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Ceywood
Monday, 6 August 2012 at 3:23am
Monday, 20 May 2013 at 11:30pm
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Thread: Deviation from conventional Michaelis-Menten kinetics experiments - algebra for fitting model.

posted
20-May-13, 23:36
edited about 25 seconds later
by Ceywood
Avatar for Ceywood
posted about 6 years ago
Hi Reenie,
thanks for taking the time to have a think about this - it's making my head hurt too! It's the derivation that's the killer but I think I may have come up with a solution during that 2am peak PhD brain function time last night actually! Got to show it to a few people but if it's confirmed as a plausible analysis method then I'll post it back here :)

Thanks again!

Thread: Deviation from conventional Michaelis-Menten kinetics experiments - algebra for fitting model.

posted
17-May-13, 10:11
edited about 9 seconds later
by Ceywood
Avatar for Ceywood
posted about 6 years ago
Hi everyone,

I'm a chem PhD student desperately trying to finish! I'm analysing and writing up my final experiments but have been having considerable problems trying to settle on a correct method of analysis.

Basically I've prepared several metal complexes that catalyse the hydrolysis of an ester. I want to determine the catalytic rate constant (kcat, Vmax) for each complex along with the Michaelis constant (Km) for the substrate-catalyst (ie ester-complex) interaction. Following conventional Michaelis-Menten kinetics you'd measure the rate of experiments with the [catalyst] constant and small and with increasing [substrate]. You can then determine kcat and Km by plotting the rate against the [substrate] using the classic Michaelis-Menten equation:

Y = Vmax*[substrate]/(Km + [substrate])

However, I've had to do my experiments by keeping the [ester] constant and small and increasing the [complex]. You can't simply substitute [substrate] for [catalyst] in the MM equation because at [substrate]=[catalyst]=0, Y will = 0. When there's zero ester to hydrolyse this is correct (because there won't be any hydrolysis at all), but when there is no catalyst present the ester will still slowly hydrolyse in solution (ie the uncatalysed rate, kuncat, Vuncat) and therefore Y does not equal 0. This uncatalysed rate is a constant - this reaction goes on in solution even when the catalyst complex is added.

The data I've collected fits with the MM method, but this is clearly not correct because the graph shouldn't pass through 0,0. (Basically my data is exactly the right curved shape but the graph starts above 0 at the Y-intercept.) I've found some linearised versions but my data does not fit with these.

Is there something I can do to the traditional MM equation above to account for this uncatalysed rate for [complex] = zero? Something like:

Y = (Vmax*[complex]/(Km + [complex])) + Vuncat

I don't think this is quite right but I'm sure there's a simple way of doing this.

Thanks so much for any advice anyone can give!

Thread: Problem finding suitable pH 9 buffer

posted
09-Aug-12, 04:47
edited about 17 seconds later
by Ceywood
Avatar for Ceywood
posted about 7 years ago
Thanks very much for your help :)

Unfortunately I've found that a lot of the Universal Buffer compositions involve either one of Good's buffers (which bind either Zn or Cu or both) or boric acid/borate and zinc borate has >0.3% solubility in water (the solvent I have to use).

I did find a good artciel about synthesising non metal binding buffers based on Good's buffers if anyone's interested though (seems like this should be a major issue for a lot of biological/physiological studies!) -

"Avoiding Interferences from Good's Buffers: A Contiguous Series of Noncomplexing Tertiary Amine Buffers Covering the Entire Range pf pH 3-11" Analytical Biochemistry 253, 50-56 (1997)

Thanks again.

Thread: Problem finding suitable pH 9 buffer

posted
06-Aug-12, 04:27
by Ceywood
Avatar for Ceywood
posted about 7 years ago
Hi everyone,
I'm new here and decided to get online to glean some info from those of you with experience/expertise in buffers.

I'm trying to find a buffer for pH 9 that will neither bind Cu2+ nor Zn2+, nor be included in beta-cyclodextrin, nor be insoluble in water at 25deg C. I'm making up solutions for UV-Vis analysis to determine the kinetics of ester hydrolysis reactions.

So far almost all of the buffers I've found contain primary or secondary amines (which will interfere with Zn-binding), bind to Cu2+, or have very low water solubility. (And I'm trying to avoid HEPBS which is $291 for 25g!)

This buffer is pretty much the only thing standing between me and finishing my PhD, so I'd greatly appreciate any suggestions anyone might have! Thanks!

Thread: Problem finding suitable pH 9 buffer

posted
06-Aug-12, 04:26
by Ceywood
Avatar for Ceywood
posted about 7 years ago
Hi everyone,
I'm new here and decided to get online to glean some info from those of you with experience/expertise in buffers.

I'm trying to find a buffer for pH 9 that will neither bind Cu2+ nor Zn2+, nor be included in beta-cyclodextrin, nor be insoluble in water at 25deg C. I'm making up solutions for UV-Vis analysis to determine the kinetics of ester hydrolysis reactions.

So far almost all of the buffers I've found contain primary or secondary amines (which will interfere with Zn-binding), bind to Cu2+, or have very low water solubility. (And I'm trying to avoid HEPBS which is $291 for 25g!)

This buffer is pretty much the only thing standing between me and finishing my PhD, so I'd greatly appreciate any suggestions anyone might have! Thanks!
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