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

paoxide and 0.2 ml of reduced glutathione. The incubation medium was made upto a final
volume of 2.0 ml with water. The tubes were incubated at 37'C for 90 and 180 minutes. The
d o n was terminated by the addition of 1.0 ml of the precipitating agent. The reaction
mixture was centrifuged and to the supernatant, 6.0 ml of disodium hydrogen phosphate was
added. One ml of DTNB reagent was added just prior to the calorimetric analysis. The
absorbance was read at 412 nm against a blank, which contained only 6.0 rnl of disodium
phosphate and 1.0 ml of DTNB reagent. Suitable aliquot5 of the standards were taken and
treated in a similar manner. The activity was expressed in terms of pgm of glutathione
utilizedlrninutelmg protein.
4.1.1.5 Glutathione reductase
Glutathione reductase was assayed according to the method of Beutler (1984).
Glutahone reductase catalyses the reduction of oxiW glutathione (GSSG) by
NADPH or NADH to reduced glutathione. The activity of the enzyme is measured at 340
mn following the oxidation of NADPH. GR is a flavin enzyme and it has been found that it
is not fully activated by FAD in normal samples. Complete activation of apoenzyme requires
the preincubation of enzyme with FAD. This is done prior to the addition of GSSG or
NADF'H to the reaction system.
Reagents
1. Tris-HC1 : 1 M with EDTA 5 mM, pH 8.0
2. FAD : l o p
3. Oxidised glutathione (GSSG) : 0.033 M
4. NADPH :2mM
To 100 p1 of Tris-HCI buffer, 20 tr] of the sample, 1.58 ml of water and 200 pl of
FAD is added and incubated at 3PC for 10 minutes. To the test sample, 200 tr] of GSSG is
lddcd and again incubated at 3PC for 10 minutes. Later 100 tr] of NADPH is added to both
the blank and the test samples and the decrease in optical density was measured against the
at 340 tun (3PC). The values are expressad as uniWgm proth