Localization of sialyl residues on cell surfaces by affinity cytochemistry

Abstract

The use of the avidin—biotin complex for the specific ultrastructural visualization and evaluation of cell surface sialyl residues was investigated. Circulating red blood cells of various mammalian species and bone marrow cells of the rabbit were treated with sodium meta -periodate and biotin hydrazide in succession, with or without prior treatment of the cells with neuraminidase. The results were compared with the distribution of cell surface anionic sites, estimated by the rate of agglutination with poly- l -lysine and the binding capacity of positively charged colloidal iron and cationized ferritin. A uniform labeling of the erythrocyte surface was obtained with variations between different animal species. Neuraminidase treatment drastically reduced the sensitivity of cells to periodate-induced biotinylation. This method represents a distinct improvement over past approaches, since it can be applied to unfixed cells at physiological pH. An additional feature of this system enabled the topographic resolution of sialic acid residues from the outer dense line of the erythrocyte membrane. A value of 50–70 Å was obtained for human erythrocytes. Developmental and species-dependent modulations in the proximity of sialic acid to the membrane were also observed. The frequency of attached ferritin—avidin conjugates to biotinylated erythroid cells in rabbit bone marrow was found to vary according to the degree of maturation in the erythroid line, corresponding to the previously described variations in colloidal iron binding.