Spectrophotometric determination of acid mucopolysaccharides

The present report describes a novel spectrophotometric method for the quantitative determination of acid mucopolysaccharides based on the interaction of these macromolecules with the zirconyl ion. The method is simple, accurate, and involves the determination of the acid mucopolysaccharide molecules rather than their hydrolytic components (as in the case of existing methods of analysis). Substances, which are normally present in the acid mucopolysaccharide preparations (such as protein, glycoprotein, and nucleic acid), do not interfere with the determination.     

The acid mucopolysaccharides of cattle retina

1. Two polysaccharides were isolated from the interstitial matrix surrounding the photoreceptor cells of cattle retina. They were liberated from this region of the tissue in a soluble form after agitation of whole retinas in 0.9% sodium chloride. One, which comprises two-thirds of the polysaccharides present, is a hyaluronidase-sensitive ;half-sulphated' chondroitin sulphate containing uronic acid, galactosamine and sulphate in the molar proportions 1.27:1.0:0.54. The other is a hyaluronidase-resistant non-sulphated heteropolysaccharide for which the name sialoglycan is proposed. It contains galactose, glucosamine and sialic acid in the molar proportions 2.4:1.0:0.4. Both polysaccharides contain only small amounts of nitrogen in excess of the amount calculated from their amino sugar and sialic acid content. 2. A similar combination of mucopolysaccharides is associated with the pigment epithelial-cell layer but in quantities only one-fifth of those present in the adjacent matrix area. 3. The ease with which they are released into aqueous media is consistent with the assumption that they are present in the extracellular spaces in both of these tissue layers. 4. The retinal residue left after removal of the two soluble polysaccharides is rich in amino sugar- and sialic acid-containing polymers, which appear to be firmly bound to the tissue fragments. 5. About one-third of the sialic acid and one-tenth of the amino sugar could be extracted with chloroform-methanol. The components in this fraction were tentatively identified as gangliosides. 6. Digestion of the chloroform-methanol-insoluble residue with Pronase yielded as the principal product a heteropolysaccharide containing 16.5% of glucosamine, 24.3% of neutral sugar (galactose plus fucose) and 18.1% of sialic acid. This substance has been classified as a sialoglycan of composition similar to (but not identical with) that of the soluble one isolated from the matrix area of the tissue.     

Electrophoretic separation and characterization of pea protoplasts

A carrier-free electrophoresis system was developed to separate protoplasts according to their zeta-potential and to study their behaviour in subsequent cell culture. Apex protoplasts from young cotyledon-free pea embryos ( Pisum sativum L. cv. Belman), including primordia, were analysed with respect to their electrophoretic mobility, viability, cell size, cell division capacity, chlorophyll content, and expression of a 50 kDa protein involved in early somatic embryogenesis. Protoplasts fractionated on the basis of their electrophoretic mobility were viable and able to divide and form microcallus in cell culture. Cell culture procedures were modified in order to handle low numbers of protoplasts (50–2000 per fraction). Electrophoretic separation revealed distinct classes of protoplasts, differing in all parameters tested except cell size. Therefore, a connection between zeta potential and other physiological parameters can be proposed.     

Electrophoretic cell separation by means of microspheres

The electrophoretic mobility of fixed human erythrocytes immunologically labeled with poly(vinylpyridine) or poly(glutaraldehyde) microspheres was reduced by approximately 40%. This observation was utilized in preparative scale electrophoretic separations of fixed human and turkey erythrocytes, the mobilities of which under normal physiological conditions do not differ sufficiently to allow their separation by continuous flow electrophoresis. We suggest that resolution in the electrophoretic separation of cell subpopulations, currently limited by finite and often overlapping mobility distribution, may be significantly enhanced by immunospecific labeling of target populations using microspheres.