Electrophoretic purification of a water-soluble guinea pig transplantation antigen.

The isolation of tissue transplantation antigens has been energetically pursued since Billingham et al.' found that cell extracts could induce homograft immunity. A major problem in this endeavor has been the solubilization of the antigens which probably reside on cellular membranes. Studies employing tissue homogenization' and pressure decompression' to disrupt cells have yielded insoluble antigenic preparations of a gross lipide composition. On the other hand, water-soluble materials have been liberated by means of low-frequency ultrasound,4 by digestion of membranous sediments,5' 6and by digestion combined with chelation of divalent cations.7 The antigen released from murine splenic cells by ultrasound has been analyzed by gel filtration on Sephadex G-200, by equilibrium ultracentrifugation, and by linear sucrose gradients.4 The active substance had a buoyant density of greater than 1.238, which was compatible with a proteinaceous rather than lipoprotein nature. It behaved on gel filtration and in sucrose gradients as if it had an approximate molecular weight of slightly less than 200,000. The present report describes the purification of sonically liberated guinea pig transplantation antigen by discontinuous polyacrylamide gel electrophoresis. This method has achieved remarkable resolution of the components of complex mixtures of macromolecules.8-"1 In the present situation the active fraction obtained by gel filtration contained 17 distinct components, and the antigenic activity was confined to a single one of this array of tissue components. Materials and Methods.-Preparation of the antigen: The spleens of inbred, histocompatible strain 2 guinea pigs"2 were excised, cleansed of fat, and minced in Tris-sucrose buffer (0.05 M Tris, 0.008 M magnesium chloride, 0.0025 M potassium chloride, 0.15 M sucrose adjusted to pH 7.45, at 25-C). Single cell suspensions were prepared either by pressing the tissue through a nylon stocking top, or by consecutive passes through nylon screening of 50 and 200 mesh. The erythrocyte contamination of the cell pellet obtained by centrifugation at 950 X g was decreased by lysis with 3% acetic acid in Tris-sucrose buffer. After two washes with Tris-sucrose buffer, the residual cells were passed through a double thickness of nylon stocking, and the cell concentration was adjusted to 8-10 X 106 cells/ml. The filtered cell suspension was sonicated in a Raytheon model DF 101 150W magnetostrictive oscillator at 10 kc/sec for 3 min at 1.0 amp and a temperature of less than 100C. After removal of the cell debris by centrifugation at 2000 rpm for 15 min, the cell membranes were sedimented by ultracentrifugation at 130,000 X g for 90 min in a model L2 65 Spinco preparative ultracentrifuge. The supernate was placed in dialysis tubing which had been boiled in 0.1 M sodium carbonate and stored in 0.01 M ethylenediaminetetraacetate (EDTA), and concentrated against Aquacide I, C grade (Calbiochem). The concentrated supernate was passed at a flow rate of 3.5-6.0 ml/hr over a 2.5 X 100-cm column of Sephadex G-200 (Pharmacia Fine Chemicals), which had been equilibrated with 0.5 Al glycine, 0.2 A! Tris, 0.5%70 mannitol, p11 8.0. Fractions were collected in 2.5-ml aliquots and read for absorbancy at 280 muA. Fractions were lyophilized following desalting through a Sephadex G-25 column which had been equilibrated with 0.05%, mannitol. Antigen was reconstituted with distilled water. Protein concentrations were estimated according to the method of Zamenhof,13 using ovalbumin as standard. Analytical disc electrophoresis: Analytical acrylamide gel electrophoresis was performed with-