Enzyme kinetics and substrate concentration

[Guest biomante]

Hi

 

I was wondering if someone knows any good electronic resource (or book) about enzyme kinetics, specially trouble-shooting.

 

I know it is simple, but I am stuck just trying to measure the concentration of a substrate I am working with using a coupled enzyme assay.

 

It is supposed to be very straightforward, but I am getting pretty weird results.

 

Any help would be really appreciated.

[Dak]

what kind of wierd results?

[ecoli]

here you go...http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html

http://www.chem.qmul.ac.uk/iubmb/kinetics/

let us know how it works out!

[Guest biomante]

Thanks, I will check the link.

 

The weird results are that I am getting (according to my calculations), extremely low substrate concentrations (silly low). Probably I am messing up the units.

[Guest biomante]

OK

 

This thing is still not working, and I am kind of running out of time (and patience).

The thing is this, I am using a coupled enzyme assay to measure the concentration of a substrate; the reaction of the first enzyme can't be followed but the second one uses NADH, so the depletion of it can be followed with a spectrophotometer.

So I just measure the absorbance (340nm) before adding enzyme (the first in the reaction) and sometime after adding it (I do it after 5 minutes), when I don't see any change in absorbance, right?

 

To get the concentration I use this formula:

(ΔA/6220)x(1000/V) = mM

 

ΔA = change in absorbance

6220 is the extinction coefficient of NADH

V= volume of substrate solution used in the 1 ml cuvette

 

So, I should get the mM concentration of the substrate right?

Well, I am still getting a very low concentration, so low I don't even think I would see any measurable enzyme activity, so it must be wrong.

If there is anyone out there that can help me with this, I would appreciate it.

Thanks

[Skye]

This does the calculation for you. The pathlength of a normal cuvette is 1 cm.

 

http://www.changbioscience.com/calculator/BeerLambert.html

 

You will often get low concentrations. Remember that you are using tiny amounts of enzyme, and a mole is a huge number.