ENZYMES ATTACKING NUCLEIC ACIDS 569 



ribonuclease were obtained. The procedure has been used for the deter- 

 mination of the concentration of ribonuclease in tissues. 



The application of this method to kinetic studies, mainly by Zittle,'^" is 

 complicated by the corrections which must be applied for the retention of 

 carbon dioxide. The necessity for this correction interferes particularly 

 with studies concerning Michaelis-Menten constants or concerning the 

 effect of phosphate esters on the action of ribonuclease. 



(d) The secondary phosphoryl groups of ribonuclease digests can also be de- 

 termined enzymically with acid prostatic phosphatase as a specific hydrolyz- 

 ing agent for secondary hydroxy groups. Schmidt et a/.'^ found that mono- 

 esterified phosphoryl groups of nucleotides are rapidly and completely 

 hydrolyzed by prostatic phosphatase, whereas diesterified phosphoryl 

 groups are resistant toward this enzyme. These observations are in agree- 

 ment with results obtained with chromatographic analyses of ribonuclease 

 digests." '^^-^ The transformation of phosphodiester groups into phos- 

 phomonoester groups by ribonuclease explains the fact that exhaustive 

 digestion with prostate phosphatase releases a much larger amount of 

 inorganic phosphate from ribonuclease digests than that formed under 

 similar conditions from ribonucleic acid.^^'^°'^' 



So far, the phosphatase method has mainly been used for analyzing 

 ribonuclease digests in the final phase of hydrolysis, whereas the others 

 are better suited for enzyme assays or kinetic studies. 



(e) Light absorption in the ultraviolet region. The action of ribonuclease on 

 ribonucleic acids is accompanied by changes in the absorption of ultraviolet 

 light for which no theoretical explanation can be given as yet. Kunitz'*- 

 found a decrease of the absorption at 290 mju which can be used for the 

 assay of purified preparations of ribonuclease. 



In the region around 260 m/x, the characteristic range of the ultraviolet 

 absorption of the bases, the action of ribonuclease causes no appreciable 

 optical changes.^" '^^ This is of interest since the quantitative degradation 

 of PNA to mononucleotides by alkali is accompanied by an increase of 

 approximately 20% in the absorption at 260 m^i (hyperchromic effect). 

 The smaller absorption of PNA samples in comparison with the sum of 

 the absorption effects of their mononucleotides is ascribed to an alteration 

 of the resonance behavior of the bases when they are bound in polynucleo- 

 tides of relatively high molecular weight. Magasanik and Chargaff'-" ob- 

 served hj^perchromic effects during the alkali hydrolysis of the high- 

 molecular, but not of the low-molecular limit polynucleotides obtained by 

 digestion of PNA with ribonuclease. They concluded from these observa- 



"> C. A. Zittle, J. Biol. Chem. 163, 119 (1946). 



^' R. A. Bolomey and F. W. Allen, J. Biol. Chem. 144, 113 (1942). 



« M. Kunitz, /. Biol. Chem. 164, 563 (1946). 



