80 NEUTRON EFFECTS ON ANIMALS 



however, since it hydrolyzes ribonucleic acid also. Studies on this enzyme 

 have been initiated in this Laboratory (17) because of its possible relation 

 to the irradiation problem. A method has been described (17, 18) for 

 distinguishing this enzyme from ribonucleinase when ribonucleic acid is the 

 substrate. In the blood and tissues studied herein the predominant en- 

 zyme is ribonucleinase (18). 



EXPERIMENTAL 



Ribonucleinase was crystallized by the method of Kunitz (19) and a 

 stock solution prepared (9). 



Several commercial brands of yeast ribonucleic acid were employed as 

 the substrate. The method of purification and the desirable properties of 

 nucleic acid to serve as substrate have been described (9, 20, 21). 



Method of Assay of Rihonudeinase. The assay method is based on the 

 liberation of CO2 from a bicarbonate solution by the acidic mononucleotides 

 and intermediate products which are formed by the action of the enzyme. 

 The components of the test system were the following: 1.0 cc. of 0.1 M 

 NaHCOs, 160 mg. of nucleic acid in solution at pH 7.5, and appropriate 

 amounts of the crystalline enzyme or the material under test. The total 

 volume of reactants was in most cases 3.5 cc. The usual Warburg equip- 

 ment was employed. The crystalline enzyme or the nucleic acid was 

 placed in the side arm of the flasks and the system thoroughly gassed at 

 37° with a 5 per cent C02-95 per cent X2 mixture. After equilibrium was 

 reached, the reactant in the side arm was tipped in and the manometers 

 read at intervals. The pH attained by this system is 7.5, the optimum 

 for ribonucleinase (19, 8). Standardization of the assay procedure with 

 crystalline ribonucleinase has been described (9). 



In the application of the above test to buffered biological fluids and 

 extracts the CO2 retained by the buffers must be determined for accurate 

 results. This was done by determining the CO2 evolved from the buffered 

 system by citric acid in comparison with an unbuffered system. Details of 

 the procedure have been described (9). 



The blood for the assays was obtained by heart puncture from male 

 rabbits and collected in a tube containing 1.0 to 2.0 cc. of 2 per cent 

 sodium oxalate to 10 cc. of blood. The oxalate did not affect the results 

 with crystalline ribonucleinase. A precipitate, probably calcium oxalate, 

 formed with the nucleic acid. The blood cells were diluted with 0.85 per 

 cent NaCl to the volume of the blood from which they were taken. The 

 bone marrow was obtained (0.5 g. sample) from the end portion of the 

 femur of the hind leg. The minced tissue was suspended in water (100 

 mg./l.O cc.) and broken up in a homogenizer. Treatment in a Pierce 

 magnetostriction oscillator, frequency about 10,000 cycles per second, for 

 5 minutes effect i^'ely dispersed the bone marrow but was not satisfactory 

 for the other tissue. 



