L-α-GLYCEROPHOSPHATE OXIDASE from Pediococcus sp.

PREPARATION and SPECIFICATION
Appearance:  Yellowish amorphous powder, lyophilized
Activity:  GradeⅢ 40 U/mg-solid or more
(containing approx. 40% of stabilizers)
Contaminant:  Lactate oxidase ≤1.0×10⁻³%
Stabilizers:  Sucrose, FAD
PROPERTIES
Stability:  Stable at -20°C for at least one year
(A decrease in activity of ca. 10% may occur at 5°C within 6 months.)(Fig.1)
Molecular weight :  approx. 76,000 (by gel filtration)
Isoelectric point:  4.1±0.1
Michaelis constants :  3.2×10⁻³M (L-α-Glycerophosphate), 6.8×10⁻³M (D, L-form)
Inhibitors:  Ionic detergents (SDS, LBS, etc.), Hg⁺⁺, Ag⁺
Optimum pH :  8.0-8.5(Fig.2)
Optimum temperature:  35-40°C(Fig.3)
pH Stability:  pH 6.5-8.5 (25°C, 20hr)(Fig.4)
Thermal stability:  below 40°C (pH 7.0, 15min)(Fig.5)
Substrate specificity:  The enzyme has the highest specificity for L-form of α-glycerophosphate.
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-301

ASSAY

Principle:

L-α-glycerophosphate oxidase

L-α-Glycerophosphate+O₂                                                  ► Dihydroxyacetonephosphate+H₂O₂


peroxidase

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

The appearance of quinoneimine dye is measured at 500nm 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 0.2M [Weigh 6.48g of D, L-α-glycerophosphate (disodium salt, MW=324.17), dissolve in 80ml of 0.125M Tris-HCl buffer, pH 8.0 contg. 0.125% of Triton X-100 and after adjusting the pH to 8.1 at 25°C with 2.0N HCl, fill up to 100ml with H₂O.] (Stable for two weeks if stored at 0-5°C)
B. 4-AA solution:  0.1% (100mg of 4-aminoantipyrine /100ml of H₂O)(Store at 4°C in a brownish bottle)
C. Phenol solution:  0.1% (100mg phenol/100ml of H₂O)(Store at 4°C in a brownish bottle)
D. Peroxidase solution:  0.025%[25mg peroxidase (110 purpurogallin units/mg)/100ml of H₂O]
(Should be prepared fresh)
E. SDS solution:  0.25% (1.25g sodium dodecyl sulfate/500ml of H₂O)
F. Enzyme diluent:  20mM Tris-HCl buffer, pH 7.5 contg. 0.2% of BSA

Procedure

Concentration in assay mixture
Tris-HCl buffer 50 mM
Glycerophosphate 98 mM
4-Aminoantipyrine 0.48mM
Phenol
 2.1 mM
Triton X-100 0.049 %
POD ca.5.4 U/ml

1. Prepare the following working solution (100 tests) in a brownish bottle and store on ice.

50 ml Substrate solution (A)
10 ml 4-AA solution (B)
20 ml Phenol solution (C)
20 ml Peroxidase solution (D)

2. Pipette 1.0ml of working solution into a test tube and equilibrate at 37°C for about 5 minutes.

3. Add 0.02ml of the enzyme solution* and mix.

4. After exactly 10 minutes at 37°C, add 2.0ml of SDS solution (E) to stop the reaction and measure the optical
density at 500nm against water (OD test).
At the same time, prepare the blank by first mixing 1.0ml of working solution with 2.0ml of SDS solution
after 10min-incubation at 37°C, followed by addition of the enzyme solution (OD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent (1.0mg/ml or more) and dilute to 0.15-0.35U/ml
with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula :

ΔOD (OD test−OD blank ) ×Vt × df

Volume activity (U/ml) =                                                               =ΔOD×2.2707×df

13.3×1/2× t ×1.0×Vs


Weight activity (U/mg)=(U/ml)×1/C

Vt
: Total volume (3.02ml)
Vs
: Sample volume (0.02ml)
13.3
: 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.
t
: Reaction time (10 minutes)
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 (10U/ml) was incubated at 25°C for 1hr.]
Chemical Concn.(mM) Residual
activity		 Chemical Concn.(mM) Residual
activity
None − 100% PCMB 2.0 100
Metal salt 2.0   MIA 2.0 93
MgCl₂   100
NaF 2.0 101
CaCl₂
   100 NaN₃ 20 95
Ba(OAc)₂   95 EDTA 5.0 105
FeCl₃   98 o-Phenanthroline 2.0 97
CoCl₂ 96 α,α′-Dipyridyl 2.0 95
MnCl₂   100 Borate 50 92
Zn(OAc)₂   100 Triton X-100 0.10% 105
NiCl₂   99 Brij 35 0.10% 103
CuSO₄   100 SDS 0.05% 1.5
Pb(OAc)₂   97 LBS 0.05% 1.4
AgNO₃   24 Na-cholate
 0.10% 106
HgCl₂   34      

Ac, CH₃CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; LBS, Sodium laurylbenzenesulfonate.

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