effect of infection of the filarial parasite brugia malayi - Pondicherry ...

Ragenb
1. Tris-HCI buffer . : 0.1 M, pH 8.2 containing 2 mM of diethylene hiamine penta
acetic acid.
2. Tris- HCI : 0.05 M, pH 7.4.
3. Pyrogallol stock solution : 25.2 rng of pyrogallol was dissolved in 1 ml of 0.05 M Tris-
HCI buffer, pH 7.4 in a test tube stoppered and wrapped with an aluminum foil.
4. Pyrogallol working solution : At the time of assay 0.5 ml was diluted to 50 ml with 0.05
M TricHCl buffer, pH 7.4 to give a 2 mM solution and shielded from exposure to light.
5. Absolute ethanol.
6. Chlomfonn.
Partially purified SOD was prepared as described by McCord and Fridovich (1969).
To 1 ml of the tissue homogenate, 0.25 ml of absolute ethanol and 0.15 ml of chloroform
was added. After 15 minutes of shalung in a mechmcal shaker, the suspension was
centrifuged and the supematant obtained constituted the enzyme extract. The reaction
mixture for autoxidation consisted of 2 ml of the buffer containing DETAPAC, 0.5 ml of 2
rnM pyrogalbl and 1.5 ml water. Irutially, the rate of autoxidation of pyrogalbl was noted at
an interval of one minute to three minutes. The assay mixture for the enzyme contained 2 ml
of 0.05 M Tris-HCI buffer, 0.5 ml pyrogallol, aliquots of the homogenate and water to give a
final volume of 4 ml. The rate of inhibition of pyrogallol autoxidation after the addition of
the enzyme was noted. Iron accelerates pyrogallol autoxidation even in trace amounts.
DETAPAC acts as a chelator and thus prevents the interference from ~ e as ~ well ' as from
CU" and hIn2*.
The enzyme activity was expressed in terms of unitsimg protein in which one unit
corn& to the amount of enzyme that mhibited the autoxidation reaction by 50%.
The activity of catalase was assayed by the method of S~nha (1972).
The caFakPc myme pnparation was allowed to split hydrogen peroxide for
~ifFcmnt paiod of time. The reaction was stopped at specifibdgme intervals by adding