Optimization of enzymatic assay of superoxid dismutase and glutation peroxidase activity in milk whey

Trajchev, Metodija and Nakov, Dimitar and Gjorgoski, Icko (2016) Optimization of enzymatic assay of superoxid dismutase and glutation peroxidase activity in milk whey. Symposium Proceedings of the 2nd International Symposium for Agriculture and food, 7-9 October, 2015, Ohrid, Republic of Macedonia.

Text Optimization of enzymatic assay of SOD and GPx activity in milk whey.pdf - Accepted Version Download (224kB)

Abstract

General spectrofotometric kinetic protocols are described to measure the antioxidant
enzyme activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in milk
whey. The SOD convert superoxide radical into hydrogen peroxide and molecular oxygen,
while the GPx convert hydrogen peroxide into water. In this way, two toxic species,
superoxide radical and hydrogen peroxide, are converted to the harmless product water.
The applicability of this methods for milk samples from dairy cows were tested. SOD
activities in the milk whey was determined by the autoxidation of pyrogallol in presence of
DTPA in TRIS-HCl buffer, pH= 8.5. The effect of reaction conditions on enzymatic
inhibition of pyrogallol autooxidation was studied when different amounts of sample were
added in reaction mixture. The reaction mixture for determination of GPx activity
contained EDTA, reduced glutathione, glutathione reductase, NADPH and cumene
hydroperoxide in phosphate buffer, pH= 7.6. The effect of reaction conditions on
enzymatic NADPH consumption was studied when different amounts of reduced
glutathione and whey sample were added in reaction mixture. The both spectrophotometric
methods were modified in order to accommodate kinetic analyses in 96-well micro plates.
The rate of pyrogallol autooxidation and NADPH oxidation linearly depend from amount
of whey sample in reactive mixture. Applying higher amounts of milk whey in reactive
mixture was decreasing optical density reading of pyrogallol autooxidation at 415 nm and
increasing the optical density readings of NADPH oxidation at 340 nm, in a linear way.
The methods could be validly applied for milk whey samples.

Item Type:	Article
Subjects:	Agricultural Sciences > Animal and dairy science Agricultural Sciences > Veterinary science
Divisions:	Faculty of Agriculture
Depositing User:	Dimitar Nakov
Date Deposited:	30 Jan 2023 07:34
Last Modified:	30 Jan 2023 07:34
URI:	https://eprints.ugd.edu.mk/id/eprint/30968

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