It includes application of michelis menton equation but i don't know how to find velocity if absorbance values are given. I used pyrogallol as substrate incase of superoxide dismutase enzyme which inhibits it's autooxidation
Hi there,
So you follow oxidization of pyrogallol at 420nm. If you want to determine the rate of oxidization, A420=f(t) has to be linear and slope is velocity of the reaction expressed in variation of absorbance by time unit. Then SOD inhibiting activity is determined by comparing the rates of pyrogallol in the presence of variable SOD. Results are expressed in terms of % of inhibition = (rate in presence of SOD/rate in absence of SOD)*100.
See the following for more details (assay of SOD activity is described in experimental section):
https://pubs.rsc.org/en/content/articlehtml/2015/ob/c5ob01702e
I think what i understood is that you are asking for the calculation of initial velocites at the substrate concentrations S1, S2 ........and so on, where the read out is absorbance. For this the best and easiest way is to perform time course (kinetics) for the absorbance at each substrate concentrations. Plot absorbance versus Time course of each substrate concentration. The slope will be your initial velocity. Later on, the inverse of this initial velocity and the concentration of the substrate could be used to determine Vmax, Km and Kcat values. Hope it will help. Gud luc
1. v = umole / min
2. v = ((delta A/min) * (Assay volume in uL)) / E b
Dear Vaishali Aggarwal ,
Your problem is not a simple one, because you are not measuring the build up of product concentration or the consumption of substrate, but the decrease in the rate of a simultaneous chemical reaction taking place in the cuvette at the same time your enzyme is acting.
You will not be able to calculate classic reaction velocities, and need to define a unit of activity. This question has been already asked in this SITE
https://www.researchgate.net/post/Pyrogallol_assay
And there you can find a detailed explanation about how to define the units.
You may also check this paper:
Kim et al.,
Measurement of Superoxide Dismutase-like Activity of Natural Antioxidants
Biosci. Biotech. Biochem., 59 (5), 822-826, 1995
Article Measurement of Superoxide Dismutase-like Activity of Natural...
It uses the same strategy of the enzyme assay to evaluate antioxidant activity of natural products, but offers a goof account of how this is done.
Best wishes,
Rogelio
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