150 TRANSFORMATION OF PNEUMOCOCCAL TYPES 



in both instances the enzymes concerned are inactivated at the same tempera- 

 ture and inhibited by fluoride provide additional evidence for the belief that 

 the active principle is a nucleic acid of the desoxyribose type. 



Serological Analysis. — In the course of chemical isolation of the active 

 material it was found that as crude extracts were purified, their serological 

 activity in Type III antiserum progressively decreased without corresponding 

 loss in biological activity. Solutions of the highly purified substance itself 

 gave only faint trace reactions in precipitin tests with high titer Type III 

 antipneumococcus rabbit serum. 4 It is well known that pneumococcal protein 

 can be detected by serological methods in dilutions as high as 1 : 50,000 and the 

 capsular as well as the somatic polysaccharide in dilutions of at least 1 : 5,000,000. 

 In view of these facts, the loss of serological reactivity indicates that these cell 

 constituents have been almost completely removed from the final preparations. 

 The fact that the transforming substance in purified state exhibits little or no 

 serological reactivity is in striking contrast to its biological specificity in 

 inducing pneumococcal transformation. 



Physicochemical Studies} — A purified and active preparation of the trans- 

 forming substance (preparation 44) was examined in the analytical ultra- 

 centrigue. The material gave a single and unusually sharp boundary 

 indicating that the substance was homogeneous and that the molecules were 

 uniform in size and very asymmetric. Biological activity was found to be 

 sedimented at the same rate as the optically observed boundary, showing 

 that activity could not be due to the presence of an entity much different 

 in size. The molecular weight cannot be accurately determined until meas- 

 urements of the diffusion constant and partial specific volume have been 

 made. However, Tennent and Vilbrandt (20) have determined the diffusion 

 constant of several preparations of thymus nucleic acid the sedimentation rate 

 of which is in close agreement with the values observed in the present study. 

 Assuming that the asymmetry of the molecules is the same in both instances, 

 it is estimated that the molecular weight of the pneumococcal preparation is of 

 the order of 500,000. 



Examination of the same active preparation was carried out by electropho- 

 resis in the Tiselius apparatus and revealed only a single electrophoretic compo- 

 nent of relatively high mobility comparable to that of a nucleic acid. Trans- 

 forming activity was associated with the fast moving component giving the 



4 The Type III antipneumococcus rabbit serum employed in this study was fur- 

 nished through the courtesy of Dr. lules T. Freund, Bureau of Laboratories, Depart- 

 ment of Health, City of New York. 



8 Studies on sedimentation in the ultracentrifuge were carried out by Dr. A. 

 Rothen; the electrophoretic analyses were made by Dr. T. Shedlovsky, and the ultra- 

 violet absorption curves by Dr. G. I. Lavin. The authors gratefully acknowledge 

 their indebtedness to these members of the staff of The Rockefeller Institute. 



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