PNP (Purine nucleoside phosphorylase)
This enzyme is useful for enzymatic determination of inorganic phosphorus, 5′-nucleotidase and adenosine deaminase when coupled with xanthine oxidase and uricase.
Preparation and specification
|
Appearance |
White amorphous powder, lyophilized |
|
Protein purity |
≥90% (from SDS-PAGE) |
|
Activity |
≥150 U/mg solid |
|
Nucleosidase |
≤0.01% |
|
Adenosine deaminase |
≤0. 1% |
Properties
|
EC number |
2.4.2.1 (Recombinant from microorganism) |
|
Molecular weight |
29 kDa (SDS-PAGE) |
|
Isoelectric point |
5.3 |
|
Michaelis constants |
1.4×10-4 M (Inosine), 7.4×10-5 M (Pi) |
|
Inhibitors |
Not inhibited by NaN3 |
|
Optimum pH |
7.0 Fig. 1 |
|
Optimum temperature |
65 ℃ Fig. 2 |
|
pH stability |
pH 5.5-10.0 (25 ℃, 16 h) Fig. 3 |
|
Thermal stability |
Below 60 ℃ (pH 8.5, 30 min) Fig. 4 |
|
Storage stability |
At least one year at -25 ～-15 ℃ Fig. 5 |
|
Stabilizers |
BSA |
Storage conditions
Store at -20°C, sealed, dry and protected from light. Valid for 1 year.
Figures
Assay principle
The assay is based on the increase in absorbance at 555 nm as the formation of quinoneimine dye proceeds in the forward reactions.
Unit definition
One unit (U) is defined as the amount of enzyme which produces 1 μmol of hypoxanthine per min under the conditions described below.
Reagents preparation
Reagent I: Add 0.0372 g EDTA·2Na, 0.07 g sodium cholate, 1mL 4-AA (50 mM) in 40 mL of 0. 1 M pH 7.5 potassium phosphate buffer, adjust to pH 7.5, then add 200 U xanthine oxidase and set to 50 mL.
Reagent II: Add 0.161 g inosine, 9.5 mL TOOS (50 mM), 0.5 mL POD (1 kU/mL) to 0. 1 M pH 7.7 potassium phosphate buffer, adjust to pH 7.7, then set to 50 mL.
Enzyme diluent: Add 0.0372 g EDTA·2Na and 0.07 g sodium cholate in 50 mL of 0. 1M pH 7.5 potassium phosphate.
Sample: Dilute the sample to 0.2-0.3 U/mL by enzyme diluent.
Procedure
1. Add 0.75 mL of Reagent I, 0.15 mL of Reagent II to a cuvette and preincubate the reaction mixture at 37 ℃ for 5 min.
2. Add 0.015 mL enzyme solution to the cuvette and mix.
3. Record the ΔAs at 555 nm in 1 minute in a spectrophotometer thermostated at 37 ℃ .
At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.
∆A = ∆As - ∆Ab
Calculation
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
1. R.E. Parks, Jr. and R.P. Agarwal; The Enzymes, Vol.7, p483 (3rd ed.) (1972).
2. P.A. Hoffe, R. May and B.C. Robertson; Methods in Enzymology, Vol.11, p70 (1972).
3. M. Sugiura, K. Kato, T. Adachi, Y. Ito, K. Hirano and S. Sawaki; Chem. Pham. Bull., 29, 1451 (1981).
4. P. Fossati; Analytical Biochemistry, 149, 62 (1985).
