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We just had an experiment to check enzyme reaction rate according to it's substrate concentration.
In the experiment, we used a varying amount of substrate and the same amount of enzyme:

(1.5mm enz, 4.0mm water, 0.5mm subs)
(1.5mm enz, 3.5mm water, 1.0mm subs)
(1.5mm enz, 2.5mm water, 2.0mm subs)
(1.5mm enz, 1.5mm water, 3.0mm subs)
(1.5mm enz, 0mm water, 4.5mm subs)

We check the rate by looking at the optical density after 3 minutes.

Upon staring at this, I couldn't help but wonder: We don't only change the substrate concentration! We also change it's amount!

While 3.0/4.5 is a higher concentration than 0.5/4.5, it's also a higher amount resulting in longer reaction time for sure. If we check after 3 minutes, there's a chance that the 0.5mm subs would already be gone.

If we really wished to look at the rate according to concentration, shouldn't we have only changed the amount of water? It also changes the concentration of the enzyme but there is there any way to isolate either of them?

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I think there are a few important answers in your question

  • The concentration of enzyme is the same in each reaction chamber. The volume of each chamber is 6ml, and you are using 1.5 ml of enzyme in a final volume of 6 ml, so the dilution factor (the same in all chambers) is 1 in 4. So if stock enzyme concentration is 4 micromolar (as an example), the concentration of enzyme is 1 micromolar in each reaction chamber.
  • The concentration of substrate varies in each reaction chamber. You MUST take the dilution factor into account when calculating the final substrate concentration. You do no tell me what the stock substrate concentration is, but if it is 1mM as an example, then in reaction chamber 1 you are taking 0.5 ml of a 1mM solution in a final volume of 6 ml, which is a 1 in 12 dilution. The substrate concentration in the reaction chamber is therefore 1/12 mM (about 83.33 micromolar). In a similar way, the concentration in the final reaction chamber (4.5 ml in a final volume of 6 ml, dilution factor of 1.33) is about 750 micromolar.

  • You are right, measuring the reaction velocity by taking a single time point after 3 minutes is dangerous! You must of course measure the initial rate if you want to analyze results according to the Michalis-Menten equation. The assumption you are making is that the rate is linear over a period of 3. minutes for all substrate concentrations. If this is not so, you will need to redesign your experiment to take a number of time points and take the tangent to the curve. I think you must be interested in determining the Michaelis constant.

  • In the language of enzyme kinetics you are doing a discontinuous assay with a single time point after 3 minutes as a measure of the initial rate at a series of substrate concentrations. A better approach if available is to use a continuous assay, for example by continuously monitoring the change of absorbance of NADH with time at 340nm. But of course continuous assay not always available.

  • I am surprised that you use water to dilute. Where is the buffer? And how control pH in your chamber? Maybe the enzyme and substrate stock solutions are made in buffer, is this the explanation?

  • You should also check that the velocity versus enzyme concentration is linear at all substrate concentrations.

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