Enzymatic Assay of Ribonuclease A (EC 3.1.27.5)
1. OBJECTIVE
To standardize a procedure for the enzymatic assay of Ribonuclease A in Analytical Services at Sigma-Aldrich St. Louis.
2. SCOPE
This procedure applies to all products that have a specification for Ribonuclease A activity. Exceptions are Ribonuclease A Insoluble Enzymes Sigma Product Numbers R4001, R1626 and R7005.
3.1. Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC
3.2. Unit Definition – One unit will cause a decrease of 100% per minute in the value of E0 – Ef at pH 5.0 at 25°C.
3.3. RNA = Ribonucleic Acid
3.4. RNase A = Ribonuclease A
4. DISCUSSION
RNA + H2O RNase A > Oligonucleotides
5. RESPONSIBILITIES
It is the responsibility of all trained Analytical Services laboratory personnel to follow this protocol as written.
6. SAFETY
Refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.
7.1. CONDITIONS: pH = 5.0, T = 25°C, A300nm, Path Length = 1 cm
7.2. METHOD: Spectrophotometric Stop Rate Determination
7.3. REAGENTS
7.3.1. 100 mM Sodium Acetate, pH 5.0 at 25°C (Buffer) Prepare a 13.61 mg/ml solution using Sodium Acetate, Trihydrate, Sigma-Aldrich Product Number S8625 in purified water. Adjust pH to 5.0 at 25°C with 2M Acetic Acid.
7.3.2. 0.1% (w/v) Ribonucleic Acid Solution (RNA)
7.3.2.1. Prepare a 1 mg/ml solution in Reagent 7.3.1 using Ribonucleic Acid, Type XI, Sigma-Aldrich Product Number R6750.
7.3.2.2. Ensure dissolution by either swirling or inversion. Do not use a stir bar. Dissolution may take up to 30 minutes.
7.3.2.3. Upon dissolution, the substrate concentration must be verified prior to running the assay.
7.3.2.3.1. Prepare a substrate cuvette consisting of 1.5 ml of the RNA solution to 1.5 ml of purified water. Mix by inversion.
7.3.2.3.2. Prepare a blank cuvette consisting of purified water and Reagent 7.3.1 mixed at a ratio of 1:1. Mix by inversion. Keep this cuvette for later in the assay.
7.3.2.3.3. Read the absorbance at A300nm of the substrate cuvette from 7.3.2.3.1 vs. the blank cuvette from 7.3.2.3.2.
7.3.2.3.4. This absorbance must be 0.73 ± 0.025 prior to beginning the assay. If necessary, adjust the absorbance using appropriate amount of Reagent 7.3.1 or R6750.
7.3.2.4. Ribonucleic Acid, Type VI from Torula Yeast, Sigma-Aldrich Product Number R6625 may also be used if it has had suitability testing performed. For R6625, bring reagent bottle up to room temperature and mix thoroughly before sampling.
7.3.3. Ribonuclease A Enzyme Stock Solution (RNase A) Prepare a solution containing 50-75 Kunitz units/ml of Ribonuclease A in cold purified water.
7.3.4. Ribonuclease A Enzyme Test Solution Step 1 (RNase A) Immediately before use, prepare a solution using 7.3.3 that contains 0.50-0.75 Kunitz unit/ml of Ribonuclease A in cold purified water.
7.3.5. Ribonuclease A Enzyme Test Solution Step 2 (RNase A)
7.3.5.1. Immediately before use, prepare a solution using 7.3.3 that contains 0.2-0.3 Kunitz unit/ml of Ribonuclease A in cold purified water.
7.3.5.2. For impurity samples, prepare a solution containing 10 mg/ml in cold purified water for solid impurity samples unless otherwise specified in assignment instructions. If the impurity sample is a liquid, run neat unless otherwise stated in assignment instructions
7.4. PROCEDURE
7.4.1. Step 1: Total Hydrolysis Determination (Ef). This need only be performed on the RNase control.
7.4.1.1. Pipette the following (in milliliters) in triplicate into suitable cuvettes and mix by inversion:
| Test | |
| RNA (Reagent 7.3.2) | 1.50 |
| RNase A (Reagent 7.3.4) | 1.50 |
7.4.1.2. Blank the spectrophotometer against the blank cuvette prepared in step 7.3.2.3.2.
7.4.1.3. At 25°C, read the absorbance of the triplicate Test cuvettes for approximately 120 minutes at 1 minute intervals, or until the Δ A300nm/min is ≤0.002. Once this rate has been maintained for 5 minutes, total hydrolysis is complete.
7.4.1.4. The agreement between the final absorbance readings of the triplicate cuvettes should be within 90% agreement.
7.4.2. Step 2: Rate Determination (E0).
7.4.2.1. Blank the spectrophotometer against the blank cuvette prepared in step 7.3.2.3.2.
7.4.2.2. Pipette the following (in milliliters) into suitable cuvettes.
| Test 1 | Test 2 | Test 3 | |
| RNA (Reagent 7.3.2) | 1.50 | 1.50 | 1.50 |
| Purified Water | 1.30 | 1.35 | 1.40 |
7.4.2.3. Mix by inversion and equilibrate to 25°C using a suitably thermostatted spectrophotometer. Monitor the A300nm until constant. Then add:
| Test 1 | Test 2 | Test 3 | |
| RNase A (Reagent 7.3.5) | 0.20 | 0.15 | 0.10 |
7.4.2.4 Immediately mix by inversion and record the decrease in A300nm for a minimum of 10 minutes at 1 minute intervals. For impurity assays in which the total decrease in A300nm over the 10 minutes is < 0.005 the limit of detection will need to be invoked. Use a ∆ A300nm/min of 0.0005 for 10 minutes starting with the initial E0 value.
7.5. CALCULATIONS
7.5.1. Plot ln(E0 – Ef) versus time (minutes) and determine the slope of the line.
| 7.5.1. | Slope = | ∆ln(E0 - Ef) |
| ∆t |
| 7.5.1. | Kunitz units/ml enzyme= | (slope)(3)(df) |
| (0.2) |
3 = Total assay volume (in milliliters)
df = Dilution factor
0.2 = Volume (in milliliters) of Reagent 7.3.5 from 7.4.2
| 7.5.1. | Kunitz units/mg solid = | Kunitz units/ml enzyme |
| mg solid/ml enzyme |
| 7.5.1. | Kunitz units/mg protein= | Kunitz units/ml enzyme |
| mg protein/ml enzyme |
7.6. FINAL ASSAY CONCENTRATION
7.6.1. From 7.4.1 in a 3.00 ml reaction mix, the final concentration is 50 mM Sodium Acetate, 0.05% (w/v) RNA, 0.75 – 1.13 Kunitz unit(s) RNase A.
7.6.2. From 7.4.2 in a 3.00 ml reaction mix, the final concentration is 50 mM Sodium Acetate, 0.05% (w/v) RNA, 0.04 – 0.06 Kunitz unit RNase A.
8. REFERENCES & ATTACHMENTS
Kunitz, M., J. Biol. Chem., 164, 568 (1946).
9. APPROVAL
Review, approvals and signatures for this document will be generated electronically using EDMS. Please print a “For Use” copy if hardcopy with signature verification is required.
