LIPOXYGENASE ASSAY

Modified from Suda, I., M. Hajika, Nishiba, S. Furuta, and K. Igita. 1995. Simple and rapid method for the selective detection of individual lipoxygenase isozymes in soybean seeds. J. Agric. Food Chem. 43:742-7.

Buffers

Sodium Borate Buffer

• 200mM (pH 9.0) 381.4 g/mole for 500ml: 0.5 x 381.4 x 0.20 = 38.14g for 250ml: 19.07g
• Adjust pH to 9.0
• Warm to dissolve if necessary
• Note: Our initial pH was about 9.7. Solute went into solution when heated, then crystals precipitated out when the solution cooled.

200mM Sodium Phosphate Buffer (pH 6.0)
Note: Sodium phosphate buffers are unusual in that they are prepared by combining stock solutions of monobasic sodium phosphate and dibasic sodium phosphate in specific proportions to obtain a solution with a given pH. The required proportion can be found in tables in manuals such as Sambrook, Fritsch and Maniatis.

Stock Solutions

1. 200mM monobasic sodium phosphate (NaH₂PO₄), FW= 119.96
   250ml: 0.25 x 0.20 x 119.96 = 6.00g
2. 200mM dibasic sodium phosphate (Na₂HPO₄), FW = 141.96
   250ml: 0.25 x 0.20 x 141.96 = 7.10g

Sodium Phosphate Solutions

		Monobasic		Dibasic
pH 6.0		87.7ml		12.3ml
pH 6.6		62.5ml		37.5ml

Sodium Linoleate Substrate

		40ml		25ml
Linoleic acid		100mg		70mg
Tween 20		100mg		70mg
0.5N NaOH		825ul		550ul

Linoleic acid comes in a 100 mg ampule, so it's most practical to prepare the quanitity of substrate that corresponds to the quanitity in the ampule.

• add Tween20 to ~30 ml dH₂O
• use above solution to rinse residual linoleic acid from ampule
• add NaOH to clarify

Methylene Blue [100 uM]

3.2 g/100 ml dH₂O

Dithiothreitol (DTT) at [200 mM]

For 10 ml volume dissolve 310 mg in 200 mM sodium phosphate buffer (pH 6.0).

b-Carotene (at 50% saturation in acetone)

b-carotene generally comes in a 25 mg ampule. To obtain a 50% saturated solution, transfer contents of ampule to a beaker containing 25 ml acetone. Mix to dissolve, then divide equally into two 25 ml Oak Ridge tubes and centrifuge at 2000 rpm for 5 minutes.

Decant the supernatant, measure volume and add an equal volume of acetone to reduce saturation to 50%. Store at 4^oC in the dark.

Seed Protein Extraction Buffer (for lipoxygenase isozymes)

500 ml 0.1 Tris-HCl buffer (pH 8.2) containing 20 mM CaCl₂. Bring to volume with dH₂O.

General comments

Many of the compounds used in the assay substrates are light and/or temperature sensitive, so the fresher the reagents, the better. This is especially true for the L-3 assay. Store sodium linoleate substrate, methylene blue, b-carotene and surplus substrate solutions at 4^oC in the dark.

Substrate solutions for tests 1 and 2 can be stored overnight at 4^oC and used the following day.

The photobleaching rate can vary from sample to sample, so recheck some of the initially scored samples to assure that significant photobleaching hasn't occurred after a sample has been scored. An additional check to verify results is to repeat assays on null seeds (e.g., those having none of the 3 lipoxygenase enzymes).

Lipoxygenase Extraction and Assays

• place quarter-seed chips in labeled wells of a 96 well deep well plate;
• add 500 ml protein extraction buffer to each well and leave overnight to soften tissue;
• mash seeds with seed press or in some other manner. Incubate at 4^oC for 30 minutes, vortexing occasionally. Prepare test substrates;
• centrifuge 5 minutes at 3600 rpm to clarify supernatant.

Test for L-1 isozyme

Substrate solution:

• 50 ml sodium borate buffer (pH 9.0)
• 10 ml methylene blue
• 10 ml sodium linoleate
• 10 ml dH₂O

Assay:

• aspirate off 70 ml substrate solution to each well of a 96 well microtitre plate;
• add 30 ml supernatnat from each sample into corresponding wells. Aspirate several times to mix;
• incubate 7 minutes. Score for color intensity (e.g., 0 = clear; 3 = dark blue). Samples remaining blue lack the L-1 isozyme.

Test for L-2 isozyme

Substrate solution:

• 20 ml sodium phosphate buffer (pH 6.0)
• 4 ml DTT (200 mM)
• 4 ml methylene blue
• 4 ml sodium linoleate substrate
• 4 ml acetone

Assay:

• transfer 80 ml sample supernatant to each well of a 96 well microtitre plate ;
• add 20 ml sample supernatnat to corresponding wells. Aspirate several times to mix;
• Score for color intensity after 5 minutes. Samples remaining blue lack the L-2 isozyme.

Test for L-3 isozyme

Note: Sample reactions in this assay are easier to score correctly if viewed from the side of the plate, so we substituted 8-tube strip tubes instead of the microtitre plates. Underestimating photobleaching can result from viewing the sample from the top rather than the side. This is due to the fact that the surface often retains a yellow color longer than the liquid beneath it.

Substrate solution:

• 25 ml sodium phosphate buffer (pH 6.6)
• 5 ml sodium linoleate substrate
• 5 ml dH₂O
• add 5 ml b-carotene (50% saturation) just prior to running assay.

Assay:

• transfer 10 ml sample supernatant to appropriate tube of an 8-tube strip;
• add 20 ml sample supernatant to corresponding wells. Aspirate several times to mix;
• Score for color intensity after 5 minutes. Samples remaining yellow lack the L-3 isozyme.