ENZYMES ATTACKING NUCLEIC ACIDS 573 



cedure of purification.'*^ Labeled ribonuclease was prepared by Anfinsen^" 

 by incubating slices of beef pancreas in the presence of C^'*02 . 



Despite the homogeneous behavior of ribonuclease during sedimentation 

 and electrophoresis (Rothen^^), recent studies of Martin and Porter^^ and 

 of Plirs, Moore, and Stein^^ with ion-exchange chromatography demon- 

 strated that crystalline ribonuclease, as well as fresh pancreas extracts, 

 contains at least two ribonucleases (Ribonuclease lA and IB). Ledoux's^^^ 

 suggestion that the chromatographic inhomogeneity of ribonuclease might 

 be attributed to different stages of oxidoreduction of its sulfur groups is 

 not in agreement with observations of other authors.'*^'' 



Chromatographically purified ribonuclease lA (the slower-moving band) 

 was obtained in crystalline form, whereas the isolation of ribonuclease IB 

 has so far not been achieved on the preparative scale. The assay methods 

 used for the activity determinations on both enzymes were based on the 

 formation of acid-soluble phosphorus compounds from yeast PNA. At 

 present, no information regarding the finer enzymic specificities of the two 

 pancreas ribonucleases is available. 



All data regarding the physicochemical properties of ribonuclease are 

 still based on measurements carried out in 1940 by Rothen^^ on ribonuclease 

 samples prepared without chromatographic resolution into ribonucleases 

 lA and IB. According to these measurements, the sedimentation rate of 

 crystalline ribonuclease I in 0.5 M ammonium sulfate is *S-^ = 1.85 X 10~'^. 

 The diffusion coefficient in 0.5 M ammonium sulfate is D = 1.36 X 10~^. 

 These data correspond to a molecular weight of 12,700, which is in good 

 agreement with the mean value of 13,000 obtained from sedimentation 

 ecjuilibria by Rothen. Kunitz^ arrived at the value of 15,000 (±1000) 

 from osmotic pressure measurements. Most of the calculations in the 

 current literature are based on the assumed value of 13,500. From the most 

 recent amino acid analyses of Hirs, the value of 14,100 for the molecular 

 weight of ribonuclease I is obtained. ^'•^^''•^^"= 



The isoelectric point of ribonuclease I was found at pH 7.8. The specific 



volume has the rather low value of 0.709. Ribonuclease I is a globular 



/ 

 protein with a dissymmetry factor r = 1.04. 



Jo 



*^ M. R. McDonald, J. Gen. Physiol. 32, 39 (1948). 

 5« C. B. Anfinsen, J. Biol. Chem. 186, 827 (1950). 

 " A. Rothen, J. Gen. Physiol. 24, 203 (1940). 

 « A. J. P. Martin and R. R. Porter, Biochem. J. 49, 215 (1951). 

 " C. H. W. Hirs, S. Moore, and W. H. Stein, J. Biol. Chem. 200, 493 (1953). 

 "» L. Ledoux, Biochim. et Biophrjs. Acta 14, 267 (1954). 

 "b C. H. W. Hirs, Federation Proc. 13, 230 (1954). 



"<= C. H. W. Hirs, W. H. Stein, and S. Moore, Abstr. 126th Meeting Am. Chem. Sac, 

 New York p. 89C (1954). 



