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Ribonuclease (RNase)

(Ribonucleate 3'-pyrimidino-oligonucleotidohydrolase; EC

Pancreatic ribonucluease (RNase) catalyzes cleavage of the phosphodiester bond between the 5'-ribose of an adjacent pyrimidine nucleotide forming 2',3'-cyclic phosphate which may then be hydrolyzed to the corresponding 3'-nucleoside phosphate. RNase is found in greatest quantity in ruminant pancreas (Barnard, E.A., 'Biological Functions of Pancreatic Ribonuclease', Nature, 240, 340, 1969) and has been extensively studied. The major component is RNase A and the minor component is RNase B which is a glycoprotein (Beintema, J.J.,et al, Eur J. Biochem., 63, 441, 1976) and have molecular weights of 13,700 and 14,700 respectfully.

The amount of enzyme which causes an increase in absorbance of 1.0 at 260 nm at 37°C and pH 5.0.

The method of Kalnitsky (Kalnitsky, et al, J. Biol. Chem., 234, 1512, 1959) is used to measure the amount of acid soluble oligonucleotide liberated under the given conditions.

  1. 0.10 M Sodium acetate buffer, pH 5.0
  2. 25% Perchloric acid containing 0.75% Uranyl acetate
  3. Enzyme: 1% yeast RNA in 0.10 M sodium acetate, pH 5.0. Preincubate at 37°C for 5-8 minutes prior to assay.

  1. Prepare stock solution at 1 mg/ml in water. Immediately prior to assay, dilute further to 2, 4 and 6 µg/ml in 0.10 M sodium acetate.
  2. Pipette 1 ml of respective enzymes dilution into centrifuge tubes. Include a blank containing 1 ml of 0.10 M sodium acetate buffer, pH 5.0.
  3. Incubate all tubes at 37°C for 5-8 minutes.
  4. At zero time and timed intervals add 1 ml of 1% RNA and incubate each tube exactly 4 minutes.
  5. Stop reaction by adding 1 ml of uranyl acetate-perchloric acid solution.
  6. Transfer to an ice bath and cool for 5 minutes.
  7. Clarify by centrifugation and dilute 0.1 ml of clear supernatant to 3.0 ml with water.
  8. Read E260 against the blank.

Activity (U/mg) = (ΔE260nm/min)(Total Vol.)(Enz. Diln.)
(Enz. Vol.)(mg Enz./ml)

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