I am working on some DNA aptamers and interested to evaluate the inhibition potency and mode of inhibition of this aptamers against particular target enzyme. Briefly, I would like to know a good method and kinetic program to determine competitive/non-competitive/uncompetitive mode of inhibition.
I have tried using Origin 8.0/MS:Excel, however it does not yield the Inhibition kinetic parameters (Ki/Kis). Further, I also wanted to make sure that the results I obtained would remain the same using different approaches/programs.
Therefore, I would highly appreciate, if you can please lend me your valuable suggestions or share your experiences.
Dear Irshad,
The simplest method that you can adopt is, just draw the substrate saturation curve with and with out inhibitor and plot the double reciprocal curve, it will give the type of inhibition, Km (Uninhibited reaction), and Ki (Inhibited reaction). From these you can calculate any other kinetic parameters.
Thaks Hari for your kind reply. Yes, i followed similar approach. I measured the rate of enzyme reaction in presence and absence of diferent inhibitor concn at various substrate Concn.The resultant rate of reaction and substrate was transformed by double reciprocal/lineweaver-burk plot (1/v Vs 1/S plot curve).
Dear Sankar,
using linear transformations introduces bias into your calculations and therefore it is not the best way to analyze enzyme kinetics. I suggest using non-linear regression analysis and to fit the original data into Michaelis-Menten or other appropriate equations. Check some dedicated software, such as Grafit or Sigmaplot, which already have a colection of built-in equations.
Dear Gerasimaite,
Thank you for your guidance. I have seen the same in the abstract of "Improved Rearrangement of the Integrated Michaelis-Menten Equation for Calculating In Vivo Kinetics of Transport and Metabolism" by Russell., 1992. But I am not able to get the full text. I hope you might have have the article and I would like to request you to get me the article there by i can have a better learning.
I would recommend you to use the dynaFit software. It is a little bit harder to use than SigmaPlot but it can directly compare how different mechanisms of inhibition fit to you data set.
@Ruta Gerasimaite . Thanks for your time and giving valuable suggestion. I have tried to fit the original data by non-linear regression using sigmaplot (evaluation version), however i did not suceed in getting the plot.I even tried to learn through online tutorials. I would appreciate if you can give me some path to generate the plot. Thanks
@Felipe Merino, Thanks for your valuable time and suggestion. I am not very familiar with dynaFit Software, however i would try to install and learn your suggested software to solve my current problem. I would appreciate if you can give me some easier path/procedure to run this software. Thanks.
Hi. You can find a description as well as a tutorial on how to use the program here.
http://www.biokin.com/dynafit/tutorial/index.html
Felipe
Well, the best way is to plot everything manually. You may need MS: Excel to help you with some calculations regarding the Lineweaver-Burk plot.
Thanks dear Felipe Merino for your kind suggestion. I am learning your suggested Software!
Thanks dear Allan for your kind suggestion. I did calculation n plot using MS excel!,however it doesnot seem to give good fit.
Prism has the best software for equations built in for simple line weaver Burke plots and has the best graphing programs. But sometimes the data does not fit to simple equations. I used to read Siegel's enzyme kinetics book and get equations derived from that reference, and try fitting the data to many different equations to determine the best fit using Sigma Plot. But dynafit might be best.
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