L-α-GLYCEROPHOSPHATE OXIDASE from Microorganism, G3O-311

PREPARATION and SPECIFICATION
Appearance:  Yellowish amorphous powder, lyophilized
Activity:  GradeⅢ 15 U/mg-solid or more
(containing approx. 60% of stabilizers)
Contaminants:  Lactate oxidase ≤2.0×10⁻⁴%|
Phosphatase ≤1.0×10⁻³%|
Stabilizers:  Sucrose, FAD
PROPERTIES
Stability:  Stable at -20°C for at least 6 months(Fig.1)
Molecular weight :  approx. 93,000 (by gel filtration)
Isoelectric point:  4.6±0.1
Michaelis constant :  2.3×10⁻³M (L-α-Glycerophosphate)
Inhibitors:  SH-reagents, ionic detergents, metal ions, etc.
Optimum pH :  6.5-7.0(Fig.3)
Optimum temperature:  40°C(Fig.4)
pH Stability:  5.0-7.5 (25°C, 60min)(Fig.5)
Thermal stability:  below 45°C (pH 6.5, 10min)(Fig.6)
Effect of various chemicals:  (Table 1)

APPLICATIONS

This enzyme is useful for enzymatic determination of triglyceride when coupled with lipoprotein lipase (LPL-311, LPL-314) and glycerokinase (GYK-301, GYK-311) in clinical analysis.

G3O-311

ASSAY

Principle:

L-α-glycerophosphate oxidase

L-α-Glycerophosphate+O₂                                                  ► Dihydroxyacetonephosphate+H₂O₂


peroxidase

2H₂O₂+4-Aminoantipyrine+EHSPT                                          ► Quinoneimine dye+4H₂O

The appearance of quinoneimine dye is measured at 555nm by spectrophotometry.

Unit definition:

One unit causes the formation of one micromole of hydrogen peroxide (half a micromole of quinoneimine dye) per
minute under the conditions described below.

Method:

Reagents
A. D,L-α-Glycerophosphate:  solution 1.5M [Weigh 48.63g of D,L-α-Glycerophosphate (disodium salt, MW= 324.17), dissolve in 60ml of H₂O and after adjusting the pH to 6.5±0.05 at 25°C with 4.0 N HCl, fill up to 100ml with H₂O] (Stable for two weeks if stored at 0-4°C)
B. PIPES-NaOH buffer, pH 6.5:  0.5M [Weigh 15.12g of PIPES (MW=302.36), suspend in 60ml of H₂O, dissolve with 10 N NaOH. After adjusting the pH to 6.5±0.05 at 25°C with 10 N NaOH, fill up to 100ml with H₂O] (Stable for two weeks if stored at 0- 4°C)
C. 4-AA solution:  28mM [569mg 4-aminoantipyrine (MW=203.25)/100ml of H₂O](Stable for one week if stored at 4°C in a brownish bottle)
D. EHSPT (TOOS) solution:  20mM [591mg N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (MW= 295.3)/100ml of H₂O] (Stable for one week if stored at 4°C in a brownish bottle)
E. Peroxidase solution:  0.05% [50mg peroxidase (110 purpurogallin units/mg)/100ml of H₂O] (Should be prepared fresh)
F. Enzyme diluent:  20mM PIPES-NaOH buffer, pH 6.5 contg. 0.5M NaCl

Procedure

Concentration in assay mixture
PIPES-NaOH buffer 193 mM
NaCl 19.2 mM
D,L-α-Glycerophosphate 577 mM
4-Aminoantipyrine 1.3 mM
EHSPT 0.96mM
Peroxidase ca.5.3 U/ml

1. Prepare the following working solution (40 tests) in a brownish bottle and store on ice.

40 ml Substrate solution (A)
40 ml PIPES-NaOH buffer, pH 6.5 (B)
5 ml 4-AA solution (C)
5 ml EHSPT solution (D)
10 ml Peroxidase solution (E)

2. Pipette 2.5ml of working solution into a cuvette (d=1.0cm) and equilibrate at 30°C for about 5 minutes.

3. Add 0.10 ml of the enzyme solution* and mix by gentle inversion.

4. Record the increase in optical density at 555nm against water for 3 to 4 minutes in a spectrophotometer
thermostated at 30°C, and calculate theΔOD 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 theΔOD test except that the enzyme diluent is added instead of enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (F), dilute to 0.05-0.20U/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×1.739×df

29.9×1/2×1.0×Vs


Weight activity (U/mg)=(U/ml)×1/C

Vt
: Total volume (2.60ml)
Vs
: Sample volume (0.10ml)
29.9
: Millimolar extinction coefficient of quinoneimine dye under the assay condition (㎠/micromole)
1/2
: Factor based on the fact that one mole of H₂O₂ produces half a mole of quinoneimine dye.
1.0
: Light path length (cm)
df
: Dilution factor
C
: Enzyme concentration in dissolution (c mg/ml)

REFERENCES

    L-α-Glycerophosphate oxidase from

    Yeast
  1. C.Gancedo, J.M.Gancedo and A.Sols; European J.Biochem, 5,165 (1968).

    2. Escherichia coli
  2. W.S.Kistler, C.A.Hirsch, N.R.Cozzarelli and E.C.C.Lin; J.Bacteriology, 100, 1133 (1969).

    3. Lactobacillus casei
  3. C.F.Strittmatter; J.Biol.Chem., 234, 2794 (1959).

    4. Streptococcus feacalis
  4. N.J.Jacobs and P.J.VanDemark; Arch.Biochem. and Biophys., 88, 250 (1960).

    5. Streptococcus feacium
  5. L.K.Koditschek and W.W.Umbreit; J.Bacteriology, 98,1063 (1969).
  6. T.W.Esders and C.A.Michrina; J.Biol.Chem., 254, 2710 (1979).
Table 1. Effect of Various Chemicals on L-α-Glycerophosphate oxidase
 [The enzyme dissolved in 0.1M dimethylglutaric acid-NaOH buffer, pH 7.0 (20U/ml) was incubated at 25°C for 10min.]
Chemical Concn.(mM) Residual
activity		 Chemical Concn.(mM) Residual
activity
None − 100% PCMB 2.0 3.3
Metal salt 2.0   MIA 2.0 3.3
MgCl₂   98
 NaF 2.0 100
CaCl₂
   107 NaN₃ 200 96
Ba(OAc)₂   99 EDTA 100 99
FeCl₂   0.3 o-Phenanthroline 2.0 95
FeCl₃   0.8 α,α′-Dipyridyl 1.0 99
CoCl₂ 103 Borate 50 10
MnCl₂   100 IAA 2.0 2.8
ZnSO₄   99 NEM 2.0 97
NiCl₂   98 Hydroxylamine 2.0 9.7
CuSO₄   7.5 Triton X-100 0.10% 106
Pb(OAc)₂   87 Brij 35 0.10% 3.9
AgNO₃   79 Tween 20
 0.10% 3.4
HgCl₂   96 Span 20 0.10% 94
      Na-cholate 0.10% 92
      SDS 0.05% 4.8
      DAC 0.05% 70

Ac, CH₃CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA, Iodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethyl-benzyl-alkyl-ammonium chloride.

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