Isolation of a Crystalline Protein Possessing the Properties of Tobacco-Mosaic Virus ; From Science, Vol. 81, No. 2113, pages 644–645
Lancaster, PA: American Association for the Advancement of Science, 1935. First Edition. Offprint, 8vo bifolium (229 x 153mm), pp. 4. Some light creasing, a short tear to the fold, near fine. Signed by Stanley. A landmark paper in twentieth-century biology, announcing the first isolation of a virus in crystalline form and demonstrating that the resulting material retained full biological activity; a result that forced a fundamental reconception of what viruses were and how they should be studied. The implications were immediate and disorienting: if a virus could be crystallized, it possessed the ordered molecular structure of a chemical substance rather than the biological organization of even the simplest cell, and the boundary between living and non-living matter was suddenly in question. Although Stanley initially characterized the material as a pure protein, later work established it as a nucleoprotein containing RNA; a correction that, far from diminishing the paper's significance, placed it at the foundation of the molecular biology of viruses and of RNA as a carrier of biological information. The paper was among the principal achievements recognized by Stanley's share of the Nobel Prize in Chemistry in 1946, shared with James Sumner and John Northrop for work on the crystallization of enzymes and virus proteins. Item #1306
Carried out at the Rockefeller Institute for Medical Research in Princeton, the paper reports the isolation of crystalline material from the juice of Turkish tobacco plants infected with tobacco-mosaic virus. Stanley brought the juice to 0.4 saturation with ammonium sulfate, removed the precipitated globulin fraction by filtration, repeatedly fractionated the dark brown globulin portion, removed residual color by precipitation with lead sub-acetate at pH 8.7, eliminated an inactive protein fraction at pH 4.5 with celite, and induced crystallization by slowly adding glacial acetic acid to a 0.5 saturated ammonium sulfate solution, when small needles approximately 0.03 mm long appeared immediately and crystallization was complete within an hour, yielding slightly more than 10 grams of active crystalline protein in total. The crystalline material contained 20 percent nitrogen and 1 percent ash, gave strongly positive results with Millon's biuret, xanthoproteic, glyoxylic acid, and Folin's tyrosine reagents, and returned negative Molisch and Fehlings tests; its infectivity, chemical composition, and optical rotation were unchanged after ten successive crystallizations, and a fractional crystallization experiment confirmed that the first small portion to crystallize from solution was as active as the mother liquor, establishing that activity resided in the crystalline protein itself rather than in a contaminant. The molecular weight, estimated by osmotic pressure and diffusion, was of the order of a few millions. Activity measurements against Early Golden Cluster bean and Nicotiana glutinosa showed the crystals to be over 100 times more active than a suspension prepared by grinding diseased Turkish tobacco leaves and approximately 1,000 times more active than twice-frozen juice from diseased plants, with one cubic centimeter of a 1:1,000,000,000 dilution of the crystals having usually proved infectious. The paper closes with the characterization of tobacco-mosaic virus as an autocatalytic protein that, for the present, may be assumed to require the presence of living cells for multiplication, and a conclusion stated with the caution that the evidence warranted, and one that subsequent decades would substantially complicate and enrich.
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