L-α-GLYCEROPHOSPHATE OXIDASE from Microorganism
sn-Glycerol-3-phosphate : oxygen 2-oxidoreductase (EC 22.214.171.124)
Glycerol-3-phosphate + O₂
► 3-Dihydroxyacetone phosphate +H₂O₂
|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|
|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)|
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.
The appearance of quinoneimine dye is measured at 555nm by spectrophotometry.
One unit causes the formation of one micromole of hydrogen peroxide (half a micromole of quinoneimine dye) per
minute under the conditions described below.
|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|
|Concentration in assay mixture|
|PIPES-NaOH buffer||193 mM|
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.
Activity can be calculated by using the following formula :
ΔOD/min (ΔOD test−ΔOD blank ) ×Vt × df
Volume activity (U/ml) =
Weight activity (U/mg)=(U/ml)×1/C
- : Total volume (2.6ml)
- : Sample volume (0.1ml)
- : Millimolar extinction coefficient of quinoneimine dye under the assay condition (㎠/micromole)
- : Factor based on the fact that one mole of H₂O₂ produces half a mole of quinoneimine dye.
- : Light path length (cm)
- : Dilution factor
- : Enzyme concentration in dissolution (c mg/ml)
MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA, Iodoacetamido; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.