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Enzymatic Assay of Lipase Type XIII from Pseudomonas Species and Pseudomonas Cepacia using a coupled enzyme system of Glycerol Kinase and Glycerophosphate Oxidase (EC 3.1.1.3)

1. Objective

To standardize a procedure for the enzymatic assay of Lipase Type XIII from Pseudomonas Species, Sigma Product Number L9518 , and Pseudomonas Cepacia, Sigma Product Number L9156, using a coupled enzyme system of Glycerol Kinase and Glycerophosphate Oxidase (EC 3.1.1.3) at Sigma-Aldrich, St. Louis, Mo.

2. Scope

The scope of this procedure applies to the following Sigma products that have a specification for Lipase Type XIII:Pseudomonas Species, Sigma Prod. No. L-9518, and Pseudomonas Cepacia, Sigma Prod. No. L9156.

3. Definitions

One unit of enzyme will produce 1.0 mmole of glycerol from a triglyceride per minute at 37°C at pH 7.0 in the presence of bovine serum albumin.

4. Discussion

Triglyceride + 3H2O    Lipoprotein Lipase   > Glycerol + 3 Fatty Acid
Glycerol + Adenosine 5’-Triphosphate   Glycerol Kinase    > Glycerol-3-Phosphate + Adenosine 5’-Diphosphate
Glycerol-3-Phosphate + O2    L-a-Glycerophosphate Oxidase   > Dihydroxyacetone Phosphate + H2O2
2H2O2 + 4-Aminoantipyrine + N,D-Dimethyl-m-toluidine    Peroxidase   >Quinoneimine + 3 H2O2

5. Responsibilities

It is the responsibility of all Analytical Services personnel to follow this protocol as written.

6. Safety

Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS:
T = 37°C, pH = 7.0, A545nm, Light path = 1 cm

7.2 METHOD:
Spectrophotometric Stop Rate Determination

7.3 REAGENTS:
Enzymatic reaction step:

7.3.1    500 mM Tris-HCl, Buffer, pH 7.8 at 25°C (Buffer A)

7.3.1.1    100 mM Potassium Phosphate, pH 7.0 at 37°C (Buffer) (Prepare 100 mL in deionized water using Potassium Phosphate, Monobasic, Anhydrous, Sigma-Aldrich Product Number P5379 . Adjust to pH 7.0 at 37°C with 5 N KOH.)

7.3.2    4% (w/v) Bovine Serum Albumin (BSA)
(Prepare 75 mL in deionized water using Albumin, Bovine,Fraction V, Sigma Product Number A4503.)

7.3.3    Sigma Lipase Substrate 
(Use Sigma Lipase Substrate, Sigma Stock No. 800-1.)

7.3.4    Substrate Reaction Mixture (SUB)
(Invert Sigma Lipase Substrate until a single layer emulsion appears. Add 10.0 mL of Sigma Lipase Substrate to a 250 mL beaker with stirring, then 25.0 mL of BSA and 10.0 mL of Buffer. With stirring, adjust to pH 7.0 at 37°C with 5 N KOH. Then stir at 600 rpms for exactly one hour at room temperature. With stirring, adjust to pH 7.0 at 37°C with KOH or HCL.)

7.3.5    200 mM Trichloroacetic Acid (TCA)
(Prepare 100 mL in deionized water using Trichloroacetic Acid Solution, 6.1 N ,Sigma Stock No. 490-10.)

7.3.6    20 mM Potassium Phosphate ,Ethylenediaminetetraacetic Acid, Tetrasodium, and Magnesium Chloride, pH 7.5 at 37°C (Enzyme Diluent)
(Add 95 mL of deionized water to 272 mg to 282 mg of Potasssium Phosphate,Monobasic, Anhydrous, Sigma-Aldrich Product Number P5379 , and 20.8 mg to 23.6 mg of Ethylenediaminetetraacetic Acid, Tetrasodium Salt,,Sigma Stock No. ED4SS Stir until dissolution and then adjust pH to 7.5 at 37°C with 1 N KOH. Then add 0.20 mL of Magnesium Chloride, 1M Solution, Sigma Product Number M1028 . With stirring,adjust pH to 7.5 at 37°C with 1 N KOH. Dilute to 100 mL with deionized water.)

7.3.7    Lipase Enzyme Solution (ENZYME)
(Immediately prior to pipetting into reaction mixtures, prepare a 1.0 mg solid / ml solution in cold Enzyme Dliuent. Immediately dilute to 1.2 units / ml with cold Enzyme Diluent.

