Technical indicators
Appearance: White amorphous powder; Protein purity: ≥ 90%; Specific activity: ≥ 150 U/mg enzyme powder; Nucleosidase: ≤ 0.01%; Adenosine deaminase: ≤ 0.1%
Enzymatic properties
Source: Microorganisms; Classification: EC 2.4.2.1; Molecular weight: 29 kDa (SDS-PAGE); Isoelectric point: 5.3; Km value: 1.4 × 10-4 M (Inosine), 7.4 × 10-5 M (Pi); Inhibitors: Not inhibited by NaN3; Optimal pH: 7.0; Optimal temperature: 65 ℃; PH stability: 5.5-10.0 (25 ℃, 16h); Thermal stability below 60 ℃ (pH 8.5,30 min); Stability: Keep at -25~-15 ℃ for 12 months and maintain over 90% activity; Protector: BSA
Purpose: Used for the development and large-scale preparation of 5 '- NT, ADA and other reagents.
Definition of enzyme activity
Unit enzyme activity is defined as the production of 1 enzyme per minute under the following conditions μ The amount of enzyme required for mol hypoxanthine.
Reagent preparation
Reagent I: Weigh 0.0372 g EDTA · 2Na and dissolve it in 40mL 0.1M pH 7.5 potassium phosphate buffer. Add 0.07 g sodium cholate, 1mL 4-AA (50mM), adjust the pH to 7.5, add 200U xanthine oxidase, and dilute to 50mL with 0.1M pH 7.5 potassium phosphate buffer.
Reagent II: In 40mL of water, add 0.39g NaH2PO4, 0.57g K2HPO4, adjust pH to 7.7, add 0.161g inosine, 9.5mL TOOS (50mM), 0.5mLPOD (1kU/mL), adjust pH to 7.7, and dilute to 50mL with water.
Enzyme diluent: Add 0.0372g EDTA · 2Na and 0.07g sodium cholate to 50mL 0.1M potassium phosphate buffer (pH7.5).
Sample: Dilute the enzyme to 0.2-0.3 U/mL using enzyme diluent.
Operating Steps
1. Add 0.75 mL of reagent I and 0.15 mL of reagent II to a 1 mL cuvette, and preheat at 37 ℃ for 5 minutes.
2. Add 0.015 mL of the enzyme solution to be tested and mix well.
React at 3.37 ℃ and detect the absorbance change of the sample within 1 minute at 555 nm using a spectrophotometer（ Δ As)
*Replace the enzyme solution with enzyme diluent, and the other steps are the same. The absorbance of the obtained solution is blank absorbance（ Δ Ab) Δ A= Δ As- Δ Ab
