AVERY, MACLEOD, MCCARTY 



low temperature in the preparation of 

 biologically active material, the purifica- 

 tion of one lot (preparation 44) was car- 

 ried out in the cold. In this instance all 

 the above procedures with the exception 

 of desoxycholate extraction and enzyme 

 treatment were conducted in a cold room 

 maintained at 0-4 °C. This preparation 

 proved to have significantly higher activ- 

 ity than did material similarly prepared 

 at room temperature. 



Desoxycholate extraction of the heat- 

 killed cells at low temperature is less 

 efficient and yields smaller amounts of 

 the active fraction. It has been demon- 

 strated that higher temperatures facilitate 

 extraction of the active principle, al- 

 though activity is best preserved at low 

 temperatures. 



Analysis of Purified Traiisforjjiifig 

 Material 



General Properties.—SaVme solutions 

 containing 0.5 to 1.0 mg. per cc. of 

 the purified substance are colorless and 

 clear in diffuse light. However, in 

 strong transmitted light the solution is 

 not entirely clear and when stirred ex- 

 hibits a silky sheen. Solutions at these 

 concentrations are highly viscous. 



Purified material dissolved in physi- 

 ological salt solution and stored at 2- 

 4°C. retains its activity in undimin- 

 ished titer for at least 3 months. How- 

 ever, when dissolved in distilled water, 

 it rapidly decreases in activity and be- 

 comes completely inert within a few 

 days. Saline solutions stored in the 

 frozen state in a CO2 ice box ( — 70°C.) 

 retain full potency for several months. 

 Similarly, material precipitated from 

 saline solution by alcohol and stored 

 under the supernatant remains active 

 over a long period of time. Partially 

 purified material can be preserved by 

 drying from the frozen state in the 

 lyophile apparatus. However, when 

 the same procedure is used for the 

 preservation of the highly purified 

 substance, it is found that the material 

 undergoes changes resulting in de- 



181 



crease in solubility and loss of activity. 



The activity of the transforming 

 principle in crude extracts withstands 

 heating for 30 to 60 minutes at 65 °C. 

 Highly purified preparations of active 

 material are less stable, and some loss 

 of activity occurs at this temperature. 

 A quantitative study of the effect of 

 heating purified material at higher 

 temperatures has not as yet been made. 

 Alio way (6), using crude extracts pre- 

 pared from Type III pneumococcal 

 cells, found that occasionally activity 

 could still be demonstrated after 10 

 minutes' exposure in the water bath to 

 temperatures as high as 90°C. 



The procedures mentioned above 

 were carried out with solutions ad- 

 justed to neutral reaction, since it has 

 been shown that hydrogen ion con- 

 centrations in the acid range result in 

 progressive loss of activity. Inactiva- 

 tion occurs rapidly at pH 5 and below. 



Qualitative Cheiuical Tests— 'Xht 

 purified material in concentrated solu- 

 tion gives negative biuret and Millon 

 tests. These tests have been done di- 

 rectly on dry material with negative 

 results. The Dische diphenylamine re- 

 action for desoxyribonucleic acid is 

 strongly positive. The orcinol test 

 (Bial) for ribonucleic acid is weakly 

 positive. However, it has been found 

 that in similar concentrations pure 

 preparations of desoxyribonucleic acid 

 of animal origin prepared by different 

 methods give a Bial reaction of cor- 

 responding intensity. 



Although no specific tests for the 

 presence of lipid in the purified ma- 

 terial have been made, it has been 

 found that crude material can be re- 

 peatedly extracted with alcohol and 

 ether at — 12°C. without loss of activ- 

 ity. In addition, as will be noted in the 

 preparative procedures, repeated alco- 

 hol precipitation and treatment with 

 chloroform result in no decrease in bi- 

 ological activity. 



