L-α-GLYCEROPHOSPHATE OXIDASE from Microorganism

G3O-321

sn-Glycerol-3-phosphate : oxygen 2-oxidoreductase (EC 1.1.3.21)
Glycerol-3-phosphate + O₂ ► 3-Dihydroxyacetone phosphate +H₂O₂

PREPARATION and SPECIFICATION
Appearance:	Yellowish amorphous powder, lyophilized
Activity:	GradeⅢ 15 U/mg-solid or more
Contaminants:	Lactate oxidase ≤2.0×10⁻⁴%\| Adenosine triphosphatase ≤2.0×10⁻⁴%\|
Stabilizers:	Amino acids , FAD

PROPERTIES
Stability :	Stable at -20°C for at least One year (Fig.1)
Molecular weight :	approx. 67,000 (by SDS-PAGE)
Isoelectric point :	4.6±0.1
Michaelis constant :	1.3×10⁻³M
Inhibitors :	SH-reagents, ionic detergents, metal ions, etc.
Optimum pH :	6.0-7.0
Optimum temperature :	45°C(Fig.3)
pH Stability :	4.5-8.5 (25°C, 20hr)(Fig.4)
Thermal stability :	below 45°C (pH6.5,15min)(Fig.5)
Effect of various chemicals:	(Table 1)(Fig.6)

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.

ASSAY

Principle:

L-α-glycerophosphate oxidase

Glycerol-3-phosphate+ O₂ ► Dihydroxyacetone 3-phosphate+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), dissolved in 60ml of H₂O and after adjusting the pH to 6.5±0.05 at 25°C with 4.0N 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 10N NaOH. After adjusting the pH to 6.5±0.05 at 25°C with 10N 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.test).

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 cuvetto (d=1.0cm) and equilibrate at 30°C for about 5 minutes.

3. Add 0.1ml 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 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 ΔOD test except that the enzyme diluent is added instead of enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (F), dilute to 0.05-0.2U/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 (2.6ml)
Vs: : Sample volume (0.1ml)
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)

**Table 1. Effect of Various Chemicals on L-α-Glycerophosphate oxidase**
[The enzyme dissolved in 0.1M PIPES-NaOH buffer, pH7.0 (10U/ml) was incubated at 25°C for 1hr.]
Chemical	Concn.(mM)	Residual activity(%)	Chemical	Concn.(mM)	Residual activity(%)
None	−	100%	NaN₃	20	100.2
Metal salt	2.0		EDTA	50	99.6
MgCl₂		100.0	o-Phenanthroline	2.0	101.6
CaCl₂		96.5	α,α′-Dipyridyl	2.0	100.0
BaCl₂		99.5	Borate	20	101.8
FeSO₄		67.4	IAA	2.0	98.7
FeCl₃		73.1	NEM	2.0	99.8
CoCl₂		99.5	Hydroxylamine	2.0	100
MnCl₂		100.1	TritonX-100	0.10%	110.6
ZnSO₄		96.4	Brij 35	0.10%	108.9
NiCl₂		97.9	Tween 20	0.10%	99.1
AgNO₃		93.1	Span 20	0.10%	103.7
CuSO₄		84.7	Na-cholate	0.10%	105.8
MIA	2.0	98.4	SDS	0.05%	3.0
NaF	2.0	99.4	DAC	0.05%	71.8

MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA, Iodoacetamido; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.

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