Sphingomyelin phosphodiesterase, Streptomyces sp., a hydrolase, is involved in the sphingomyelin metabolism process. Sphingomyelin phosphodiesterase hydrolyzes the conversion of sphingomyelin to phosphocholine and ceramide. Sphingomyelin phosphodiesterase also plays an important role in cellular differentiation, various immune and inflammatory responses, and intracellular cholesterol trafficking and metabolism. Has very little activity on phosphatidylcholine.
Principle
The amount of quinoneimine dye generated by the reaction can be detected with a spectrophotometer at 555 nm.
Reagent Preparation
Reagent Ⅰ: 0.1 M Tris-HCl, pH 8.0
Reagent Ⅱ: 10 mM MgCl₂ solution
Reagent Ⅲ: 6 mM Sphingomyelin solution: Weigh 45.8 mg of sphingomyelin and dissolve it in 10 mL of sterile water (containing 5% Triton X-100)
Reagent Ⅳ: 500 U/mL ALP solution
Reagent Ⅴ: 120 U/mL COD solution
Reagent Ⅵ: 1 kU/mL POD solution: Dissolve 1000 U of POD in 1 mL of double-distilled water
Reagent Ⅶ: 50 mM 4-AA solution
Reagent Ⅷ: 50 mM TOOS solution
Reagent Ⅸ: 1.0 M NaCl solution
Reagent Ⅹ: 1% (W/V) Triton X-100 solution Enzyme dilution buffer: 10 mM Tris-HCl (pH 8.0), containing 0.1% Triton X-100 and 10 mM NaCl
Prepare the reaction mixture as follows: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, Make up the volume to 90 mL with Reagent Ⅰ
Sample: Dilute the enzyme to 0.01 U/mL with the enzyme dilution buffer.
Analytical Procedure
1. Add 900 μL of the reaction mixture to a cuvette.
2. Preincubate the reaction mixture at 37 ℃ for 5 min.
3. Add 20 μL of Reagent Ⅳ, 80 μL of Reagent Ⅴ and 50 μL of enzyme solution to the reaction mixture, mix well, incubate at 37 ℃ for the reaction, and record the absorbance change (ΔAₛ) within 1 min at 555 nm with a spectrophotometer.
Replace the enzyme solution with the enzyme dilution buffer and perform the same procedures; the absorbance of the resulting solution is the blank absorbance (ΔAᵦ).
ΔA=ΔAs−ΔAb
Detection Results
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Notes:
Vt: Total volume of the reaction solution (1.05 mL)
Vs: Volume of the enzyme solution (0.05 mL)
1: Reaction time (min)
1/2: 1 mole of hydrogen peroxide generates 1/2 mole of quinoneimine dye
df: Dilution factor
C: Enzyme concentration (mg/mL)
39.2: Molar absorptivity of the chromophore at 555 nm under standard reaction conditions (cm²/μmol)
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