SPC (Sphingomyelinase)
This enzyme is useful for enzymatic determination of small and density low density lipoprotein when coupled with alkaline phosphatase and choline oxidase.
Specification
|
Appearance |
White amorphous powder |
|
Purity(SDS-PAGE) |
≥90.0% |
|
Specific activity |
≥220.0 U/mg (Enzyme Powder) |
|
glucose oxidase |
≤0.01% |
|
ATPase |
≤0.005% |
|
catalase |
≤0.001% |
Properties
|
Source |
Microorganism, Sphingomyelinase |
|
|
EC number |
EC 3.1.4.12 |
|
|
Molecular weight |
36 kDa (SDS-PAGE) |
|
|
Isoelectric point |
8.6 |
|
|
Michaelis constants |
2.4× 10-5 M (Sphingomyelin) |
|
|
Inhibitors |
EDTA |
|
|
Optimum pH |
8.0 |
Fig. 1 |
|
Optimum temperature |
45 ℃ |
Fig. 2 |
|
pH stability |
pH 6.0 – 8.0 (37 ℃, 60min) |
Fig. 3 |
|
Thermal stability |
Below 40 ℃ (pH 7.5, 30 min) |
Fig. 4 |
|
Storage stability |
Store at -25～-15℃ for 12 months could maintain more than 90% activity |
Fig. 5 |
Figures
Purpose
For the development and mass formulation of small and low-density lipoprotein reagents.
Assay Principle
The appearance of quinoneimine dye is measured at 555 nm by spectrophotometry.
Unit definition
One unit (U) is defined as the amount of enzyme which produces 1 μmol of H2O2 per min under the conditions described below.
Reagents preparation
Reagent I：0. 1 M Tris-HCl,pH 8.0.
Reagent II: 10 mM MgCl2.
Reagent III： 6 mM Sphingomyelinase: Weigh 45.8 mg of sphingomyelin and dissolve in 10mL of sterile water.(Contains 5% Triton X- 100)
Reagent IV：500 U/mL ALP.
Reagent V：120 U/mL CHO.
Reagent VI：1 kU/mL POD, take 1000 U of POD and dissolve in 1mL of double steaming.
Regent VII: 50 mM 4–AA.
Regent VIII: 50 mM TOOS.
Regent IX: 1.0 M NaCl.
Regent X: 1 %（W/V）Triton X- 100.
Enzyme diluent ： 10 mM Tris-HCl, pH 8.0. Contains 0. 1% Triton X- 100 and 10 mM NaCl.
Reaction mix:
|
Reagent II |
20 mL |
|
Reagent III |
10 mL |
|
Reagent VI |
0.5 mL |
|
Reagent VII |
3 mL |
|
Reagent VIII |
3 mL |
|
Reagent IX |
1 ml |
|
Reagent X |
10 mL |
|
Reagent I |
Set to 90 mL |
Sample: The enzyme was diluted to 0.01 U/mL with enzyme dilution buffer.
Procedure
1. Add 900uL reaction mixture to the colorimetric dish.
2. Preheat the reaction mixture at 37℃ for 5 min.
3. Add 20ul reagent IV, 80ul reagent V, 50μL enzyme to the cuvette and mix. In 37 ℃, record the ΔAs at 555 nm in 1 minute in a spectrophotometer thermostated.
* Replace the enzyme solution with enzyme dilution solution, other steps are the same, the absorbance of the resulting solution is the blank absorbance(∆Ab)
∆A= ∆A5-∆Ab
Calculation
Weight activity（U/mg）=Volume activity × 1
Vt: total volume of reaction solution (1.05mL);
Vs: volume of enzyme solution (0.05mL);
1: Reaction time (min);
1/2: 1 mole of hydrogen peroxide produces 1/2 mole of quinoneimine dye;
Df: dilution factor;
C:enzyme concentration ( mg/mL);
39.2: Millimolar absorbance coefficient
of the chromophore at 555 nm under
standard reaction conditions (cm2/umol).
