Glycerol-3-phosphate oxidase is an enzyme that catalyzes the oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate, producing hydrogen peroxide in the process. Glycerol-3-phosphate oxidase is located in the mitochondria of microorganisms and is involved in the glycerol-3-phosphate cycle, regulating cellular energy metabolism. Glycerol-3-phosphate oxidase can be used in conjunction with Lipoprotein Lipase and Glycerol Kinase to determine triglyceride levels.
Preparation and Specification
Appearance : Yellowish amorphous, lyophilized
Specific activity: More than 40 U/mg solid
Contaminants:
Acetate kinase Less than 0.1 % (U/U)
Lactate oxidase Less than 0.001% (U/U)
Properties
Substrate specificity : See Table 1
Molecular weight : 180 kDa (Sephacryl S–200); 130 kDa (Sephadex G200); 67 kDa (SDS–PAGE)
Isoelectric point : pH 4.03
Michaelis constants : L-α-Glycerophosphate 2.23 mM (pH 6.5); 4.18 mM (pH 7.5)
Optimum pH : 6.5 and 8.5–9.0 Figure 1
pH stability : 5.0–7.0 (37℃, 30 min) Figure 2
Optimum temperature : 37℃
Thermal stability : Stable at 55℃ and below (100 mM Phosphate buffer pH 6.5, 5 min.) Figure 3
Effect of various chemicals : See Table 2
Stabilizers : FAD, Sucrose
Table 1. Substrate specificity
| Substrate (300 mM) | Relative activity (%) |
|---|
| L-α-Glycerophosphate | 100 | | Glucose-1-phosphate | 0 | | Glucose-6-phosphate | 0 | | Glycerol | 0 | | Glucose | 0 |
|
Table 2. Effect of various chemicals
| Additive | Concentration | Relative activity (%) |
|---|
| None | 2 mM | 100 | | MgCl₂ | 2 mM | 101 | | MgSO₄ | 2 mM | 102 | | ZnCl₂ | 2 mM | 102 | | ZnSO₄ | 2 mM | 102 | | NaCl | 2 mM | 103 | | NH₄Cl | 2 mM | 103 | | BaCl₂ | 2 mM | 103 | | Ba(CH₃COO)₂ | 2 mM | 101 | | NiCl₂ | 2 mM | 103 | | CoCl₂ | 2 mM | 103 | | MnCl₂ | 2 mM | 114 | | LiCl | 2 mM | 103 | | KCl | 2 mM | 102 | | CaCl₂ | 2 mM | 103 | | EMULGEN 810 | 0.1% | 98 | | EMULGEN 911 | 0.1% | 98 | | RHEODOL TWL-106 | 0.1% | 99 | | RHEODOL 460 | 0.1% | 99 | | ADEKANOL NP-720 | 0.1% | 99 | | Triton X-100 | 0.1% | 99 | | Triton X-305 | 0.1% | 98 | | Tween 80 | 0.1% | 100 |
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Applications
This enzyme is useful for enzymatic determination of triglyceride.
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TG: Triglyceride, DHAP: Dihydroxyacetone phosphate
Assay
Principle
The assay is based on the increase in absorbance at 600 nm as the formation of quinoneimine dye in the following reactions:
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Unit definition
One unit is defined as the amount of enzyme which generates 1 μmole of H₂O₂ per minute at 37℃ under the conditions specified in the assay procedure.
Reagents
Enzyme solution
1. Reaction mixture
Dissolve 6.05 g of PIPES and 9.45 g (purity calculation) of Disodium Glycerophosphate with 70 ml of distilled water and adjust pH to 6.5 with 4 N NaOH at 25℃. Add all reagents listed below and confirm pH is 6.5 at 25℃. Add distilled water to make a total of 100 ml.
100 U/ml POD* solution: 5.0 ml
15 mM 4-AA solution: 10.0 ml
100 mM DAOS (salt sodium) solution: 1.0 ml
5% (W/V) Triton X-100 solution: 1.0 ml
*100 U/ml POD solution: Dissolve 1,000 U (PPU) of POD with 10 ml of distilled water.
2. Reaction stopper
0.5% (W/V) SDS solution
SDS: Sodium dodecyl sulfate
3. Enzyme dilution buffer
10 mM PIPES – NaOH buffer pH 6.5
PIPES: Piperazine-1,4-bis(2-ethanesulfonic acid)
Enzyme solution
Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. Dilute it with enzyme dilution buffer to adjust the concentration as required.
Procedure
1. Pipette accurately 1.0 ml of reaction mixture into a small test tube and preincubate at 37℃.
2. After 5 min, add exactly 20 μl of enzyme solution and mix to start the reaction at 37℃.
※ In the case of a test blank, add 20 μl of enzyme dilution buffer in place of enzyme solution.
3. At 5 min after starting the reaction, add 2.0 ml of the reaction stopper to stop the reaction.
4. Measure the absorbance at 600 nm.
Absorbance
sample : As
blank : Ab
0.050 Abs ≤ △A (As − Ab) ≤ 0.250 Abs
Calculation
Activity (U/mg of powder) = (△A/5) / (16.8 × 1/2) × (3.02 / 0.02) × (1 / X)
16.8: millimolar extinction coefficient of quinoneimine dye at 600 nm (cm²/μmole)
1/2: a multiplier derived from the fact that 2 mole of H₂O₂ produces 1 mole of quinoneimine dye
5: reaction time (min)
3.02: final volume (ml)
0.02: volume of enzyme solution (ml)
X: concentration of the sample in enzyme solution (mg/ml)
ZJ26F1039831
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