Ribonuclease A Assay

Method: Standardization of ribonuclease activity has been difficult due to varying rates at which reactions occur as well as to the significant differences in nucleotide patterns in RNA isolated from biological sources. Crook et al. (1960) have published an assay using a synthetic substrate, cytidine 2', 3'-phosphate. Zimmerman and Sandeen (1965) described a sensitive assay using polycytidylic acid.

The method of Kalnitsky et al. (1959) is used at Worthington. The rate of hydrolysis of yeast RNA at pH 5.0 is determined by measuring the amount of acid soluble oligonucleotide released under defined conditions. One unit causes an increase in absorbance of 1.0 at A₂₆₀ at 37°C and pH 5.0 under the specified conditions.

Reagents

• 0.10 M Sodium acetate buffer, pH 5.0
• 25% Perchloric acid containing 0.75% Uranyl acetate
• 1% Worthington yeast RNA in 0.10 M sodium acetate, pH 5.0. Equilibrate to 37°C prior to assay.

Enzyme

Prepare stock solution at 1 mg/ml in reagent grade water. Immediately prior to assay, dilute further to 2, 4, and 6 micrograms per milliliter in 0.10 M sodium acetate, pH 5.0.

Procedure

Pipette one ml of respective enzyme dilution into centrifuge tubes. Include a blank containing one ml of 0.10 M sodium acetate buffer, pH 5.0. Incubate all tubes at 37°C for 5-8 minutes. At timed intervals, add one ml of 1% RNA. Incubate each tube exactly 4 minutes and stop reaction by the addition of one ml of uranyl acetate-perchloric acid solution. Transfer to an ice bath and cool for 5 minutes. Clarify by centrifugation and dilute 0.1 ml of clear supernatant to 3.0 ml with reagent grade water. Read A₂₆₀ versus blank.

Calculation