Re: Re: How do I calculate extinction coefficient of my product pNP

Hi Feng,

Spectral absorption follows a simple equation:

Abs = Emole * C * L

Where Abs is the absorbance at a given wavelength, Emole is the Extinction Coefficient at the same wavelength, C is the Concentration of the absorbing molecule and L is the path length of the cuvette(usually 1cm).

You are entirely correct. Most scientists will calculate the Emole from a graph of the Abs vs C of the product, pNP at around 405nm. The slope is the Emole. The offset is a graph of the Substrate Concentration vs Abs, it will have an Abs, albeit low. Subtract the AbspNPP from the AbspNP to get the final slope.

If you chose to use 410nm, that will be fine, but you risk being on a spectral slope. Anytime you are measuring on a spectral slope in a dynamical system(lipase is certainly a dynamical system), expect high Standard Deviations.

For the graph, I would expect at least five points with five determinations for each point. Construct error bars and see how tight the fit is.

Feng, your research sounds very exciting! I hope this helps. I’ll be looking for your paper in the journals.

Thank you very much for your question.
Peter

References:
http://www.fisk.edu/~aburger/Published03_06/Introduction/Optical/Extinction_coe fficient/extinction_coefficient.html
http://www.ruf.rice.edu/~bioslabs/methods/solutions/mixtures.html
http://www.piercenet.com/Products/Browse.cfm?fldID=01041001
http://www.piercenet.com/files/TR0006dh5-Extinction-coefficients.pdf