Enzymatic Assay of Lipase Type XIII from Pseudomonas sp. Using a Coupled Enzyme System of Glycerol Kinase and Glycerophosphate Oxidase (EC 3.1.1.3)

Objective and Scope

To standardize a procedure for the enzymatic assay of Lipase Type XIII from Pseudomonas sp., Product No. L9518 , and Pseudomonas cepacia, using a coupled enzyme system of glycerol kinase and glycerophosphate oxidase (EC 3.1.1.3).

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

Definitions

One unit of enzyme will produce 1.0 mmol of glycerol from a triglyceride per minute at 37 °C at pH 7 in the presence of bovine serum albumin.

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

Procedure

CONDITIONS:
T = 37 °C, pH = 7.0, A_545nm, Light path = 1 cm

METHOD:
Spectrophotometric Stop Rate Determination

REAGENTS:
Enzymatic reaction step:

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

100 mM Potassium Phosphate, pH 7.0 at 37 °C (Buffer) (Prepare 100 mL in deionized water using Potassium Phosphate, Monobasic, Anhydrous, Product No P5379. Adjust to pH 7.0 at 37 °C with 5 N KOH.)

4% (w/v) Bovine Serum Albumin (BSA)
(Prepare 75 mL in deionized water using BSA, Fraction V, Product No A4503.)

Lipase Substrate

Substrate Reaction Mixture (SUB)
(Invert Lipase Substrate until a single layer emulsion appears. Add 10 mL of Lipase Substrate to a 250 mL beaker with stirring, then 25 mL of BSA and 10 mL of Buffer. With stirring, adjust to pH 7 at 37 °C with 5 N KOH. Stir at 600 RPM for exactly one hour at room temperature. With stirring, adjust to pH 7 at 37 °C with KOH or HCL.)

200 mM Trichloroacetic Acid (TCA)
(Prepare 100 mL in deionized water using Trichloroacetic Acid Solution, 6.1 N.)

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, Product No. P5379 , and 20.8 mg to 23.6 mg of Ethylenediaminetetraacetic Acid, Tetrasodium Salt. 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, 1 M Solution, Product No. M1028. With stirring,adjust pH to 7.5 at 37 °C with 1 N KOH. Dilute to 100 mL with deionized water.)

Lipase Enzyme Solution (ENZYME)
(Immediately before 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):

50 mM MES (MES1)
(Prepare 250 mL in deionized water using MES, free acid, hydrate, Product No. M8250 . Adjust to pH 6.5 at 37 °C with 1 N NaOH.)

N,N-Diethyl-m-toluidine (N,N-DMT), Product No. 102571.

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

50 mM MES, 0.021% (v/v) N,N-Diethyl-m-toluidine, and 1.05 mM Magnesium Chloride (MES3) (Immediatelly before use, add 0.20 mL of magnesium chloride, 1 M Solution, Product No. M1028 to MES2 with stirring.)

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, Product No. A4382 , in 2 mL of MES1. Add 1 mL to MES3 with stirring.)

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, Product No. A2383 in 2 mL of MES1. Add 1 mL to MES4 with stirring.)

50 mM MES, 0.021% (v/v) N,N-diethyl-m-toluidine,1.02 mM magnesium chloride, 0.10 mM 4-aminoantipyrine, and 0.2 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, Product No. P8250. Add 1.0 mL to MES5 with stirring.)

50 mM MES, 0.021% (v/v) N,N-diethyl-m-toluidine, 1.01 mM magnesium chloride,0.10 mM 4-aminoantipyrine, and 0.2 mM adenosine 5’-triphosphate, peroxidase, and glycerol kinase (MES7)
(Prepare a minimum of 2 mL at 500 units/mL in MES1 using glycerol kinase³ from E. Coli, Product No. G4509. Add 1 mL to MES6 with stirring.)

50 mM MES, 0.020% (v/v) N,N-diethyl-m-toluidine,1 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 sp., Product No. G9637. Add 1 mL to MES7 with stirring.)

TEST METHOD

Enzymatic Assay:

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

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

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

Mix by inversion and incubate at 37 °C for exactly 10 minutes.

Centrifuge all reaction mixtures for a minimum of 4,000 rpm for 15 minutes. Remove 1.0 mL 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, Product No. T2906. Make sure that outside surface of pipette tip is wiped off.

Centrifuge at 14,000 rpm for 10 minutes.

Remove 0.5 mL from the middle layer of each reaction mixture and place into a 1.7 mL, polypropylene, microcentrifuge tube Product No T2906. This centrifugation step may or may not have a visible TCA precipitate pellet.

Centrifuge at 14,000 rpm for 10 minutes. Remove 0.25 mL 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.

Colorimetric Determination Assay:

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

Mix by inversion and record the increase of A_545nm 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 A_545nm versus air

CALCULATIONS

Net A_545nm (Test_{n-1 to n-3}) = Final A_545nm (Test_{n-1 to n-3}) - (Final A_545nmBlank)

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

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.

Notes

1. The weight to prepare an original 2 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 milligram purpurogallin from pyrogallol in 20 sec. at pH 6 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.