Assay Procedure for Lipoxidase (Ref.
No. FGAP042)
Based on that of Theorell et al in which the rate of increase in
absorbance caused by the oxidation of linoleic acid is measured at 234 nm and 25°C.
|
Lipoxidase |
|
| Linoleate + O2 |
 |
13 hydroperoxyoctadeca -9,11 dienoate |
Unit Definition
That amount of enzyme which causes an increase in absorbance of
0.001 per minute at 234 nm and 25°C.
Reagents
| A |
1M NaOH Dissolve 4 g of
NaOH [MM40] in ± 60 ml ice cold distilled H2O.
Caution This solution becomes very hot and is extremely
corrosive.
Carefully adjust volume to 100 ml with distilled H2O.
|
| B |
0.2 M Borate Buffer pH 9.0 Dissolve 6.18 g Boric Acid (H3BO3 in ± 400 ml
distilled H2O. Adjust pH to 9.0 with 1 M NaOH [3A] and dilute to 500 ml with
distilled H2O. Store diluent on ice.
Store assay buffer at 25°C, in the water bath.
|
| C |
0.01% [v/v] Linoleic Acid (Substrate) Dissolve 0.01 ml linoleic acid [Sigma product L1376] [MM 280.4] in 6 ml
95% [v/v] ethanol [MM 46.07] and then add 10 ml distilled H2O. Further dilute
this solution 1/6 with buffer [A] and check that 0.2 £ A234 nm £ 0.21.
Shake before use. STORE ON ICE.
|
| D |
Enzyme Solution Dissolve enzyme at a concentration of 5 mg enzyme/ml ice-cold buffer [A].
Immediately before assay, dilute solution with ice-cold H2O to yield 270-360
u/ml. [0.09 £ DA
234/min £ 0.12]
|
|
|
Procedure
Into a 10 mm cuvette, pipette the following:
Equilibrate at 25°C and monitor DA 234/min
| Add enzyme at t = 0 |
1.0 ml |
| TOTAL |
3.00 ml |
Zero the spectrophotometer and then record rate of increase of
absorbance at 234 nm as soon as reaction mixture reaches 25°C. Check
temperature in cuvette.
AFTER EACH ASSAY, CLEAN THE CUVETTE WITH 0.5 M HCl OR WITH
CHROMIC ACID. RINSE CUVETTE THOROUGHLY WITH DISTILLED H2O BEFORE USING AGAIN
FOR ASSAYING.
Caclulation
|
DA 234/min
x Vt x dilution |
| Activity [u/ml solution] = |
 |
|
e x Vs |
Where
Vt= final volume of reaction mixture [3.0 ml]
Vs= sample volume [1.0 ml]
e = 0.001
| Activity [u/mg material] = |
u/ml solution
|
|
mg enzyme/ml original solution |
Reference
Theorell H. ; Bergstrom S. and Abeson A. : (1946), Pharm. Acta.
Helv. 21, 318] |
Specialists in the Isolation and Purification of Horseradish
Peroxidase and a Select Range of Enzymes