Exercise No. 9
Effect of ions on elimination of free radicals in biological material
Principle
In many pathological conditions free radicals form in the organism. These radicals are mostly
derived from oxygen, e. g. superoxide anion O2-. . The enzyme superoxide dismutase catalyzes its
decomposition into oxygen and H2O2, and in this way it decreases its toxicity. Some metallic ions (e.
g. Cu(II), Mn(III), Fe(III) and mainly their biocoordination compounds have the ability to react with
superoxide and so to eliminate its increased production in the organism.
Superoxide formed by the system xanthine - xanthine oxidase reduces the detector of superoxide a
tetrazolium salt ( INT ) into monoformazane that can be determined spectrophotometrically at 510 nm.
Superoxide dismutase or Cu(II) complex take up superoxide, and so they decrease reduction of the
detector.
Reagents
a) 0.05 mol/1 phosphate buffer solution, pH 7.8
b) xanthine, 5 x 10-5 mol per 1 of reaction mixture
c) xanthine oxidase (XO), 10 U per 1 of reaction mixture
d) INT 9.8 x 10-5 mol per 1 of reaction mixture
e) SOD 1.33 x 10-7 g per 1 of reaction mixture
f) Cu(II) complex (potassium N-salicylidene-L-glutamatocuprate (monohydrate) K [Cu(sal-Lglu) ( H20 )] 4 x 10-7 g per 1 of reaction mixture
Reagent
Buffer solution, pH 7,8 (ml)
Xanthine (ml)
SOD (ml)
Cu(II) complex (ml)
INT (ml)
Test tubes
1
1,2
0,1
0,1
2
1,1
0,1
0,1
0,1
3
1,1
0,1
0,1
0,1
Start the reaction and add xanthine oxidase in 20s intervals
Xanthine oxidase (ml)
0,1
0,1
0,1
Incubation 10’ at room temperature. Measure absorbance at 510 nm in 20s intervals,
using Buffer soln. as reference.
A510
% of INT reduction
100
% inhibition of INT reduction
0
Units of dismutase activity (U)
Calculation: inhibition of INT reduction by the formed superoxide (in %) is calculated from
absorbance values:
INT reduction by superoxide................................................................... 100 %
INT reduction by superoxide in the presence of SOD............................... x %
I [ % ] = 100 - x
One unit of SOD activity ( U ) is defined as the ability to inhibit INT reduction by 50%.