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initial velocity in an enzyme reaction


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10 replies to this topic

#1 claire84

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Posted 14 November 2012 - 07:20 AM

Hallo everybody,
I'm going to start the set up an enzymatic inhibition assay, but I'm not an expert and I'm having some problems to understand some key points of these assays.
In particular, I understood I need to run the reaction under initial velocity condition, but how can I measure it?
I know it's the initial linear portion of the enzyme reaction when less than 10% of the substrate has been deplcted, but esperimentally how can I decide it?

I'll really appreciate your help and suggestions
thanks a lot!

#2 mdfenko

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Posted 14 November 2012 - 08:10 AM

it was probably easier with older technology.

first of all, how are you measuring enzyme activity? are you following it on a chart or doing timed assays?

if you follow on a chart then you can (if you mix fast enough) determine initial velocity by drawing a tangent to the beginning of the reaction (on the chart) and determining the slope of the tangent (like i said, older technology). that's how we did it ~40 years ago.

since then, initial velocity can be determined with a rapid mixing stopped flow spectrophotometer (or a spectrophotometer with the right attachments). if the reaction can be followed spectrophotometrically (like nad/nadh assays).

for timed assays, you have to determine the reaction time that will deplete 10% of the substrate and incubate for less time (be consistent with all of the conditions being tested). this will give an approximation of initial velocity.
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#3 claire84

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Posted 14 November 2012 - 09:30 AM

I'm working with a protease and I'm going to monitor the cleavage of a peptidic substrate. After cleavage there will be an increase of fluorescence at a certain wavelenght. I'd like to monitor the reaction in continuos using a fluorometer.
So, I just need to consider the linear part of the curve and to perform a linear regression, is it right?

#4 mdfenko

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Posted 14 November 2012 - 11:43 AM

not exactly, the initial velocity would be determined by drawing a tangent to the beginning of the "burst phase" (not the tangent they show in the illustration, that's a regression line from the linear part of the reaction). once the reaction is linear you are past the initial velocity.

Edited by mdfenko, 14 November 2012 - 11:45 AM.

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#5 PhDinAcronyms

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Posted 14 November 2012 - 02:31 PM

You might find these links helpful in understanding the assays and analysis:
http://assay.nih.gov...me_General_2010
http://www.ucl.ac.uk.../inhibition.htm
http://www.worthingt...enzymeConc.html

#6 claire84

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Posted 15 November 2012 - 03:12 AM

Thank you for the links, they seem very usefull.
Maybe I'm wrong, but the burst kinetics is in the pre-steady state kinetics, while in my assay I need to be in a steady state condition to measure the Km and Vmax and then the IC50 of my compounds..

#7 PhDinAcronyms

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Posted 15 November 2012 - 04:06 PM

Yes, you want the steady state kinetics. The burst kinetics is usually very brief (and often happens before you get the plate in the plate reader). Even if you do see the burst phase on your reading, you should be able to differentiate it from the longer steady state phase which will be linear and then start tapering off (where it is no longer steady state). For your particular protease, I would look through published papers on it to decide appropriate starting concentrations for the enzyme and a chromogenic substrate. You can then optimize from there.

#8 mdfenko

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Posted 16 November 2012 - 09:23 AM

i used the page for "burst kinetics" for the illustration of initial velocity. initial velocity is not determined from the steady state, it is determined from the initial burst of activity upon mixing.
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#9 claire84

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Posted 19 November 2012 - 02:27 AM

I'm a little bit confused now..I was thinking the same of PhDinAcronyms

#10 mdfenko

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Posted 19 November 2012 - 09:10 AM

i stand corrected. according to the first reference submitted (from 2010) by phdinacronyms, initial velocity is determined from steady state with less than 10% of substrate utilized.

i guess that invalidates the thousands of kinetics assays i performed in the 70's.
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#11 PhDinAcronyms

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Posted 19 November 2012 - 02:22 PM

I was starting to get confused as well but I think we were essentially referring to the same part of a kinetics assay just with different terminology. It's because of today's assay sensitivity and rapid measurements that it can capture the single turnover of an enzyme which occurs in something like milliseconds (the burst kinetics). Then you see the initial velocity portion.

My guess would be that you were still measuring the correct initial velocity mdfenko and you wouldn't have seen the actual burst kinetics, so I don't think it invalidates any results from the 70's. From my knowledge, I think standard chromogenic or fluorescent protease assays (assuming you're hand pipetting) would be unlikely to see the actual burst phase, at least in my experience. Not sure if there any other enzyme biochemists out there who might be able to clarify this better for us?




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