Cross-linked Polyampholytes

In connection with an investigation of the staining of intracellular antigens with ferritin-antibody conjugates (1), we required an embedding material which was extensively ionic. Although several hydrophilic embedding materials have been described (cf. reference 2), to our knowledge no satisfactory ionic ones have been reported. We sought to prepare an ionic embedding material by the bulk polymerization of a suitable monomer or monomers. The simplest kind of ionic polymer is a polyelectrolyte (3), which may be prepared by poly-merizing an ionizable monomer such as metha-crylic acid (MA). A polyelectrolyte thus contains ionic charges of only one sign. It occurred to us that it would be more advantageous, for a variety of reasons, to prepare a polyampholyte (4), made by the copolymerization of comparable molar quantities of two monomers of opposite ionic charge. A polyampholyte is studded with more or less alternating negative and positive charges which confer on it a large total ionic charge but only a small net ionic charge. In this respect, a polyampholyte resembles a protein molecule, or the surface of a biological membrane. Fortunately, the copolymerization of a number of such pairs of vinyl monomers has already been carefully investigated (4-6). Such copolymers are usually water-soluble, so that for tissue embedding it would ordinarily be necessary to add a cross-linking agent to the monomers to render the co-polymer insoluble. We have made cross-linked vinyl-type polyampholytes from the anionic monomer MA and the cationic monomer di-methylaminoethylmethacrylate (DMA), together with a small amount of a cross-linking monomer, tetramethylenedimethacrylate (TMA). In preliminary trials, these cross-linked polyampholytes have exhibited satisfactory and reproducible properties as embedding materials for electron microscopy. The MA-DMA monomer mixture is miscible with water in all proportions. In recent years increasing attention has been given to water-soluble embedding media for various kinds of electron microscope investigations (2). Our limited experience with these polyampholytes is therefore described in this paper in the hope that these systems will be of some general interest. ~[heir utility in conjunction with ferritin-antibody staining techniques will be discussed in a separate communication (7). MATERIALS AND METHODS The monomer MA (Rohm and Haas Co., Phila-delphia) was distilled at l0 mm pressure; the fraction boiling at 60°C was collected and stored at 4°C. DMA (Rohm and Haas) and TMA (Mono-mer-Polymer Laboratories, Philadelphia) tended to polymerize on vacuum distillation, and were therefore used as supplied by the manufacturers. The water content of the monomers was not …