ISOLATION AND COMPOSITION OF PENTOSE NUCLEIC ACIDS 385 



sion and sedimentation measurements on highly charged particles to their 

 size and shape is discussed in Chapter 13. In many instances physical 

 measurements clearly indicate the inhomogeneity of PNA preparations. 

 So far no method has been developed which permits the fractionation of 

 the molecular species contained in such heterogeneous preparations. 



The methods used for the isolation of PNA aim at the complete recovery 

 of the PNA contained in the biological specimen under investigation. This 

 is usually an organ, such as liver or pancreas, a homogeneous collection of 

 cells, such as yeast paste, or a cell fraction, such as mitochondria or micro- 

 somes. The composition and properties of the isolated preparation will be 

 a cross-section of the properties of the various pentose nucleic acids present 

 in this material. A comparison of the nature of the PNA obtained from 

 different biological specimens will reflect the differences in the types of 

 PNA predominating in each specimen. It is important to keep in mind 

 that most methods of isolation do not, however, result in the complete 

 recovery of the PNA contained in the material. Fractionation may thus 

 take place in the course of isolation, and PNA samples isolated from the 

 same starting material by different isolation procedures may actually 

 differ in composition and properties. 



As mentioned before, it cannot be decided whether a given preparation 

 of PNA has been degraded in the course of isolation as long as the biologi- 

 cal function of PNA is not known. Therefore, only the study of the action 

 of the chemical and physical agents employed in the course of the isolation 

 procedures on purified samples of PNA is helpful in choosing the least 

 destructive path. Obviously, any treatment that causes degradation of 

 purified PNA samples must be avoided in their preparation. In earlier 

 work PNA was extracted from tissues by dilute alkali; however, the ex- 

 treme ease with which PNA is hydrolyzed by alkali to a mixture of nucleo- 

 tides shows clearly that alkali should not be used in the preparation of 

 PNA. Dilute acid hydrolyzes PNA in a similar manner; in addition, the 

 purine-pentose bond is labile to acid and, although heat is usually neces- 

 sary to accomplish these transformations, prolonged contact with acid 

 even in the cold should be avoided. The most troublesome factor in the 

 isolation procedure is the presence of highly active and sturdy nucleolytic 

 enzymes in most tissues. The best known of these enzymes is pancreatic 

 ribonuclease;" it was found to accompany PNA through most of the stages 

 of isolation and was clearly responsible for the degraded state of the PNA 

 isolated.'^ This enzyme is still active at low temperatures and is not easily 

 inactivated by heat. The activity of the enzyme may be competitively 



" M. Kunitz, J. Gen. Physiol. 24, 15 (1940). 



38 J. E. Bacher and F. W. Allen, J. Biol. Cheni. 183, 641 (1950). 



