[AAM92]

Hello everyone, I was looking around for some help online with a lab report I have and your website looks very helpful, so I decided to see if anyone here would be willing to give me a hand with some work I have. To give some background, I am a Psychology student specialising in Neuroscience and am currently studying a Biochemistry course in Molecular Neuroscience in addition to my more Cognitive Neuroscience courses in the Psychology department. For this reason I have not covered many of the basics of biochemical reactions, so could do with some help with a few calculations.

I have completed the lab work and am just trying to calculate various things with my data set right now. The main thing I am stuck on is measuring total AChE activity in some samples. I have the equation:

Total activity=ΔA/ε*l*3*total volume/0.1*dilution factor*10^3

I believe change in absorbance is just the change in absorbance over 1 minute, but the lab book gives no details about ε*l and I do not believe it was mentioned in the lab. Not knowing this factor is the main point I am stuck on as I think I know what the other terms mean. I have seen that it refers to the extinction coefficient, but do not know how to work this out (or if ε on its own refers to this, what l is).

Any help or comments would be greatly appreciated! Also, if anyone could recommend a textbook, website or any other resource that has details of how to calculate and graph enzyme and protein assays that would be extremely helpful. I have looked in many of the biochemistry books in the library, but most of them seem to cover the theory more than the actual calculations.

Thanks, 

Alex

[yesway]

Hi,

l is the path length of the cuvette - most student lab photometers I have seen use 1 cm path length cuvettes. And epsilon is in fact the molar extinction coefficient. You need to bring up the reaction scheme and the employed wavelength(s) to find out what you actually measured in order to find out the applicable epsilon value. As it was a time-dependent activity assay, most likely the decay/buildup of a substrate/product was measured. Sometimes the primary reaction can be coupled to an analytic reaction (which allows photometric detection of the reaction progress via coupling enzymes). So you need to show the full assay conditions.

It is very important that you catch up on Lambert-Beer's law !

[yesway]

e.g. for AChE activity indication I have seen the following coupled system

AChE will deacetylate
Ac-Thiocholine iodide -> Thiocholine
which in turn generates from
5,5'-Dithiobis-2-nitrobenzoic acid -> 5-Mercapto-2-nitrobenzoic acid (which appears yellow, and therefore absorbs blue light at approx 435-480 nm)

[Babcock_Hall]

The TNB anion has a molar absorptivity of about 14,150 at 412 nm, but I am not sure whether you used the DTNB assay or not.  With respect to your equation, I compared it with the general one that I use, where the volume of the assay solution is in liters, the rate is in ΔA/Δt, and activity is in micromoles per minute.
rate·(vol/ε)·(1,000,000 μmol/1 mol) = activity.

How long did you run the assay?  The time it took to generate the ΔA you observed is understood to be in the denominator.  I am not sure whether the volume is in the numerator or the denominator in the equation you provided, but it is in the numerator in the equation above.  I make use of Ninfa, Ballou, and Benore's textbook quite a bit (Fundamental Laboratory Approaches for Biochemistry and Biotechnology).