Protocol for N7000 Nuclease P1 (Nuclease 5'-Nucleotidehydrolase, 3'-Phosphohydrolase)
Enzymatic Assay of Nuclease P1
RNA as Substrate:
Principle:
Nuclease P1
RNA + H2O - - - - - - - - - - > Acid Soluble Polynucleotides
Conditions: T = 37°C, pH = 5.3, A260nm, Light path = 1 cm
Method: Spectrophotometric Stop Rate Determination
Reagents:
A. Barbital Buffer: 60mM sodium barbital, pH 8.6.
B. 0.59% Barbital Acetate Buffer, pH 5.3 at 37°C: Prepare 25ml in deionized water using Barbital Buffer. Adjust to pH 5.3 at 37°C with 1M HOAc.)
C. 0.2% RNA: Prepare 4ml in deionized water using RNA, Torula Yeast Catalog No: R2040
D. 2.5% Perchloric Acid Solution (HClO4): Prepare 5ml in deionized water using Perchloric Acid.
E. 0.25% Uranyl Acetate Solution (Uran Acet): Prepare 5ml in Reagent D using Uranyl Acetate. Store on ice.
F. Nuclease P1 Enzyme Solution: Immediately before use, prepare a solution containing 0.1-0.2u/ml of Nuclease P1 in cold deionized water.
Procedure:
1. Pipette (in ml) the following reagents into suitable containers:
| Test | Blank | |
| Reagent B (Buffer) | 0.20 | 0.20 |
| Reagent C (RNA) | 0.20 | ------ |
| Mix by swirling. | ||
| Equilibrate to 37°C. | ||
2. Add:
| Test | Blank | |
| Reagent F (Enzyme Solution) | 0.10 | ------ |
| Deionized Water | ------ | 0.10 |
| Immediately mix by swirling. | ||
| Incubate for exactly 15 minutes at 37°C. | ||
3. Add:
| Test | Blank | |
| Reagent E (Uran Acet) | 0.50 | 0.50 |
| Mix by swirling. | ||
| Incubate in an ice bath for 20 minutes. | ||
4. Centrifuge for 5 minutes.
Remove 0.8 ml of the supernatant.
Dilute to 2.80ml with deionized water.
Transfer the solutions to suitable cuvettes.
Obtain the A260nm for the Test and Blank.
Calculations:
Units/ml enzyme = (A260nm Test - A260nm Blank)(1)(2.8)(df)
(10.6)(15)(0.1)(0.8)
1 = Volume (in ml) of stopped reaction
2.8 = Final volume (in ml) of assay
df = Dilution factor
10.6 = Millimolar extinction coefficient of hydrolyzed ribonucleic acid at 260nm
15 = Time (in minutes) of assay as per the Unit Definition
0.1 = Volume (in ml) of enzyme used
0.8 = Volume (in ml) of stopped reaction used in the spectrophotometric determination:
Units/mg solid = units/ml enzyme
mg solid/ml enzyme
Units/mg protein = units/ml enzyme
mg protein/ml enzyme
Unit Definition (RNA as Substrate):
One unit will liberate 1.0umole of acid soluble nucleotides from RNA per minute at pH 5.3 at 37°C.
Final Assay Concentrations:
In a 0.50ml reaction mix, the final concentrations are 0.24% barbital, 0.08% ribonucleic acid, 0.01-0.02 unit nuclease P1.
Note for complete digestion:
Optimal conditions for complete digestion of RNA or heat-denatured DNA are around pH 5.3 and 50ºC. Under such conditions, 1mg of the enzyme should completely hydrolyze 2g of RNA or 0.2g of heat-denatured DNA into 5'-mononucleotides in one hour. Addition of 0.1mM ZnCl2 to the reaction mixture is effective for stabilization of the enzyme during incubation.
