A recording double-beam UV spectrophotometer is used to determine the conjugated diene absorbance of product fatty acid hydroperoxide at 234 nm as a function of time. Although this method is used by a majority of investigators, it has limitations regarding (1) the clarity of the enzyme and substrate solutions, (2) UV-absorbing substances in the solutions, and (3) the presence of hydroperoxide-metabolizing enzymes that destroy the conjugated diene moiety. The method works best with purified LOXs, especially type 1 LOXs. With type 2 LOXs, it is especially important to use low substrate concentrations solubilized with nonionic, non-UV absorbing detergents, like Tween 20. Compared to the polarographic method, the UV method is over three-fold more sensitive over the measurable span utilized by the two methods (Figure C4.2.2).
As for Basic Protocol 1, the LOX enzyme is prepared according to the Support Protocol. However, more care should be taken to reduce turbidity and other UV-absorbing materials caused by suspended lipid and pigments, especially when low activity is expected (requiring more enzyme addition). Triton X-100 is a UV absorber, and it causes more lipid and pigments to be suspended. Partial purification may be necessary. HEPES and PIPES buffers are not used in this method because of excessive absorption at 234 nm (0.5 to 0.6 absorbance for 0.1 M solutions); MES is useful with limitations (0.2 absorbance for 0.1 M solution).
Additional Materials (also see Basic Protocol 1) Linoleic acid solution (UVSUBS; see recipe)
Double-beam UV spectrophotometer with continuous recording capability 1-cm quartz cells
1. Add 2.97 ml of 0.1 M assay buffer at 25°C to both a sample and a reference quartz cuvette.
Preferably, the cuvette holders should be temperature controlled; otherwise special attention should be given to the temperature of the reaction, such as use of reagents equilibrated at room temperature.
2. Add 30 |l linoleic acid solution (UVSUBS) to both cuvettes and stir.
Linoleic acid solution is added to the buffered reference cuvette if one anticipates turbidity caused by substrate at low pH; otherwise, LOX solution and buffer only can be added to the reference cuvette, especially if the LOX solution has turbidity or high absorbance.
3. Add an appropriate amount of LOX enzyme solution to the sample cuvette (usually 1 to 10 |l; dilution of the LOX solution may be required), stir, and immediately start UV measurement at 234 nm as a function of time.
At the extremes of activity rates, the measured activities may not be accurate; therefore, one should measure varying amounts of enzyme and plot the quantity of enzyme added against activity. The linear portion of the curve should determine the range of LOX solution to be used.
Optional: Certain LOXs are more peroxidative in character, as assessed by the amount of conjugated oxodiene compounds produced. These products can be assessed by monitoring at 280 nm (e = 22,000) and comparing this rate with that obtained at 234 nm.
4. Use the most rapid activity portion after the short lag to determine activity (usually a straight line; see Figure C4.2.2). To determine the amount of hydroperoxide produced, use a molar extinction coefficient of 26,800 cm-1mol-1 liter and the equation:
A234 (AU/min) = e (cm-1mol-1 liter) x c (mol/liter) x cell length (cm)
For a 3-ml sample (do not forget to include the amount of enzyme added), a 1-cm path length, and an activity of 1.0 AU/min, hydroperoxide produced = [1.0/min x 3.0 ml] / [26,800 cm~1moV1 liter x 1 cm] = 0.112 umol/min. If a 10-ul volume of LOX initiated the reaction, the activity would be 0.0112 jumol/min/jul LOX. Preferably, specific activity would be calculated on the basis of protein (see Basic Protocol 1, step 13).
The molar extinction coefficient used here is higher than most literature values (23,00025,000). The reasons for using the higher value are discussed in a review by Gardner (1997).
5. Determine pH optimum as described (see Basic Protocol 1, step 14), but do not use HEPES or PIPES buffers.
The usefulness of 0.1 M MES is questionable as this buffer gives an absorption of ~0.2 at 234 nm; MES may be more useful as a 50 mM solution. Phosphate buffer may be used if the ionic strength does not exceed 0.1 with the caveat that the buffering power may not be sufficient. More flexibility can be obtained with a cuvette with a shorter path length (see Critical Parameters).
BASIC PROTOCOL 2
Analysis of Lipoxygenase Activity and Products
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