580 G. SCHMIDT 



digests is likewise more accurate than that in ribonuclease digests owing 

 to the practically complete insolubility of DNA even in very dilute solu- 

 tions. The colorimetric determination of acid-soluble deoxyribose com- 

 pounds by means of Dische's reagent is particularly convenient according 

 to Allfrey and Mirsky since the time-consuming ashing procedure required 

 for determination of the acid-soluble phosphorus is eliminated. Optical 

 changes: Kunitz^^ found that the action of deoxyribonuclease on DNA is 

 accompanied by an increase of absorption at 260 m/x amounting maximally 

 to 30%. Under standard conditions, the initial increase per minute of the 

 optical density is proportional to the amounts of deoxyribonuclease, and 

 its determination is a useful assay method particularly for purified prepara- 

 tions of deoxyribonuclease. Similar optical changes were observed by 

 Kurnick^^ during the nonenzymic depolymerization of DNA by heat. 

 Viscosity changes: The hydrolysis of DNA is accompanied by a rapid 

 decrease of the viscosity of the enzyme-substrate mixtures. This pheno- 

 menon is a useful basis for enzyme determinations since the initial rates of 

 decrease are proportional to the amounts of deoxyribonuclease." '^^-^^ 

 Since the viscosity of DNA solutions is influenced by various other factors, 

 such assays must be carried out under strictly comparable conditions, 

 particularly in regard to electrolyte concentrations. Binding of methyl 

 green {Kurnick's method) •?'^ '^"^ Kurnick found that methyl green (ethylated 

 hexamethylpararosaniline) combines only with highly polymerized DNA, 

 and that the color of this compound is stable at pH 7.5 in contrast to free 

 methyl green which fades at this pH. When DNA containing methyl 

 green is used as substrate solution, the depolymerization of DNA by 

 deoxyribonuclease (or by heat) is accompanied by the liberation of a 

 corresponding amount of methyl green. The rate of decrease of absorption 

 at 640 m/x at pH 7.5 can be used as a measure of deoxyribonuclease activity. 

 Since the fading of the color of the "liberated" methyl green is not instan- 

 taneous, the enzyme action is stopped by the addition of citrate, and the 

 colorimetric readings are carried out after four hours' standing. 



The behavior of another basic dye, methylene blue, during the action of 

 deoxyribonuclease on DNA was studied by Vercauteren.^^-^^ The binding 

 of this dye, unlike that of methyl green, does not require a very high degree 



88 N. B. Kurnick, /. Gen. Physiol. 33, 243 (1950). 



89 J. P. Greenstein and W. V. Jenrette, Cold Spring Harbor Symposia Quant. Biol. 9, 

 236 (1941). 



s" J. P. Greenstein, J. Natl. Cancer Inst. 2, 357 (1942). 



" M. McCarty, J. Gen. Physiol. 29, 123 (1946). 



« N. B. Kurnick, Arch. Biochem. and Biophys. 43, 97 (1953). 



93 R. Vercauteren, Nature 165, 603 (1950). 



" R. Vercauteren, Enzymologia 14, 134 (1950). 



" R. Vercauteren, Arch, intern, physiol. 57, 214 (1949). 



