Proteinase K Powder (GMP）
PREPARATION And SPECIFICATION
| Appearance | White amorphous powder, lyophilized |
| Activity | ≥40 U/mg |
| Nucleic acid residue | ≤5pg/mg |
| DNase | None detected |
| RNase | None detected |
PROPERTIES
| EC number | 3.4.21.64 | |
| Molecular weight | 29 kDa (SDS-PAGE) | |
| Isoelectric point | 7.81 | |
| Optimum pH | 7.5-12.0 | Fig. 1 |
| Optimum temperature | 65 ℃ | Fig. 2 |
| pH stability | pH 4.5-12.5 (25 ℃, 16 h) | Fig. 3 |
| Thermal stability | Below 50 ℃ (pH 8.0, 30 min) | Fig. 4 |
| Activators | SDS, urea | |
| Inhibitors | Diisopropyl fluorophosphate; phenylmethylsulfonyl fluoride |
Applications
Genetic diagnostic kit; RNA and DNA extraction kits; Extraction of non-protein components from tissues, degradation of protein impurities, such as DNA vaccines and preparation of heparin; Preparation of chromosome DNA by pulsed electrophoresis; Western blot; Enzymatic glycosylated albumin reagents in vitro diagnosis.
Precautions
Wear protective gloves and goggles when using or weighing, and keep well ventilated after use. This product may cause a skin allergic reaction. Cause serious eye irritation. If inhaled, it may cause allergy or asthma symptoms or dyspnea. May cause respiratory irritation.
100 mM buffer solution: pH 6.0-8.0, Na-phosphate; pH 8.0-9.0, Tris-HCl; pH 9.0-12.5, Glycine-NaOH. Enzyme concentration: 1 mg/mL.
Reaction in 20 mM K-phosphate buffer pH 8.0. Enzyme concentration: 1 mg/mL.
25 ℃, 16 h-treatment with 50 mM buffer solution: pH 4.5-5.5, Acetate; pH 6.0-8.0, Na-phosphate; pH 8.0-9.0, Tris-HCl. pH 9.0-12.5, Glycine-NaOH. Enzyme concentration: 1 mg/mL.
30 min-treatment with 50 mM Tris-HCl buffer, pH 9.0. Enzyme concentration: 1 mg/mL.
ASSAY
Unit definition
One unit (U) is defined as the amount of enzyme required to hydrolyze casein to produce 1 μmol tyrosine per minute under the following conditions.
Reagents Preparation
Reagent I: 1 g milk casein was dissolved in 50 mL of 0.1 M sodium phosphate solution (pH 8.0), incubated in 65-70 ℃ water for 15 min, stirred and dissolved, cooled by water, adjusted by sodium hydroxide to pH 8.0, and fixed volume 100ml.
Reagent II: 0.1 M trichloroacetic acid, 0.2 M sodium acetate, 0.3 M acetic acid. Reagent III: 0.4 M Na2CO3 solution. Reagent IV: Forint reagent diluted with pure water for 5 times. Reagent V: enzyme diluent: 0.1 M sodium phosphate solution (pH 8.0). Reagent VI: tyrosine solution: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/mL tyrosine dissolved with 0.2 M HCl.
Procedure
0.5 ml of reagent I is pre-warmed to 37℃, add 0.5 ml of enzyme solution, mix well, and incubate at 37℃ for 10 min.
Add 1 ml of reagent II to stop the reaction, mix well, and continue incubation for 30 min. Centrifuge the reaction solution. Take 0.5 mL supernatant, add 2.5 mL reagent III, 0.5 ml reagent IV, mix well and incubate at 37℃ for 30 min.
OD at 660 nm was determined as OD1; blank control group: 0.5 ml reagent V is used to replace enzyme solution to determine OD at 660 nm as OD2, △ OD=OD1-OD2.
L-tyrosine standard curve: 0.5mL different concentration L-tyrosine solution, 2.5mL Reagent III, 0.5mL Reagent IV in 5mL centrifuge tube, incubate in 37℃ for 30min, detect for OD at 660 nm for different concentration of L-tyrosine, then obtained the standard curve Y=kX+b, where Y is the L-tyrosine concentration, X is OD at 600 nm.
Calculation
Volume activity (𝑈/𝑚𝑙) = (𝑘 △ OD+b) × df × 2 / 10 × 0.5 × 0.5
Weight activity(𝑈/𝑚𝑔) = Volume activity × 1/𝐶
- 2: Total volume of reaction solution (mL)
- 0.5: Volume of enzyme solution (mL)
- 0.5: Reaction liquid volume used in chromogenic determination (mL)
- 10: Reaction time (min) Df: Dilution multiple C: Enzyme concentration (mg/mL)
References
- Wieger U & Hilz H. FEBS Lett. (1972); 23:77.
- Wieger U & Hilz H. Biochem. Biophys. Res. Commun. (1971); 44:513.
- Hilz, H. et al. Eur. J. Biochem. (1975); 56:103–108.
- Sambrook J et al. Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989).
