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Assay Procedure for Lipase

Principle

The appearance of quinoneimine dye is measured at 545nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of glycerol (half a micromole of quinoneimine dye) per minute under the conditions described below.

Method

Reagents

 

A. Olive oil emulsion :Sonicate the mixture of 5.0g of olive oil[reagent grade (highly refined, low acidity)]and 5.0ml of 5.0% Triton X-100 solution (B) for 10 minutes (20KHz). To the oil emulsion, add 25ml of 4.0% BSA solution (C) and 15ml of 0.1M Kphosphate buffer, pH 7.0 (D), and mix. (Should be prepared freshly)
B. Triton X-100 solution :5.0% (5.0ml Triton X-100/100ml of H2O)
C. BSA solution :4.0%[4.0g bovine serum albumin/100ml of H2O]
D. K-phosphate buffer, pH 7.0 :0.1M
E. TCA solution :0.2M (33g trichloroacetic acid/1,000ml of H2O)
F. MES-NaOH buffer :50mM MES buffer, pH 6.5[Dissolve 9.76g of 2-(N-morpholino)-ethanesulfonic
acid (MW=195.23) in ca. 850ml of H2O and, after adjusting the pH to 6.5 with 5.0N NaOH, fill up to 1,000ml with H2O]
G. Color developing reagent
:Dissolve the following chemicals and enzymes into 200ml of 50mM MES buffer
(F) in the following order:
4.0 ml
0.04 ml
4.0 mg
24.2 mg
40.7 mg
200 units
500 units
300 units
Triton X-100 solution (B)
N,N-Diethyl-m-toluidine (Stir until completly dissolved)
4-Aminoantipyrine
ATP・Na2・3H2O
MgCl2・6H2O
Glycerol kinase
L-α-Glycerophosphate oxidase
Peroxidase (Purpurogallin units)
  (Stable for one week if stored at 4℃ in an amber bottle)
H. Enzyme diluent :20mM K-phosphate buffer, pH 7.5 containing 2.0mM MgCl2 and 0.5mM EDTA-Na3

Procedure

(1st step)

  1. Pipette 2.0ml of olive oil emulsion (A) into a test tube and equilibrate at 37℃ for about 5 minutes.
Concentration in assay mixture
K-Phosphate buffer 29.1 mM
Olive oil 90.9mg/ml
MgCl2 0.18 mM
Triton X-100 9.1 %
EDTA 45 μM
BSA 1.8 %
  1. Add 0.2ml of the enzyme solution* and mix.
  2. After exactly 15 minutes at 37℃, add 2.0ml of TCA solution (E) to stop the reaction and remove the precipitate by filtration through filter paper (Toyo-Roshi No.131 or Whatman No.42).

(2nd step)

  1. Pipette 0.05ml of the filtrate thus obtained into a test tube.
  2. Add 3.0ml of color developing reagent (G) and incubate at 37℃ for 15 minutes.
  3. Measure the optical density at 545nm against water (OD test).

At the same time, prepare the blank by first mixing 2.0ml of the olive oil emulsion (A) after 15minincubation at 37℃ with 2.0ml of TCA solution, followed by the addition of the enzyme solution (1st step). By using the filtrate obtained from the mixture, carry out the 2nd step using the same procedure as the test and measure the optical density at 545nm (OD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent (H) and dilute to 0.4-1.2U/ml with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula:

 

Weight activity (U/mg)=(U/ml)×1/C

 

Vt-1 :Total volume in 1st step (4.2ml)
Vt-2 :Total volume in 2nd step (3.05ml)
Vs-1 :Sample volume (0.2ml)
Vs-2 :Sample volume in 2nd step (0.05ml)
28.2 :Millimolar extinction coefficient of quinoneimine dye under the assay condition (F/micromole)
1/2 :Factor based on the fact that one mole of H2O2 produces half a mole of quinoneimine dye
1.0 :Light path length (cm)
t :Reaction time in 1st step (15 minutes)
df :Dilution factor
C :Enzyme concentration in dissolution (c mg/ml)

 

This procedure is for informational purposes. For a current copy of Sigma’s quality control procedure contact our Technical Service Department.

Materials

     
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