome h. Purification and physical properties

Abstract

Helicorubin is a natural hemochromogen found free in the gastro-intestinal tract of snails and some other invertebrates. Since its discovery all studies were carried out on crude or very impure material. A method is described for the purification of this hemoprotein from the gastro-intestinal fluid of the edible snail (Helix pomatia). A very similar hemochromogen, present in the tissues of the gastric gland (hepatopancreas) of the snail, was also isolated in a greatly purified state. In view of the intracellular localization of this hemochromogen and of some of its properties, it is called cytochrome h. Preparations of helicorubin and cytochrome h were obtained which gave single symmetrical boundaries of the hemoproteins in the ultra-centrifuge and on electrophoresis in the Tiselius apparatus. Although these preparations still contain variable concentrations of a poly-saccharide material, its presence is not revealed in these experiments. Evidence that polysaccharide does not form part of the hemoprotein molecules, and a possible method for the complete separation of polysaccharide from the hemoproteins, are discussed. The molecular weight of helicorubin and cytochrome h, calculated from the sedimentation and diffusion constants, and iron and protein contents of the preparations, is about 18,500. Helicorubin and cytochrome h are both acidic hemoproteins, with isoelectric points below pH 4.3; they can be separated electrophoretically from a mixture, helicorubin being the more acidic of the two. Helicorubin and cytochrome h each contain one atom of iron/molecule. Both hemoproteins undergo oxidation and reduction: in the reduced and oxidized states the absorption spectra are of typical hemochromogen and parahematin types respectively. Their absorption bands, compared with those of the cytochrome components, lie much nearer those of cytochrome b than of cytochrome c. The absorption bands of helicorubin lie at longer wave lengths than the corresponding bands of cytochrome h. Helicorubin and cytochrome h, like cytochrome c but unlike other known components of cytochrome, are heat-stable. Unlike cytochrome c, they do not combine with CO at any pH. Helicorubin, which is always found free in solution in the gastro-intestinal fluid of snails, is intimately related to the cytochrome h present in the cells of the digestive gland, and probably represents a slightly modified form of cytochrome h liberated with other substances during the normal secretory activity of this gland.