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Enzyme Kinetics using Absorbance to make a Lineweaver Burk plot

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So here's the deal, I have an exam tomorrow and one of the topics is enzyme kinetics. I need to know how to derive a Lineweaver Burk plot using an absorbance over time graph for both a control and in the presence of an inhibitor.

So, in the practice lab, I have five concentrations for the control: 6.25uM, 12.5uM, 25uM, 50uM, 100uM. I also have four for the inhibitor: 25uM, 50uM, 100uM, 200uM.

I've plotted the absorbance readings over time (at 15 second intervals for 120 seconds) for each concentration.

I need to know in detail:

How do I find the rate, in this case v? (It just says to plot 1/v not 1/V0 or 1/Vi which is usually the case). Do I simply use the change in absorbance between the two points, which have the greatest difference and multiply this figure by 4 (it says the formula is change in absorbance divided by change in time and that they want it to be represented as change in Absorbance/minute)? Or do I just take the initial absorbance from the final absorbance (at 120 seconds) and divide it by 2 to get the the rate/v?

To get 1/v I assume I just divide 1 by the rate for each substrate concentration.

Also, for the X axis of the Lineweaver Burk plot, do I simply plot 1/6.25uM, 1/12.5uM etc.? I know this sounds silly, but the reason I ask is that when I do this I get 0.16, 0.08, 0.04 etc., where as in lecture example, the X axis had negative 100,00 all the way up to 300,000!

I also need to calculate the Vmax and Km values, which I'm guessing are just the reciprocals of the X and Y Axes?

I can give you the actual figures if you like, both the ones I observed experimentally and the ones that were in the example. To start, I've just attached the example Lineweaver Burk plot (on page 4 of the pdf). I have a feeling this is really simple, and I've done Lineweaver Burk plots years ago that used concentration instead of absorbance, but what the lecturers' example shows doesn't make a lot of sense to me.

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Solution Preview

1. For large values on 'x' axis, most likely the unit is 1/M not 1/uM. Since 1uM = 10^(-6)M, 1 / M = 1x10^6 /M. So you can end up with values of 10^6 on the x-axis.

2. The point at which the line intersects the 'y' axis is equal = 1 / Vmax. So, the reciprocal of the y-intercept is the ...

Solution Summary

The enzyme kinetics using absorbance to make a Lineweaver Burk plot is examined.

See Also This Related BrainMass Solution

Lineweaver-Burk Plot from Absorbance Data (with and without inhibitor) and Michaelis-Menten Kinetics

I carried out a practical last week and need to write it up but canot remember how to do the calculations from the Michaelis-Menten graph plotted which I guess helps me to plot a Lineweaver-Burk.
I have attached the practical requirements.
I have to produce a table (attached) but cannot fill in certain columns until I can work out calculations. Could someone please show me how to calculate using one example so I can complete table and write my practical?

I had 12 test tubes the 1st six were control second six had inhibitor added. The substrate concentration in each six test tubes were (for first six, then following six) 0.1mM, 0.2mM, 0.4mM, 0.8mM 1.6mM and 2.4mM. Absorbance was measured at 700nm and recorded in order as follows: 1st six - 0.038, 0.064, 0.094, 0.206, 0.199 and 0.238 2nd set was 0.039, 0.067, 0.064, 0.110, 0.119 and 0.230.

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