Colorimetric Reaction Step (Prepare the following reagents in the following order):

7.3.8    50 mM MES (MES1)
(Prepare 250 mL in deionized water using MES,Free Acid, Hydrate, Sigma Product Number M8250 . Adjust to pH 6.5 at 37°C with 1 N NaOH.)

7.3.9    N,N-Diethyl-m-toluidine (N,N-DMT)
(Use N,N-Diethyl-m-toluidine, Aldrich Prod. No. 102571.)

7.3.10    50 mM MES and 0.021% (v/v) N,N-Diethyl-m-toluidine (MES2)
(Approximately 30 minutes prior to preparing reagents 7.3.11 through 7.3.16, add 0.04 mL of N,N-DMT to 190 mL of MES1 with stirring. Stir vigorously for thirty minutes.)

7.3.11    50 mM MES, 0.021% (v/v) N,N-Diethyl-m-toluidine, and 1.05 mM Magnesium Chloride (MES3) (Immediatelly prior to use, add 0.20 mL of Magnesium Chloride, 1M Solution, Sigma Product Number M1028 to MES2 with stirring.)

7.3.12    50 mM MES, 0.021% (v/v) N,N-Diethyl-m-toluidine,1.04 mM Magnesium Chloride, and 0.10 mM 4-Aminoantipyrine (MES4) 
(Dissolve 8.0 to 8.8 mg of 4-Aminoantipyrine,Sigma-Aldrich Product Number A4382 , in 2.0 mL of MES1. Add 1.0 mL to MES3 with stirring.)

7.3.13    50 mM MES, 0.021% (v/v) N,N-Diethyl-m-toluidine,1.03 mM Magnesium Chloride,0.10 mM 4-Aminoantipyrine, and 0.21 mM Adenosine 5’-Triphosphate (MES5)
(Dissolve 24.1 mg to 26.5 mg of Adenosine 5’-Triphosphate, Disodium1, Sigma Product Number A2383 in 2.0 mL of MES1. Add 1.0 mL to MES4 with stirring.)

7.3.14    50 mM MES, 0.021% (v/v) N,N-Diethyl-m-toluidine,1.02 mM Magnesium Chloride,0.10 mM 4-Aminoantipyrine, and 0.20 mM Adenosine 5’-Triphosphate, and Peroxidase (MES6) 
(Prepare a minimum of 2.0 mL at 400 units /mL in MES1 using Peroxidase2 Type II from Horseradish, Sigma Product Number P8250 . Add 1.0 mL to MES5 with stirring.)

7.3.15    50 mM MES, 0.021% (v/v) N,N-Diethyl-m-toluidine,1.01 mM Magnesium Chloride,0.10 mM 4-Aminoantipyrine, and 0.20 mM Adenosine 5’-Triphosphate, Peroxidase, and Glycerol Kinase (MES7)
(Prepare a minimum of 2.0 mL at 500 units/mL in MES1 using Glycerol Kinase3 from E. Coli, Sigma Product Number G4509 . Add 1.0 mL to MES6 with stirring.)

7.3.16    50 mM MES, 0.020% (v/v) N,N-Diethyl-m-toluidine,1.00 mM Magnesium Chloride,0.10 mM 4-Aminoantipyrine, and 0.20 mM Adenosine 5’-Triphosphate, Peroxidase, and Glycerol Kinase,and L-a-Glycero-3-phosphate Oxidase (COCKTAIL)
(Prepare a minimum of 2.0 mL at 500 units/mL in MES1 using L-a-Glycero-3-phosphate Oxidase4 from Pediococcus Species, Sigma Product Number G9637 . Add 1.0 mL to MES7 with stirring.)

7.4 TEST METHOD

7.4.1    Enzymatic Assay:

Pipette (in milliliters) the following reagents in the following sequence into 15 ml, plug seal, screw cap, polypropylene centrifuge tubes :

  Test-1 Test-2 Test-3 Blank
SUB 0.02 0.02 0.02 0.02
Buffer 0.10 0.10 0.10 0.10

 

Mix by swirling and equilibrate to 37°C for a minimum of five minutes. Then add:

ENZYME 0.10 0.10 0.10 -----

 

Immediately mix by inversion and incubate for exactly 15 minutes at 37°C. Then add:

TCA 2.0 2.0 2.0 2.0
ENZYME ----- ----- ----- 0.10

 

