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Glucose Oxidase Assay

I was given

1.2ml ODAD sol(.42mM ODAD in .1M MES buffer pH 5.5)
.1ml horse radish peroxidase (1mg/ml)
0.56M Glucose sol (substrate)
1.5ml H20
.01ml glucose oxidase (aspergillus niger; 25 units/ml ... erm wtf?)

Aim was to mix all these solutions use different volumes of glucose and measure using spectrophotometer, the absorption over time. So intially, I used .05ml glucose and after adding the oxidase, the absorption was recorded every 15 secs, and absorption increased.

Now ultimate aim is to find Km of glucose after plotting a graph of the oxidation of glucose versus time at various concentration that I used.
So I have to use michealis-menton curves etc to find Km but Im not sure how you get the concentrations because I was changing glucose volume ... how would I workout concentrations? Also how do I work out velocity of reaction?

Theres further info about glucose oxidase; 160kDa etc and the reaction equations ... Im quite confused, Im sure someone knows how to do this ... please help, will rep.

cheers

Reply 1

Kyle_S-C

I haven't done Michaelis-Menten kinetics since last year, but the Wiki entry on enzyme kinetics gives information on how to plot things and calculate important properties (like the reaction velocity).

Oh, and I think the concentration of glucose should be given by:

0.56 \times \frac{V_{glucose}}{V_{total}} = conc_{glucose} / M

Reply 2

gyrase

Thanks for reply

Kyle_S-C

Oh, and I think the concentration of glucose should be given by:

0.56 \times \frac{V_{glucose}}{V_{total}} = conc_{glucose} / M

This is what I really need although I have to be 100% sure that it is correct... V(total) will change because I'm adding different amounts of glucose each time .. so will this be correct? Also WHat is M?

cheers

Reply 3

Kyle_S-C

Oh, M is just to let you know that it's in the same units as the glucose (i.e. M = mol dm^-3).

V(total) should be the total volume of the solution, including everything else and V(glucose) should be the volume of glucose solution you're putting in.

Reply 4

gyrase

Cool so Im making slow progress.

I need to plot a graph of product formed versus time I think, but all I've got is absorbance values for different concs of glucose over time. :s-smilie:

help!

Reply 5

Kyle_S-C

Don't worry, you just need the Beer-Lambert law. It basically says that concentration of whatever is doing the absorbing is proportional to absorbance (and other things which should have been kept constant).