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My experiment, where I am investigating the effect of increasing substrate concentration of hydrogen peroxide on enzyme activity hasn't worked out as I hoped. The last result, highest concentration of hydrogen peroxide, shows a decrease in enzyme activity compared to the lower substrate concentration and I don't understand why?

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    $\begingroup$ We need more details to be able to help you. What specific concentrations of hydrogen peroxide did you try? Nanomolar? Or molar? It makes a big difference in possible explanations for what is happening. $\endgroup$ – Curt F. Nov 29 '15 at 14:55
  • $\begingroup$ It was 1, 3, 5, 10, 15 and 20% solutions of hydrogen peroxide with 2% catalase. I put 10cm^3 of hydrogen peroxide and 5cm^3 of catalase solution in a measuring cylinder with o.1cm^3 of washing liquid. The volume of bubbles produced corresponds to enzyme activity. Its a pretty basic high school experiment $\endgroup$ – George Nov 29 '15 at 15:15
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20% hydrogen peroxide sounds HUGE. But something can happen that is called substrate inhibition. It can be detected if the substrate acts as a non-competitive reversible inhibitor (i.e. binds only to the active ES Michaelis complex). In the case I studied during my PhD, the inhibition constant ($K_\mathrm{i}$) was roughly 1000 times higher that the Michaelis constant ($K_\mathrm{M}$). The steady-state rate was well fit with : $$r = \frac{r_\infty}{1+\frac{K_\mathrm{M}}{s} + \frac{s}{K_\mathrm{i}}}$$ So the activity would go through a maximum ($s$ is substrate concentration).

Tip : if it is possible, do you assays with substrate concentrations growing exponentially, like $1\,\mathrm{\mu M}$, then 2, 5, 10, 20, etc.

A good way to decide between enzyme denaturation and substrate inhibition would be to know if it is reversible : denaturation must not be reversible. You could first make a baseline assay (at low conc.), expose the enzyme to a high concentration, then change the composition of the solution (eg dialysis, dilution) and run another assay at low concentration.

A "volume of bubbles" does not sound very accurate. Could you use a Clark electrode to measure the activity instead ?

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the simplest answer for me is that;(at a higher concentration) there was not enough enzyme for all the hydrogen peroxide to react with which slowed down the process. I did the same experiment and the lab report I read gave me this explanation. And my biology teacher approved.

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  • $\begingroup$ If there is not enough enzyme, this would imply (in first approximation) a constant, maximum value. The reaction slowing down requires further explanation. $\endgroup$ – Linear Christmas Nov 19 '17 at 19:06

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