A glycoprotein of molecular weight 18,000 was purified from saline extracts of cured tobacco leaves by ammonium sulfate fractionation, chromatography on Sephadex G-25, and continuous-flow preparative electrophoresis on polyacrylamide gel. Twelve of 31 volunteers (%5 smokers and 6Yi6 nonsmokers) exhibited immediate cutaneous hypersensitivity (wheal and flare reactions) when injected intracutaneously with 2 fig of this material. Immunochemically similar material was demonstrated in, and purified from, cigarette smoke condensate and cigarette smoke. The concentration in cigarette smoke condensate ("tar") was determined to be 1.8-3.6 mg/g. Antigenically crossreactive material was also demonstrated in eggplants, green peppers, potatoes, and tomatoes which, like tobacco, are members of the family Solanaceae.Immediate cutaneous hypersensitivity to extracts of defatted tobacco leaves has been reported previously in smokers and in nonsmokers. Harkavy has suggested that allergy to constituents of tobacco may underly the relationship between tobacco smoking and coronary artery disease and peripheral vascular disease (1,2 Purification of Tobacco Glycoprotein Antigen (TGP). (i) Virginia Bright strips (100 g) were powdered in a Waring blender and defatted by stirring for 24 hr with petroleum ether. The leaves were air-dried after removal of petroleum ether and extracted for 24 hr with 1 liter of phosphate-buffered physiological saline at pH 7.4, which contained 0.4% phenol as preservative. Solid material was removed by filtration through Whatman no. 3 filter paper and the solution further clarified by centrifugation at 43,000 X g in a Sorvall RC-2B centrifuge.(ii) The clarified solution was concentrated to 200 ml by pressure dialysis through Amicon PM-10 membranes (Amicon Corp., Lexington, Mass.) and brought to 50% (NH4)2SO4 saturation. The brown precipitate was collected by centrifugation, redissolved in, and dialyzed against distilled water, and lyophilized. (iii) Approximately 50 mg of the lyophilized powder was then redissolved in 5 ml of phosphate-buffered saline and applied to a fine Sephadex G-25 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) column measuring 2.5 X 100 cm, equilibrated with phosphate-buffered saline containing 0.1% NaN3. Brown material emerging with the void volume was collected, concentrated by pressure dialysis and rechromatographed. On the second passage a single symmetrical peak as measured by absorbance at 280 nm emerged with the void volume with little or no trailing. This peak, containing brown material, was dialyzed against distilled water and lyophilized. (iv) The lyophilized powder (30-40 mg) was dissolved in 3 ml of Tris-HCl buffer at pH 8.9 which was made 20% in sucrose, and applied to the top of a polyacrylamide gel column consisting of a 7.5% separating gel and a 3.5% stacking gel. The buffers in upper and lower chambers, and in the separating and stacking gels, were identical to those described by Davis and Ornstein (4). The dimensions of the separating gel and stacking gel ...
