ENZYMES ATTACKING NUCLEIC ACIDS 567 



PNA quantitatively in flocculent form even from very dilute solutions, 

 but does not precipitate mononucleotides and oligonucleotides of low 

 molecular weight. Care must be taken to have the uranyl chloride present 

 in excess when concentrated solutions of PNA are used as substrate. The 

 acid-soluble fraction contains the mononucleotides and probably the 

 dinucleotides quantitatively; some oligonucleotides of higher molecular 

 weight, however, are difficultly soluble in acids and are partially precipi- 

 tated together with PNA. 



(6) Formation of titratable acidic groups. The cleavage of each nucleotide 

 interlinkage involving a diesterified phosphoryl group results in the appear- 

 ance of a titratable secondary phosphoryl group. The appearance of acidic 

 groups during ribonuclease action was established by Kunitz^ and by 

 Allen and Eiler.^^ When ribonucleates are used as substrates, the accuracy 

 of titration is limited by the possibility of pK shifts during their enzymic 

 degradation. In addition, the interpretation of the titration curves is 

 impeded by the overlapping of the dissociation ranges of the amino groups 

 with the beginning, and of those of the phenolic groups with the end, of 

 the dissociations of the secondary phosphoryls. According to Schmidt, 

 Seraidarian, and Thannhauser," this difficulty can be overcome by calcu- 

 lating the amounts of secondary phosphoryl groups from the slope of the 

 titration curve between two close pH values in the neighborhood of pH 6 

 (e.g., between pH 5.9 and pH 6.2). In this range, which is the region of 

 the p/^2 values of the nucleotides, the dissociation of the amino groups is 

 negligible. 



A simple transformation of the Henderson-Hasselbach eciuation shows 

 that the amounts of secondary phosphoryl groups (T) in a nucleotide 

 mixture can be calculated from the e(]uivalents of alkali consumed between 

 (pH)i and pH 8 (Ui), and those consumed between (pH)2 and pH 8 (U2) 

 provided (pH)i and (pH)2 are in the range between pH 5.8 and pH 6.4. 



The equation is: 



T = 



The validity of this equation can easily be checked with pure nucleotides 

 for which the T values obtained for arbitrary pairs of (pH)i and (pH)2 

 within the range from pH 5.8 and 6.4 are constant. For nucleic acids or 

 nucleotide mixtures which contain different secondary phosphoryl groups 



" F. W. Allen and J. J. Eiler, J. Biol. Chem. 137, 757 (1941). 



" M. Seraidarian, Thesis, Science Faculty, Tufts College, 1952. 



