Jul 16, 2020

Public workspaceLipoxygenase activity determination V.2

DOI
dx.doi.org/10.17504/protocols.io.bhqmj5u6
  • 1University of Manitoba
Neilier Junior
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DOI: dx.doi.org/10.17504/protocols.io.bhqmj5u6
Protocol Citation: Neilier Junior 2020. Lipoxygenase activity determination. protocols.io https://dx.doi.org/10.17504/protocols.io.bhqmj5u6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 19, 2020
Last Modified: July 16, 2020
Protocol Integer ID: 38381
Abstract
Lipoxygenase activity on linoleic acid was determined according to the method described by Axelrod et al. (1981). This method determines the increase in absorbance at 234 nm, resulting from the formation of a conjugated double bond system in the formed hydroperoxide.
Materials
MATERIALS
ReagentSodium phosphate monobasic monohydrateSigma AldrichCatalog #S9638
ReagentSodium phosphate dibasicSigma AldrichCatalog #S3264
ReagentTween 20Sigma-aldrichCatalog #P1379-500ml
ReagentSodium hydroxideSigma – AldrichCatalog #S8045
ReagentLinoleic acidCatalog #L1012
Safety warnings
Wear personal protective equipment: gloves, lab coat and mask.
Before start
Organize your workspace

Make sure all solutions and equipment are available.
Reagent Preparation
Reagent Preparation
10 mM sodium linoleate stock solution In a 150 mL Erlenmeyer add: 10 mL distilled water (previously boiled) 78 μL linoleic acid 90 μL of tween 20 Keep the solution protected from light by wrapping the Erlenmeyer in aluminum foil. Mix the solution with the aid of a pipette, taking care not to form bubbles. Add 0.5 M NaOH until the solution is clarified (approximately 100 µL). Transfer the solution to a 25 mL volumetric flask protected from light. Make up the volume to 25 mL. Divide the stock solution of sodium linoleate into 1.5 mL amber microtubes and store at -20 ° C.
Critical
50.0 mM phosphate buffer, pH 6.0

Mix sodium phosphate monobasic and dibasic solutions in the proportions indicated below, and dilute to 200 mL with deionized water.

  • 6.15 mL of 0.2 M sodium phosphate, dibasic dihydrate (Na2HPO4•2H2O FW = 178.05)
  • 43.85 mL of 0.2M sodium phosphate, monobasic, monohydrate (NaH2PO4•H2O FW = 138.01)

Note: The dibasic stock sodium phosphate may be somewhat harder to dissolve; adding a little heat may help.

Complete the final volume to 200 mL with deionized water.

Adjust the final pH to 6.0.
Procedure
Procedure
Pipette (in microliters) the following reagents into 1.5 mL microtubes

BlankTest
Phosphate Buffer1002 L1000 L
Sodium Linoleate Stock Solution10.0 L10.0 L
Enzymatic Extract (sample)-2.0 L

Mix by inversion
Zero the spectrophotometer with Blank content at A234 nm.
Immediately after enzyme addition (Test), mark the time and pour the contents into a suitable cuvette. After 30 s of reaction onset, monitor readings at A234 nm for 120 s.
Calculations
Calculations
Calculate velocity from absorbance values obtained

V0= ΔA234 nm (ε l Δt)-1

  • V0: enzymatic activity
  • ΔA234 nm: absorbance variation at 234 nm
  • ε: molar extinction coefficient of linoleic acid hydroperoxides at 234 nm
  • l: optical path
  • Δt: time (120 s)