THE BIREFRINGENCE OF THE HUMAN RED CELL GHOSTS* BY ERIC PONDER A~m

Schmitt, Bear, and Ponder (1936, 1938) observed that the rabbit red cell ghost, prepared by freezing and thawing, is slightly birefringent. Extraction of the lipids and suspension of the ghosts in media of varying refractive indices led to the conclusion that a positive intrinsic birefringence, due to the radial orientation of lipid, is nearly balanced by a negative form birefringence due to the tangential orientation of proteins. The net birefringence is very small, and, looking back on the experiments, the ghosts were not as Hb-free as was thought; in 1938, the possibility of the complexing of Hb with lipoproteins had not even been suggested. Mitchison (1950, 1953) and Swarm and Mitchison (1950) have described a technique, excellent in itself, for making quantitative measurements of the birefringence of the human red cell ghost, prepared by adding red cells to glycerol. Their results led them to the conclusion that there is a birefringent region, about 5000 A thick, at the red cell surface, and Mitchison has proposed a structure for the mammalian red cell or ghost in which bundles of protein, arranged like Chinese crackers, occupy a layer about 5000 A thick. Such a layer would make up about half the volume of the red cell. Mitchison does not think that the birefringence measured by his method is dependent on the admitted presence of Hb in the surface ultrastructure of his ghosts, but the reasons given (of. Perutz and Mitchison, 1950) have been criticised by Dervichlan (1955). The complexing of Hb with the lipoproteins of the red cell ultrastructure has now been demonstrated experimentally (Bessis, Bricka, Breton-Gorius, and Tabuis, 1954; Ponder, Ponder, and Barreto, 1955). This paper is concerned with the question as to how much of the birefringence of the red cell ghost is due to its containing complexed Hb. Starting with the observation (Ponder, 1952) that there is a large variation in the amount of residual IIb contained in ghosts prepared in glycerol, by freezing and thawing, and by many other methods, we propose to show that only those ghosts which confain enough Hb to render them visible with the ordinary microscope show birefringence of the type described by Mitchison, while ghosts which contain so little Hb that they are invisible with the ordinary microscope, although