Mix by inversion and allow to sit at 37°C for exactly 10 minutes. Then centrifuge all reaction mixtures for a minimum of 4000 rpms for 15 minutes. Remove a 1.0 ml aliquot from the middle layer of each reaction mixture (upper layer: Olive Oil Substrate, middle layer: aqueous, and lower layer: TCA precipitate) and place into a 1.7 ml, polypropylene, microcentrifuge tube, Sigma Product Number T2906 . Make sure that outside surface of pipette tip is wiped off. Centrifuge at 14,000 rpm for 10 minutes in a suitable centrifuge. Remove a 0.5 ml aliquot from the middle layer of each reaction mixture and place into a 1.7 mL, polypropylene, microcentrifuge tube, Sigma Product Number T2906 . This centrifugation step may or may not have a visible TCA precipitate pellet. Centrifuge at 14,000 rpms for ten minutes. Remove a 0.25 mL aliquot from the lower layer of each reaction mixture. If any aliquot from any reaction mixture appears slightly hazy repeat the centrifugation step, if all aliquots are clear, proceed with colorimetric determination step.

7.4.2 Colorimetric Determination Assay:

Pipette (in milliliters) the following reagents in the following sequence into pre-equilibrated at 37°C acrylate, 3.5 ml, cuvettes:

  Test-1 Test-2 Test-3 Blank
COCKTAIL 3.0 3.0 3.0 3.0
Test-1from Enzymatic Assay 0.05 ----- ----- -----
Test-2 from Enzymatic Assay ----- 0.05 ----- -----
Test-3 from Enzymatic Assay ----- ----- 0.05 -----
Blank from Enzymatic Assay ----- ----- ----- 0.05

 

Mix by inversion and record the increase of A545nm until the rate is < or = to 0.0020 / minute using a suitable thermostatted spectrophotometer at 37°C. Continue monitoring the rate for another five minutes. Measure the final A545nm versus air

7.5 CALCULATIONS

7.5.1    Net A545nm (Testn-1 to n-3) = Final A545nm (Testn-1 to n-3) - (Final A545nmBlank)

7.5.2 Units/ml enzyme = (Net A545nm Testn=1 to n=3 ) (3.05)(4.20)(df)
(28.2)(0.50)(0.05)(0.1)(15)

 

df = dilution factor of enzyme solution
4.20= volume (in milliliters) of total enzyme reaction mixture in step-1
3.05= volume (in milliliters) of total colorimetric reaction mixture in step-2
0.10= volume (in milliliters) of enzyme used in enzyme reaction mixture of step-1
0.05 = volume (in milliliters) of Step-2 enzymatic reaction used in step-2
15.0 = Incubation time in minutes of enzymatic reaction in Step-1 per Unit Definition
28.2= Millimolar extinction coefficient of quinoneimine dye (reduced)
0.50= 0.50 micromoles of quinoneimine dye produced per micromole of glycerol

Units/mg solid = Units/mL enzyme
mg solid/mL enzyme

 

Units/mg protein = Units/mL enzyme
mg protein/mL enzyme

 

7.6 FINAL ASSAY CONTENTRATION:
In a 4.20 mL enzymatic reaction mix, the final concentrations are 22 mM Potassium Phosphate,2.2% (w/v) Album,Bovine, 11.1%(v/v) stabilized Olive Oil Emulsion, and 0.12 units of Lipase.

8. References

T. Saki. Y. Takagi, T. Suzuki, T. Narasaki, G. Tamura and K. Arima (1969) Agr. Bio. Chem(Tokyo).
T. Yamaguchi, N. Muroya, M. Isobe and M. Sugiura (1973) Agr. Bio. Chem(Tokyo).

Notes

1. The weight to prepare an original 2.0 ml solution of Adenosine 5’-Triphosphate should be corrected for the percent water, percent solvent, and percent purity by HPLC analysis.

2. The unit definition of Peroxidase is one unit will form 1.0 milligram Purpurogallin from Pyrogallol in 20 seconds at pH 6.0 at 20°C.

3. The unit definition of Glycerol Kinase is one unit will convert one micromole of glycerol and adenosine 5’-Triphosphate to L- a-glyerol-3-phosphate and adenosine 5’-Diphosphate per minute at pH 9.8 at 25°C in a coupled system of Pyruvate Kinase and L-Lactic Dehydrogenase.

4. The unit definition of Glyerol-3-phosphate Oxidase is one unit will oxidize one micromole of L- a-glyerol-3-phosphate to dihydroxyacetone phosphate with the formation of hydrogen peroxide per minute at pH 8.1 at 37°C.

9. Approval

  Print Name Sign Name Title Date
Prepared by: Marvin D. Rice Marvin D. Rice Originator 09/12/04
Approved by: David Lintz David Lintz Supervisor, Analytical Services 10/05/04
Approved by: Gene McNaughton Gene McNaughton Quality Assurance 10/06/04

Materials

     
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