PURINE-NUCLEOSIDE PHOSPHORYLASE from Microorganism

PNP-311

PREPARATION and SPECIFICATION
Appearance:  White amorphous powder, lyophilized
Activity:  GradeⅢ 15U/mg-solid or more
Contaminants:  Catalase ≤20%
5'-Nucleosidase ≤1.0×10⁻³%
Adenosine deaminase ≤1.0×10⁻³%
ATPase ≤1.0×10⁻²%
Stabilizers:  K-Gluconate, mannitol, EDTA
PROPERTIES
Stability:  Stable at -20°C for at least One year (Fig.1)
Molecular weight :  approx. 120,000
Isoelectric point :  4.1±0.1
Michaelis constants:  6.4×10⁻⁵M (Inosine), 3.2×10⁻⁴M (Pi)
Inhibitors:  p-Chloromercuribenzoate, SDS, Hg⁺⁺, Ag⁺
Optimum pH :  7.5-8.0(Fig.3)
Optimum temperature :  65°C(Fig.4)
pH Stability :  pH 6.0-9.0 (30°C, 16hr)(Fig.5)
Thermal stability :  below 60°C (pH 7.7, 30min)(Fig.6)
Substrate specificity :  (Table 1)
Effect of various chemicals :  (Table 2)

APPLICATIONS ³~⁵⁾

This enzyme is useful for enzymatic determination of inorganic phosphorus, 5'-nucleotidase and adenosine deaminase when coupled with xanthine oxidase (XTO-212) and uricase (UAO-201, UAO- 211)

ASSAY

Principle:

purine-nucleoside phosphorylase

Inosine+Pi                                                  ► Hypoxanthine+α-D-Ribose 1-phosphate

xanthine oxidase

Hypoxanthine+2H₂O+O₂                                                  ► Uric acid+2H₂O

The appearance of uric acid is measured at 293nm by spectrophotometry.

Unit definition:

One unit causes the formation of one micromole of uric acid per minute under the conditions describe below.

Method:

Reagents
A. K-Phosphate buffer, pH7.7:  50mM
B. Inosine solution:  32mM [Dissolve 85.8mg of inosine (MW=268.23) in 10ml of H₂O with heating] (Stable for at least two weeks if stored at 4°C)
C. Xanthine oxidase solution :  ca. 6.6U/ml [Dissolve xanthine oxidase (XTO-212) to ca.6.6U/ml with ice-cold buffer A] (Should be prepared fresh)
D. Enzyme diluent :  buffer A

Procedure

Concentration in assay mixture
K-Phosphate buffer ca.47 mM
Inosine 2.1 mM
Xanthine oxidase ca. 0.2U/ml

1. Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 37°C for about 5 minutes.

2.7 ml K-Phosphate buffer, pH 7.7 (A)
0.2 ml Substrate solution (B)
0.1 ml Xanthine oxidase solution (C)

2. Add 0.05ml of the enzyme solution* and mix by gentle inversion.

3. Record the decrease in optical density at 293nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 37°C, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test). At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (D), and dilute to 0.1−1.5U/ml with the same buffer and store on ice.

Calculation

Activity can be calculated by using the following formula :

ΔOD/min (ΔOD test-ΔOD blank)×Vt×df

Volume activity (U/ml) =                                                               = ΔOD/min×4.88×df

12.5×1.0×Vs


Weight activity (U/mg)=(U/ml)×1/C

Vt
: Total volume (3.05ml)
Vs
: Sample volume (0.05ml)
12.5
: Millimolar extinction coefficient of uric acid under the assay condition (㎠/micromole)
1.0
: Light path length (cm)
df
: Dilution factor
C
: Enzyme concentration in dissolution (c mg/ml)
 

REFERENCES

  1. R.E.Parks, Jr. and R.P.Agarwal; The Enzymes,Vol.7, p483 (3rd ed.)(1972)
  2. P.A.Hoffee, R.May and B.C.Robertson; Methods in Enzymology,Vol.11, p70 (1972)
  3. Y.Machida and T.Nakanishi; Agric.Biol.Chem.,45, 1801 (1981)
  4. M.Sugiura, K.Kato, T.Adachi, Y.Ito, K.Hirano and S.Sawaki; Chem.Pharm.Bull.,29, 1451 (1981)
  5. P.Fossati; Analytical Biochemistry.,149, 62 (1985)
Table 1. Substrate Specificity of Purine-nucleoside phosphorylase ³ ⁾
[Inosine: Purine-nucleoside phosphorylase - Xanthine oxidase system, pH 7.7 Guanosine, Adenosine, ATP, Thymidine: UV-system, pH 7.4 ]
Substrate(0.2mM) Relative activity(%) Substrate(0.2mM) Relative activity(%)
Inosine 100 ATP 0
Guanosine 41 Thymidine 0
Adenosine 0    

Table 2. Effect of Various Chemicals on Purine-nucleoside phosphorylase
[The enzyme dissolved in 50mM PIPES buffer, pH 7.0 (10U/ml) was incubated with each chemical at 30°C for 1hr.]
Chemical Conc.(mM) Residual
activity(%)		 Chemical Conc.(mM) Residual
activity(%)
None − 100 N-ethylmaleimide 2.0 91.9
Metal salt 2.0   NaF 2.0 90.9
MgCl₂
  90.1
 NaN₃ 20 95.7
CaCl₂   96.7 EDTA 5.0 96.8
Ba(OAc)₂   93.4
 o-Phenanthroline 2.0 98.3
FeCl₃   73.9 Borate 50 9.0
MnCl₂   95 Iodoacetamide 2.0 98.7
ZnCl₂   77.6 TritonX-100 0.10% 138.4
NiCl₂   94.1
Na-cholate 0.10% 124.4
CuSO₄   9.8
SDS 0.10% 0.1
Pb(OAc)₂   9.1
Span 20 0.10% 128.9
AgNO₃   0.5
      
HgCl₂   0.1      

EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate.

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