568 ^- SCHMIDT 



with slightly varying pK values, constancy of the T values prevails only 

 over a slightly narrower range of pH values, and the T values tend to de- 

 crease with the shift of the selected pair of (pH) values toward the alkaline 

 range. It is easy, however, to find the range in which the decrease of T is 

 minimal. The T value obtained in this range represents a close approxima- 

 tion to the correct value for the amount of secondary phosphoryl groups 

 in the titrated sample. 



These remarks will be sufficient to show that the increment of acidic 

 groups during the hydrolysis of ribonucleic acid by ribonuclease can only 

 be calculated from complete titration curves. This procedure is obviously 

 too slow for measurements in the initial phases of the hydrolysis, but it is 

 suitable for the determination of the final extent of hydrolysis. If only 

 approximate measurements are required, the titrimetric technique can be 

 adapted to determinations of hydrolysis by adjusting the pH of the sub- 

 strate solution to a value around 8. The action of added ribonuclease 

 results in a gradual decrease of the pH of the digest. At certain time inter- 

 vals, the initial pH is reestablished by the addition of measured amounts 

 of 0.1 A^ alkali. These amounts correspond to the newly formed acidic 

 groups if pK shifts during the hydrolysis remain negligible, and if, in the 

 pH range covered by the measurements, no titratable groups other than 

 phosphoryl groups are liberated. The occurrence of pK shifts cannot be 

 excluded, and the selection of pH 8 as end-point is arbitrary, particularly 

 in view of the fact that the range of the inflection point of nucleotides in 

 the region is usually very narrow and that the slopes of the titration curves 

 of most oligonucleotides around these inflection points are rather steep. 



Vandendriessche^^ as well as Cavalieri ei aU^ reported titrimetric observa- 

 tions from which they concluded that the action of ribonuclease results in 

 the liberation of phenolic hydroxy groups the titration of which overlaps 

 with that of secondary phosphoryl groups. The existence of internucleotide 

 linkages involving phenolic hydroxy groups is by no means excluded by 

 the wealth of recent evidence in favor of the concept that the majority of 

 the internucleotide linkages are phosphoric acid ester bonds with 3'- and 

 5'-hydroxy groups, respectively. 



(c) Manometric determination of ribonuclease according to Bain and Rusch.^^ 

 The manometric determination of the liberated acidic groups ofi'ers the 

 advantage that the initial stages of the hydrolysis can be quantitatively 

 studied. Bain and Rusch reported a straight-line time-activity curve 

 during the first 30 minutes of hydrolysis when sufficiently dilute enzyme 

 solutions were used. Under such conditions, proportionality between the 

 amounts of carbon dioxide developed and between the concentrations of 



38 L. F. Cavalieri, S. E. Kerr, and A. Angelos, J. Am. Chevi. Soc. 73, 2567 (1951). 



39 J. A. Bain and H. P. Rusch, /. Biol. Chem. 153, 659 (1944). 



