Spectrophotometric Protein (Absorbance?) Ratios at UV

[CMbodybuilder]

Hello everybody, I am a new member here and recently joined not only because I love science but also because I have this question to which I cannot find an answer. I recently conducted a research study on quantification of protein powders (like the ones advertised to bodybuilders/athletes). I am a high school student who worked in a lab, so I began with Bradford, but upon a background reading of the protein solutions alone (no assay), the absorbance was too great on the plate reader to even warrant further attempts. I then moved onto many different methods, such as gel filtration, ammonium sulfate precipitation, and lipid extraction. Essentially, the other ingredients (flavorings, colorings, thickeners, etc.) would get in the way of the method.

 

Eventually, I decided to dilute the solutions to a very low concentration in attempts to effectively make the other ingredients so diluted that they wouldn't interfere in the readings. I put the solutions in a spectrophotometer and attempted to read the absorbance at 280 nm and 260 nm, then use the equation Concentration = 1.55*A280 - 0.76*A260, which, from my research, I know is a method to estimate concentration of unknown proteins (peak absorbance at 280nm due to aromatic aminos like Tyrosine and Tryptophan) with possible nucleic acid contamination (peak absorbance at 260nm). What I could not understand, or find for that matter, is why the ratios of 1.55 and 0.76 are used. I assume they are absorbance coefficients, maybe even the average absorbance coefficients at that wavelength for all proteins, but I can't find an answer wherever I look.

 

SHORTER VERSION: In the equation for protein quantification, Concentration = 1.55*A280 - 0.76*A260, where do the coefficients 1.55 and 0.76 come from?

 

Thank you all for the help!

Edited September 1, 2015 by CMbodybuilder

[CharonY]

IIRC the conversion factors are based on an old paper from Warburg (40s-50s) on enolase. The values are reasonably close to BSA (~1.5, I think) so that it is still in common use as a rough estimate. If done accurately you would derive factors for each of your proteins of interest, but typically UV is used to get rough estimates, anyway.

A more precise method involves using factors based on quantity of aromatic AAs in your protein.

[BabcockHall]

I teach a biochemistry laboratory course, and we use various protein shakes as our unknowns. I prepare them at 5 mg of solid per milliliter in DI water. I gravity filter them, and next I filter them through 0.45 micron syringe filters to remove suspended matter. These two steps are very slow, and the 0.45 micron filters have to be changed several times (possibly centrifugation would clarify them adequately). However, the resulting solutions generally behave well in Bradford assays. Sugars should not interfere with a Bradford assay, but they could interfere with other colorimetric assays.

 

Some vitamins absorb in the UV region as well, and these might also interfere with a UV assay.