186 



Similarly it has been found that fluo- 

 ride in the same concentration also in- 

 hibits the enzymatic depolymerization 

 of desoxyribonucleic acid. 



The fact that transforming activity 

 is destroyed only by those prepara- 

 tions containing depolymerase for de- 

 soxyribonucleic acid and the further 

 fact that in both instances the enzymes 

 concerned are inactivated at the same 

 temperature and inhibited by fluoride 

 provide additional evidence for the be- 

 lief that the active principle is a nucleic 

 acid of the desoxyribose type. 



Serological Analysis.— In the course 

 of chemical isolation of the active ma- 

 terial it was found that as crude ex- 

 tracts were purified, their serological 

 activity in Type III antiserum progres- 

 sively decreased without correspond- 

 ing loss in biological activity. Solutions 

 of the highly purified substance itself 

 gave only faint trace reactions in pre- 

 cipitin tests with high titer Type III 

 antipneumococcus rabbit serum.^ It is 

 well known that pneumococcal pro- 

 tein 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 pneu- 

 mococcal transformation. 



Physicochemical Studies.^— A pur- 

 ified and active preparation of the 

 transforming substance (preparation 

 44) was examined in the analytical 



4 The Type III antipneumococcus rabbit 

 scrum employed in this studv was furnished 

 through the courtesy of Dr. Jules T. Freund, 

 Bureau of Laboratories, Department of 

 Health, City of New York. 



AVERY, MACLEOD, MCCARTY 



ultracentrifuge. 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 asym- 

 metric. 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 Vil- 

 brandt (20) have determined the dif- 

 fusion constant of several preparations 

 of thymus nucleic acid the sedimenta- 

 tion 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 mo- 

 lecular weight of the pneumococcal 

 preparation is of the order of 500,000. 



Examination of the same active 

 preparation was carried out by elec- 

 trophoresis in the Tiselius apparatus 

 and revealed only a single electropho- 

 retic component of relatively high 

 mobility comparable to that of a nu- 

 cleic acid. Transforming activity was 

 associated with the fast moving com- 

 ponent giving the optically visible 

 boundary. Thus in both the electrical 

 and centrifugal fields, the behavior of 

 the purified substance is consistent 

 with the concept that biological ac- 

 tivity is a property of the highly poly- 

 merized nucleic acid. 



Ultraviolet absorption curves 



5 Studies on sedimentation in the ultracen- 

 trifuge were carried out by Dr. A. Rothen; 

 the electrophoretic analyses were made by 

 Dr. T. Shedlovsky, and the ultra-violet ab- 

 sorption curves by Dr. G. I. Lavin. The 

 authors gratefully acknowledge their in- 

 debtedness to these members of the staff of 

 The Rockefeller Institute. 



