You are not my quantum mechanic

November 4, 2009 7:57 AM

I'm looking for something like Feynman's explanation for how a difraction grating works only for the absorbance of a colored chemical in solution.

In biochemistry there is a technique where one measures protein concentration by looking at how much UV light the protein absorbs. Unfortunatly this technique doesn't see agregates (big blobs of amorphous protein) the same way it sees properly folded protein in solution.

The traditional explanation has to do with things being in the shadow of other things. But recently I've heard people making arguments about the wavelength of the light and how big the particles are. I can tell that both sides are way off base in terms of modern physics.

Can anyone point me at something that would explain this phenomenon the way it really happens, but with out too much (any) tensor calculus?

posted by Kid Charlemagne to science & nature (2 comments total)

i'm not sure this is what you're getting, but my understanding of why it's not ok to measure the concentrationof proteins by light absorbance is because the light absorbance gives a read of the average optical density of the solution. If the protein has aggregated, the solution does not have a uniform optical density and so, an absorbance reading through one pathway, that for instance contains an aggregate particle, will be markedly different from a reading through a pathway that doesn't contain an aggregate particle.

see http://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law for a better explanation of how the whole absorbance thing works.
posted by Tandem Affinity at 9:40 AM on November 4

There are two phenomena.

Native proteins are much smaller than the wavelength of light (nm vs um) so they absorb light according to their color. Specific chemical structures (chromaphores) in the protein absorb specific wavelengths of light giving an absorbance peak with low absorbance either side of it and the peak height is proportional to concentration.

When you denature proteins their individual size doesn't change that much but they begin to aggregate and form large blobs with sizes approaching the wavelength of light. The larger blobs can scatter light so the absorbance goes up with decreasing wavelength. Its the same reason that any suspension of fine particles (chalk dust, paint, clouds) apppears white. In a spectrum you don't get a single peak just the steady increase with decreasing wavelength. Investigating the shape of that curve allows you to calcualte the size and number of the particles.

In many real systems and especially aggregated proteins you get both factors at the same time and making sense of it all is hard. You are generally better to measure concentration (by nitrogen analysis) and size (by dynamic light scattering) independently.
posted by Fiery Jack at 9:49 AM on November 4