[batty2279]

I purchased from a bottle of GAP from sigma that comes as a racemic mixture, with an estimated 45-55 mg/mL.

http://www.sigmaaldrich.com/catalog/product/sigma/g5251?lang=en&region=US


To determine the exact concentration of this substrate for my enzyme kinetics, i need to run an assay with GAPDH.  This is the cited assay:

Determination of D-GAP concentration.  Briefly, reaction mixtures containing
30 mM pyrophosphate buffer, pH 8.6, 200 μM NAD+, 200 μM sodium arsenate, 5 mM L-cysteine, and 10 U of GAPDH
were preincubated at 30°C for 10 minutes. D,L-GAP was added to a final concentration not exceeding 200 μM (100 μM DGAP),
and the reaction was monitored spectrophotometrically at λmax = 340 nm until the reaction was complete. The
concentration of D-GAP was calculated from the concentration of NADH formed (extinction coefficient = 6220 M-1•cm-1).

1) They say the concentration of substrate used is not to exceed 200 uM, but then it says 100 uM in parentheses.  Did they just estimate some amount between 100-200?

2) The spec assay part is a bit confusing.  NADH will be formed, so I should get an increase in absorbance at 340.  Do I just wait till it plateaus, and then use the vo value (slope) with Beers law?  Sub it for A and solve for c using the ε = 6220 and l = 1 cm?

Thanks for any insight.

[Yggdrasil]

1) They say the concentration of substrate used is not to exceed 200 uM, but then it says 100 uM in parentheses.  Did they just estimate some amount between 100-200?

A racemic mixture contains approximately equal amounts of D- and L- enantiomers, so a 200µM solution of the mixture would contain ~100µM of the D-enantiomer.

2) The spec assay part is a bit confusing.  NADH will be formed, so I should get an increase in absorbance at 340.  Do I just wait till it plateaus, and then use the vo value (slope) with Beers law?  Sub it for A and solve for c using the ε = 6220 and l = 1 cm?

I think the way the assay works is that it assumes complete conversion of GAP to glycerate 1,3-bisphosphate, so that the amount of NADH formed at the end is equal to the initial amount of D-GAP in your reaction.  So, there's no need to look at the kinetics, just at the total amount of NADH formed.

[batty2279]

yes, the amount of GAP converted is directly proportionate to the amound of NADH formed, however i'm still unclear on how to calculate this.  I'm assuming some form of Beers law since I'm looking for concentration and they provided the molar extinction coefficient.  I just don't know what parameter to use.  Final absoption value?  Or some kinetic value, such as vo?

[batty2279]

   i think i just answered my own question....

haha i thought when they said "monitored spectrophotometrically" they were observing the change in absorbance over time, as in, some type of vo. 

i guess they mean they just watched the absorbance increase until it stopped rising (full conversion of GAP) and used this as A in A = Elc.

no?

[Yggdrasil]

i guess they mean they just watched the absorbance increase until it stopped rising (full conversion of GAP) and used this as A in A = Elc

Yup, that sounds about right to me.  Good luck with the experiments.

[Babcock_Hall]

Glyceraldehyde 3-phosphate decomposes slowly with time.  IIRC there is a base-promoted elimination of phosphate.  This link discusses freezing the solution:  http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/7/g5376dat.pdf