I have done enzyme assay for my purified protein, according to the previously reported protocol one proton will be released per reaction and it was monitored by change in the pH of the reaction mixture directly with absorption of the dye(phenol red) at 430nm.
After adding substrate i have taken OD at regular intervals (5,10,15,20 min),but every for five minutes OD was decreasing to some extent.
Why it is happening with increase in time?
Please provide your suggestion.
I can think of 4 possibilities. (1) The enzyme is unstable under the assay conditions. (2) The enzyme activity decreases because of the change in pH. (3) The substrate is being used up. (4) An accumulating product is inhibiting the enzyme.
Most of the enzyme active under a specific range of pH. In your case, as the pH changes, the activity might be changing which results in lesser conversion of substrate into product..
I have found that the disappearance of subtrate concentration initialy follow the following relation:
delta[s]=-At (A activity and t is time)
for more detail you can follow the michaelis-menten .
I understood from the question that the examined enzyme activity (substrate amount is saturating) in term of OD (absorbance) decreases as pH decreases. Thus, OD at 20 min < OD at 10 min < OD at 5 min. Two phenomena can be considered as the pH decreases:
(i) the molar absorption coefficient of the phenol red (maximal at pH 6.5) decreases
(ii) the enzyme reaction velocity decreases, as enzyme activity is always function of pH.
Consequently, the total (I say the total) decrease in the enzyme action (activity) is apparent; the enzyme assay system is incorrect.
Remedy: First, the pH zone where the enzyme reaction velocity is constant (theoretically e.g. 6.5-7.0), must be determined. Second, enzyme activity in this zone must be considered by following the pH decrease only.
Hope this comment can be useful.
How Joseph said, your system can simply be incorrect. It would be good to verify too if your rates are initial rates.
It seems that in his case the pH changes are required to measure the enzyme activity, so the buffer would interfere with the assay.
You should be able to do the same assay with a pH electrode instead of a dye and then you can determine if the issue is a change in molar absorptivity of the dye or enzyme activity changing with pH.
I haven't used phenol red as an indicator......Instead, change in NADH concentration (@340nm) was used to measure the activity.
May be the pH change with increasing substrate concentration effected the initial velocity.
Absorbance of NADH decreases as it is converted to NAD. Is that what you saw?
In this reaction, NADH is used simultaneously as the substrate is consumed by enzyme. In other words, the reaction rate is measured based on the rate of consumption of NADH, and the reaction continues till the enzyme is not saturated and deactivated. Consequently, the concentration of NADH decreases over time. Concentration of NADH is fixed when either the enzyme is completely saturated or the substrate is completely consumed.
Hi , I had the same problem . Did you figure it out if that was due to pH ?
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