This procedure may be used for determination of Ribonuclease A (RNase A) activity. The Spectrophotometric Stop Rate Determination [Absorbance at 300 nm (A300), Light path = 1 cm] is based on the following reaction:
Ribonucleic Acid + water –––––––––––> Oligonucleotides
Unit Definition: One unit of Ribonuclease A activity will cause a decrease of 100% per minute in the value of E0 – Ef at pH 5.0 at 25 °C.
Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.
Use ultrapure water (≥18 MΩxcm resistivity at 25 °C) for the preparation of reagents.
Buffer (100 mM Sodium Acetate, pH 5.0 at 25 °C) – Prepare a 13.61 mg/mL solution using Sodium acetate, trihydrate (Prod. No. S8625) in ultrapure water. Adjust pH to 5.0 at 25 °C with 2 M acetic acid.
RNA Solution [0.1% (w/v) Ribonucleic Acid Solution] – Prepare ~1 mg/mL solution in Buffer using Ribonucleic Acid (Prod. No. R6750). Ensure dissolution by either swirling or inversion. Do not use a stir bar. Dissolution may take up to 30 minutes. Once the RNA has dissolved, the RNA concentration must be verified prior to running the assay. Pipette the following into 3.0 mL acrylate disposable cuvettes and mix by inversion:
Determine the ΔA300 Substrate = A300 Substrate – A300 Blank. ΔA300 Substrate must be 0.73±0.025. If necessary, adjust the absorbance using appropriate amount of Buffer or solid Ribonucleic Acid.
Enzyme Solution (RNase Solution) – Prepare a RNase Stock Solution containing 50–75 Kunitz units/mL in cold ultrapure water.
In a 3.00 mL reaction mix, the final concentration is 50 mM Sodium Acetate, 0.05% (w/v) RNA, and 0.75–1.13 Kunitz unit(s) of RNase A.
1. Pipette the following into 3.0 mL acrylate disposable cuvettes. Prepare the Test in triplicate. Mix by inversion.
2. Equilibrate a suitably thermostatted spectrophotometer to 25 °C. Zero the spectrophotometer using the Blank cuvette.
3. Monitor the absorbance at A300 of the Test cuvettes for ~120 minutes at 1 minute intervals, or until the ΔA300/min is ≤0.002. Once the rate has been maintained for 5 minutes, total hydrolysis is complete.
4. The final absorbance readings for the three Tests must be within 90% agreement.
In a 3.00 mL reaction mix, the final concentration is 50 mM Sodium Acetate, 0.05% (w/v) RNA, and 0.04–0.06 Kunitz unit RNase A.
1. With the spectrophotometer set at 25 °C, zero the instrument using a Blank cuvette, prepared according to Total Hydrolysis Determination (Ef), step 1.
2. Pipette the following into 3.0 mL acrylate disposable cuvettes:
3. Mix by inversion and equilibrate to 25 °C in the spectrophotometer.
4. Then add:
5. Immediately mix by inversion and record the decrease in A300 for a minimum of 10 minutes at 1 minute intervals.
1. Pipette the following into 3.0 mL acrylate disposable cuvettes and mix by inversion:
2. Determine the ΔA300 Substrate = A300 Substrate – A300 Blank.
3. The ΔA300 Substrate must be within 20% of the value found in the RNA Concentration Verification, see Preparation Instructions, RNA Solution for the assay to be valid. Differences >20% indicate there has been contamination of the RNA substrate.
Plot ln(E0 – Ef) versus time (minutes) and determine the slope of the line.
|Slope =||Δln(E0 – Ef)|
|Kunitz units/mL enzyme =||–(Slope) (3) (df)|
|(mL of enzyme)|
3 = Total assay volume (in mL)
df = Dilution factor
mL of enzyme = Volume of Enzyme Solution added in Rate Determination (E0), step 4
|Kunitz units/mg solid =||Kunitz units/mL enzyme|
|mg solid/mL enzyme|