Hello Everyone,
I would like to ask, how to analyze and understand the pre-steady state enzyme kinetics using stopped flow instrument in easy way. Although the instrument (Stopped flow instrument, Biologic company) has default analysis software to predict the data.I didn't understand how analyze enzyme kinetics like reaction intermediates and other steps like association and dissociation rate constants.
Does anyone have experience in using the Stopped flow system please share your knowledge regarding how to analyze and understand the data. Although i have gone through many papers i didn't understand analysis and their way of presenting data is quite complicated. I am new to this stopped system i seek some help in analyzing the data.
Thank you very in advance for your kind help and assistance.
Hi there,
I think the following paper and reference therein should be helpful:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1134586/?page=1
The stopped flow instruments is aimed to collect the reaction kinetics in a relatively short time. It does not interpret the experimental data. You should have a minimum background in chemical kinetics to understand what you see using a stopped flow instrument.
It depends on what you are interested in. Generally, stopped flow is used to measure the rate of formation and decay of the intermediates of a reaction. Depending on the intermediates that you observe, you will need to write kinetics models and then use those models to obtain a fit to your experimental data. The resulting fit will give you the kinetic rate constants.
The answers you have received should prove helpful but as already pointed out, youy need to get acquainted with chemical kinetics in order to understand the information that stopped-flow methods may provide about your enzyme.. I recommend having a look at
'Structure and Mechanism in Protein Science ' by Alan Ferst (published by W H Freeman New York)
The book contains a chapter that deals with the kinetics.
Peter
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