MadSci Network: Biochemistry

Re: rate of reaction equation for hydrogen peroxide and catalase.

Date: Mon Mar 26 08:55:25 2007
Posted By: Alex Tobias, Ph.D., Scientist
Area of science: Biochemistry
ID: 1174819791.Bc

Hi Amirah,

Sounds like a great experiment I wish I did in high school. I have good news for you: the rate of reaction is right under your nose. All those measurements youíve been taking on how much oxygen is being produced for each different concentration of H2O2 Ė thatís where youíre going to find the reaction rate.

Your table should have columns for starting concentration of H2O2 (food for thought: how do you really know the concentration? H2O2 is unstable and can decompose by itself so do you have an independent measurement of concentration besides measuring a volume from a bottle?), time, catalase concentration, and volume of O2 evolved.

What you need to do with the data in your table is calculate (estimate is probably a better word) the initial rates of O2 evolution for each initial H2O2 concentration. In some systems, it is very difficult to measure these initial rates because the reactions are so fast that the rate of mixing of the ingredients introduces a lot of error. Iím assuming that in this case, the rate of O2 evolution is slow enough that you can obtain an accurate estimate of the initial rates (maybe a time scale of 1 or 5 minutes is sufficient).

Letís assume that within the first minute, a measurable amount of O2 has been produced. On your table, in the initial rate column, you would enter the volume of O2 produced per unit time. The units would be in ml of O2 per minute or per second.

You can then convert from volume of O2 to moles of O2 using the ideal gas law (PV = nRT) which I assume youíve covered before. Then you can have another column for initial rate in units of moles of O2 per unit time. In this experiment, even though reaction rate appears on the left side of the equation, you are not going to use the equation to calculate reaction rates. Rather, you are going to input reaction rates that come directly from your measurements of O2 evolution over time, and you are going to be calculating Vmax and KM.

Now that you have all your data, both ďrawĒ (direct measurements) and processed (the calculated initial rates), you can perform a regression to obtain the desired parameters, KM and Vmax. Your question shows that you havenít grasped the inputs and outputs of this experiment: Vmax is not an input, it is an output.

The Michaelis-Menten equation you listed is almost correct for this situation. We just have to be more careful about the units.

The equation you want to use is V = Vmax*[S]/(KM + [S])

Where V represents the reaction velocity (another word for the initial rate) in units of moles of product per unit time. This V is not to be confused with the V in the ideal gas equation I wrote above; there it represents the volume of gas.

The equation you listed in your question had a problem with units, because generally, Vmax is expressed in units of moles of product per unit time. It is optional to decompose Vmax as Vmax = kcat * Eo, so in the equation you listed in your question, you would need to replace Vmax with kcat. Since Eo, the initial (or total) concentration of enzyme is in units of moles, and since Vmax is in units of moles/time, kcat is in units of inverse-time (like sec-1). I would just use the equation I listed above. Later, when you have an estimate for Vmax, you can calculate kcat by dividing by Eo.

I mentioned something above that might be confusing you even more than you already are: regression. Essentially, you need to take the proper data from your table and plot it on a graph in a particular way. You will actually need to do more processing of the data to get it in the right form for plotting. Iím including two links for common ways to plot your data in order to extract the parameters Vmax and KM. Both have their flaws, but the Eadie-Hofstee method is probably the more accurate of the two. Even better is to use nonlinear regression, but that is a technique beyond high school and even most undergraduate studies. Note that unless your experiment covered a sufficiently wide margin of different concentrations of substrate, your data may not make it possible to obtain decent estimates of Vmax and KM. Ideally, you want to have data at high enough substrate concentration such that you achieve Vmax in your experiment. This is the maximum possible rate of reaction under the given conditions, which cannot be increased any further by adding extra substrate. KM, the Michaelis constant, also has a physical meaning. It represents the concentration of substrate that yields a rate (V) that is exactly one-half of Vmax. In nature, values of KM range over more than six orders of magnitude.

Here are the links to the regression methods:

-Alex Tobias

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