266 



coefficient of about 8 S, resembling the 

 preparation of Cohen and Stanley^ 

 The protein, in pH 9 borate (0.01 M) 

 or in 0.01 M NaCI at that pH, ex- 

 hibited largely a single boundary with 

 a sedimentation coefficient of 4.5 S. 

 At lower pH values larger compo- 

 nents, presumably due to aggregation, 

 were observed. 



TMV-RNA 

 (59 r/ml) ' 

 Max 



Q 



Q. 



O 



0.5 



.0.0 



TMV - Protein 

 (830 r/m\) 

 Max/ Min = 2.4 



220 



260 

 m PL 



300 



Fig. 1. Ultraviolet absorption spectrum 

 of purified virus protein and nucleic acid. 



Re constitution of Active Virus.— 

 For reconstitution of the virus, 1 ml. 

 of approximately 1 per cent protein 

 solution is mixed with 0.1 ml. of a 1 

 per cent nucleic acid solution. Opales- 

 cence appears after addition of a suit- 



7 Cohen, S. S., and Stanley, W. M., /. Biol. 

 Chem. 142:863, 1942. 



FRAENKEL-CONRAT AND WILLIAMS 



able buffer; 0.01 ml. of pH 6 acetate 

 (3 M) has given the best results, but 

 phosphate (pH 6.3 and 7.0) and pH 6 

 ammonium acetate have also been used 

 successfully. The samples are held at 

 3° for at least 24 hours. They may 

 then be directly diluted and assayed. 

 A4ore often they were ultracentri- 

 fuged, the pellets redissolved in water, 

 traces of insoluble material separated 

 by centrifugation, and aliquots of the 

 opalescent supernatants diluted for 

 spectrophotometry. Most of the pro- 

 tein and 40-60 per cent of the nucleic 

 acid * were in the pellet, and the spec- 

 trum was that expected for a nucleo- 

 protein such as TMV (max. /min. = 

 about 1.17). On the basis of the O.D. 

 of TMV (0.27 at 260 m^i for an 0.01 

 per cent solution), approximate con- 

 centrations were calculated, and the 

 sample was diluted to a ransre of 100 

 to 10 |ig./ml. for assay. For pellets com- 

 posed of protein only (absorption 

 maximum at 280 m|i), calculations 

 were based on the O.D. of the virus 

 proteins (0.13 for an 0.01 per cent 

 solution at that wave length). Assays 

 were performed on groups of 8-12 

 plants {Nicotimm glutinosa), distrib- 

 uting each of an equal number of sam- 

 ples (including at least one standard 

 and often a solvent blank) over 10 

 equivalent half-leaves. It was indeed 

 quite surprising to find that the recon- 

 stituted nucleoprotein preparations 

 produced local lesions (from 2 to 30 

 per half-leaf), when tested at 10-100 

 ^Ag./ml., which were indistinguishable 

 in appearance from those of TA4V 

 at 0.1 [ig./ml. In contrast, no activity 

 was observed when the protein or nu- 

 cleic acid alone was tested up to 1,800 



* When less than 6 per cent of nucleic acid 

 is added to the protein, almost all is incor- 

 porated in the pellet, but lower infectivitv is 

 the result. Protein alone also forms pellets 

 upon ultracentrifugation at pH 6. 



