Enzymatic Assay of Ribonuclease A (EC 3.1.27.5)

Description

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:

RNase A
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 – E_f at pH 5.0 at 25 °C.

Precautions

Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Reagents and Equipment Required

Sodium acetate, trihydrate (Prod. No. S8625)
Ribonucleic Acid (Prod. No. R6750)

Preparation Instructions

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:

Reagent	Substrate (mL)	Blank (mL)
RNA Solution	1.50	–
Ultrapure water	1.50	1.50
Buffer	–	1.50

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.

• For Total Hydrolysis Determination (E_f) – Prepare a solution by diluting the RNase Stock Solution with cold ultrapure water to a final concentration of 0.50–0.75 Kunitz unit/mL.
• For Rate Determination (E₀) – Immediately before use, prepare a solution by diluting the RNase Stock Solution with cold ultrapure water to a final concentration of 0.20–0.30 Kunitz unit/mL.

Procedure

Total Hydrolysis Determination (E_f)

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.

Reagent	Test (mL)	Blank (mL)
RNA Solution	1.50	–
Enzyme Solution	1.50	–
Ultrapure water	–	1.50
Buffer	–	1.50

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.

Rate Determination (E₀)

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 (E_f), step 1.

2. Pipette the following into 3.0 mL acrylate disposable cuvettes:

Reagent	Test 1 (mL)	Test 2 (mL)	Test 3 (mL)
RNA Solution	1.50	1.50	1.50
Ultrapure water	1.30	1.35	1.40

3. Mix by inversion and equilibrate to 25 °C in the spectrophotometer.

4. Then add:

Reagent	Test 1 (mL)	Test 2 (mL)	Test 3 (mL)
Enzyme Solution	0.20	0.15	0.10

5. Immediately mix by inversion and record the decrease in A₃₀₀ for a minimum of 10 minutes at 1 minute intervals.

Determination of Substrate Contamination

1. Pipette the following into 3.0 mL acrylate disposable cuvettes and mix by inversion:

Reagent	Substrate (mL)	Blank (mL)
RNA Solution	1.50	–
Ultrapure water	1.50	1.50
Buffer	–	1.50

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.

Results

Calculations

1.

Plot ln(E₀ – E_f) versus time (minutes) and determine the slope of the line.

Slope =	Δln(E0 – Ef)
	Δt

2.

Kunitz units/mL enzyme =	–(Slope) (3) (df)
	(mL of enzyme)

where:
3 = Total assay volume (in mL)
df = Dilution factor
mL of enzyme = Volume of Enzyme Solution added in Rate Determination (E₀), step 4

3.

Kunitz units/mg solid =	Kunitz units/mL enzyme
	mg solid/mL enzyme

References

1. Kunitz M. 1946. A spectrophotometric method for the measurement of ribonuclease activity . Journal of Biological Chemistry. 164(2):563-8.