GK (Glycerol kinase)
This enzyme is used for diagnostic tests for the determination of triglycerides together with Glycerol-3-phosphate Oxidase.
Specification
|
Appearance |
White amorphous powder, lyophilized |
|
Purity(SDS-PAGE) |
≥90.0% |
|
Enzyme powder specific activity |
≥30.0 U/mg |
|
Catalase |
≤0.001% |
|
uricase |
≤0.01% |
|
Hexokinase |
≤0.01% |
|
ATPase |
≤0.005% |
Properties
|
EC number |
2.7.1.30 (Recombinant from microorganism) |
|
|
Molecular weight |
55 kDa (SDS-PAGE) |
|
|
Isoelectric point |
5.0 |
|
|
Michaelis constants |
4.4× 10-5 M (Glycerol), 3.7× 10-5 M (ATP) |
|
|
Inhibitors |
Ag+, Hg2+ |
|
|
Optimum pH |
7.5 |
Fig. 1 |
|
Optimum temperature |
45 ℃ |
Fig. 2 |
|
pH stability |
pH 5.5- 10.0 (25 ℃, 16 h) |
Fig. 3 |
|
Thermal stability |
Below 45 ℃ (pH 7.5, 30 min) |
Fig. 4 |
|
Storage stability |
At least one year at -25 ~ - 15 ℃ |
Fig. 5 |
|
Stabilizers |
BSA |
|
Assay Principle
The assay is based on the increase in absorbance at 555 nm as the formation of quinoneimine dye proceeds in the forward reactions.
Unit definition
One unit is defined as the amount of enzyme which consumes 1 μmol of glycerol per minute at 37 ℃ under the conditions specified in the assay procedure.
Reagents preparation
Reagent I: 0.3 M glycerol.
Reagent II: 1 kU/mL POD.
Reagent III: 50 mM TOOS.
Reagent IV: 50 mM 4-AA.
Reagent V: 20 U/mL L-α-glycerophosphate oxidase.
Reagent VI: 200 mM HEPES, contains 20 mM MgCl2 and 40 mM ATP (pH 7.9).
Reaction mixture:
|
Reagent I Reagent II Reagent III Reagent IV Reagent V Pure water |
1.67 mL 400 μL 3 mL 3 mL 40 mL Set to 100 mL |
Enzyme dilution buffer: 20 mM, pH 7.5 potassium phosphate buffer.
Sample: Dilute the sample to 0.2 – 0.4 U/mL by enzyme dilution buffer.
Procedure
1. Add 1mL reaction mixture to the 1 mL cuvette.
2. Preincubate at 37 ℃ for 2 min.
3. Add 0.02 mL enzyme solution to the cuvette and mix.
4. Record the ΔAs at 555 nm in 1 minute in a spectrophotometer thermostated at 37 ℃ .
At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.
∆A = ∆As - ∆Ab
Calculation
1.02: Total volume (mL)
0.02: enzyme volume (mL)
1.0: Light path length (cm)
df: dilution factor
C: Enzyme concentration (mg/mL)
1/2: 1mol H2O2 will react to 1/2 mol Quinoneimine dye
39.2: Millimolar extinction coefficient of quinoneimine dye under 555nm (cm2/μmol)
References
1. Sakasegawa, S., et al. (1998) Biosci. Biotechnol. Biochem., 62, 2388-2395.
