I have performed enzyme (protease) activity assay using flouregenic peptide (containing AMC at c terminal) as substrate at different concentrations of substrate. I have RFU values with respect to time. Now how to plot michelics menten plot. How can I convert RFU into Velocity for making Velocity vs substrate concentration graph for michelics menten plot. Help me by explaining the entire process for finding these values and to form plot.
Hi there,
For each kinetics at a given substrate concentration, variation of RFU versus time has to be linear or at least linear during a sufficient laps of time to determine the velocity which will be deltaRFU/delta t. Then you may plot velocity versus substrate concentration. If the system obeys the MM equation then the curve will be hyperbolic (v=VmS/(Km+S). Using LB linear plot (1/v vs 1/S) you may determine Vm (1/Vm being y for x=0) and Km (-1/Km being x for y=0).
Adding to Dominique Liger 's explanation, an essential aspect of measuring the rate of the reaction according to the textbook* method for Michaelis-Menten kinetics is to measure the initial rate of the reaction. This is the rate at the start, which will also be the fastest part of the reaction. If you draw a line tangent to the reaction progress curve (fluorescence versus time) starting at time zero, the part of the progress curve before the tangent line diverges from the progress curve is the part you should use to calculate the slope, which is the rate (delta RFU/delta time). If your progress curves don't have such a linear portion at the start, then you have one or more technical problems to fix.
*This is described in every introductory biochemistry textbook.
Of course, RFU (relative fluorescence units) is not a direct measure of product concentration. To express Vmax in terms of product formation, you will have to convert fluorescence intensity to product concentration. To do this, you can run the reaction at each substrate concentration to completion by using a high enzyme concentration. That will show you how the fluorescence is related to the product concentration. Alternatively, you can prepare a standard curve of pure AMC versus fluorescence intensity.
Hi Dominique Liger is it necessary to keep enzyme concentration high to study kinetics, as kinetics is independent of enzyme concentration. i am getting saturation in RFU vs time. But not getting saturation of enzyme when plotting RFU/sec vs substrate concentrate. But GraphPad is showing that km is near around 300 micromolar. And I am using substrate concentration between 10 t0 140 micromolar. what should i do?
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