The interaction of DNA with pancreatic ribonuclease A

1. 1. The complex formation of calf thymus DNA with pancreatic ribonuclease A (ribonucleate pyrimidinenucleotido-2′-transferase (cycling), EC 2.7.7.16) was studied through ultracentrifugation, competitive inhibition of ribonuclease activity and gel filtration on Sephadex G-200. Ultracentrifugation data indicated that under low ionic strength ribonuclease formed a soluble complex with DNA at least in the range of pH 7.5 to pH 8.5 and this ability to form the complex was strongly affected by the presence of 0.05 M NaCl. 2. 2. The activity of ribonuclease was competitively inhibited by DNA when assayed in 0.01 M phosphate buffer (pH 7.5). The denatured DNA was much more effective than native DNA in inhibiting ribonuclease activity. 3. 3. Through gel filtration on Sephadex G-200, ribonuclease was found to form a complex with DNA in a definite combining ratio in 0.01 M phosphate buffer (pH 7.5). The combining ratios were calculated to be about one ribonuclease molecule per 500 000–1000 000 daltons of native DNA and 8000–10 000 daltons of denatured DNA. In the presence of 0.1 M NaCl the complex dissociated into free enzyme and DNA. The combining ratio of ribonuclease to native DNA was about 100 times smaller than that of denatured DNA and the discrepancy is discussed in connection with specific sites on DNA. The thermal denaturation profile of DNA was not affected by ribonuclease in the amount of this combining ratio.