Molar extinction coefficent calculation

[vande060]

Homework Statement

I am to calculate the molar extinction coefficient for Bovine serum albumin

Homework Equations

ε = (nW×5500) + (nY×1490) + (nC×125) for coefficients around 280nm

The Attempt at a Solution

I know how to solve this by adding up the tryptophan, tyrosine, and cytosine for the peptide sequence of BSA, but for the lab I am running we are testing absorbance at 595 nm, not 280nm. Also I know that the Practical Handbook of Biochemistry and Molecular Biology cites most extinction coefficients at 280. Can I still use my calculation for the formula that is supposed to be at 280nm.

When I do the math I get: (3*5500) + (21*1490) + ( 34*125) = 52040 M ^-1 cm^ -1

this seems too high though, as most sources I'm finding say the extinction coefficent should be around 44,000.

I used an amino acid sequence my prof gave me.

 

[Ygggdrasil]

Perhaps your professor forgot that the first 25 amino acids of BSA are a signal peptide that gets cleaved off and is not present in the mature BSA protein (which has an extinction coeff ~44,000).

Absorption at 595nm is used to measure protein concentration in a Bradford assay, where protein binding to a coomassie blue dye creates a color change that allows you to measure the protein concentration. Unlike measuring the absorbance at 280nm, you cannot calculate an extinction coefficient for the Bradford assay from the primary sequence of the protein. In order to convert an absorbance measurement at 595nm to a protein concentration in the Bradford assay, you need to make a standard curve with solutions of known concentration.

 

[vande060]

WE are doing it by making a standard curve, I think this was more of an exercise. I'll try to find good literature to cite the correct calculation of the EC by, otherwise I'm going with using the peptide sequence he gave us. Thanks so much for clearing that up