APX activity was estimated by the method of Nakano and Asada (1981). Ascorbate peroxidase was assayed by recording the decrease in optical density due to ascorbic acid at 290 nm . The 3 ml reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbic acid, 0.1 mM EDTA, 0.1 mM H2O2, enzyme and water. The reaction was run for 5 min at 25 °C.
how to calculate APX activity by using coefficient of absorbance 2.8 mM-1 cm-1 ??????
One unit of enzyme determines the amount necessary to decompose 1 μmol of ascorbate per min.
Hi there,
the two crucial things for determining enzyme activity is to first access to initial velocity data (ie working in condition where product is formed proportionally versus time) and to work with saturating subsrate concentration in order to observe maximal velocity. Guessing you are working in this condition, initial velocity v0 is the ratio between product and time. In your case, you observe disappearance of substrate which correspond numerically to appearance of product if stoechiometry is 1:1... So v0=deltaDO/delta time (unit is min-1) then you can express this in terms of concentration of substrate consumed per min v0=[(delta(DO)/2.8)/5)] in mM per minute then transform it into µmol/min by multiplying the result by 3.10-3 (volume of the assay) and then multiplying by 1000 (converting mmol into µmol). The result will be expressed in U...
Well what you can do is to have a standard curve of ascorbic acid with various concentrations by taking readings at 290nm. Do the enzyme assay and correspond the absorbance with the standard curve and get to know the decrease in ascorbic acid content. Initial concetration you know and final shall be calculated. There by you con conventionally get the enzyme activity in terms of umoles of ascorbic acid decomposed/min
BUT i am not following standard graph method...i am taking change in OD and want to calculate using coefficient of absorbance 2.8 mM-1 cm-1
but that method will help and has accuracy for your lab conditions (standardized).
The starting OD before enzyme addition during the kinetics experiment will give you the actual concentration in your sample as there is a standard epsilon for ascorbate. What Priyanka is actually looking for is activity which is directly related to the variation of concentration versus time and not concentration itself...
Here it is @Jitendriya Panigrahi.
Sorry for the delayed response.
http://pcp.oxfordjournals.org/content/42/4/433.long
Ascorbate Peroxidase (AsPOX)
- 0.2 M Tris/HCL buffer (pH 7.8)
- 0.25 mM ascorbic acid
- 0.5 mM H2O2
Can anyone help me how to make reaction mixture?
In 3ml, how can I make all these buffers, [The 3 ml reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbic acid, 0.1 mM EDTA, 1.5 mM H2O2 and 0.1 ml enzyme]
Any one can help me?
Aqleem Abbas you should calculate it: m= M (mol) x Mm (g/mol) x V (L)
Ihsein Rokia Amine-Khodja Kindly help me now I have calculated APX values 242 for 30 seconds, 192 for 30 seconds, 160 for 1 minutes, 669 for 3 minutes. Where to put these spectroscopic values/
Thans
It is based on decrease of O.D. to 290 nm as ascorbate was oxidized. The reaction mixture contains 0.1 ml potassium buffer,0.5 mM ascorbic acid, 0.1 mM EDTA, 1.5 mM H2O2 and 1 ml enzyme. In blank ascorbic acid was not added.T he reaction was started by adding the enzyme or H202 and the decrease in absorbance was recorded 30 seconds after this addition at 430 nm.
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