using UV absorbance scans to 'measure' homogeneity of proteins
pxpst2 at vms.cis.pitt.edu
Wed Sep 8 10:43:18 EST 1999
In article <david-0809991507400001 at mac355.nmus.pwf.cam.ac.uk>,
david at cryst.bioc.cam.ac.uk (David) wrote:
> I'm sorry for not making it clear, but the kind of homogeneity I was
> thinking about was with respect to proteins rather than nucleic acids, and
> I guess in particular, whether a protein that appears pure on, e.g. a gel
> or gel filtration columnm, has higher-order oligomers or aggregates
> present. As suggested, good ways to study this include gel filtration and
> analytical ultracentrifugation, which we have done for our system, but I
> had recently heard the advice that one should always as a first step, look
> at the UV280 scan to see if there are aggreagates, which was what prompted
> my original question.
Remember that absorption of light in mainly due to only 3 aa's(
trp(~280nm)>tyr(~275nm>phe~258nm)). If you have a solution of a monomeric
protien in solution or a multimeric protein in solution, the absorption
spectra will not reveal it. It will just tell you the concentrations of
Other methods that can be used to explore the secondary structure of your
protein and can yield information on whether or not you have mutimeric
proteins are Light scattering and anisotopic spectroscopy. Light
scattering uses a laser to measure the angle at which the light comes off
a macro molecule. A derivation of this is called dynamic light scattering
and measures the frequency shift of the laser light after hitting a target
in solution. The information that is yielded from these methods are
diffusional coefficents and molecular weight.
Another more esoteric method but quite useful is flouresence anistrophy.
The same molecules that absorb light, flouresce light. Lifetimes of
flourescence is in the order of 1-100ns which is the same time frame for
rotational diffusion of a molecule. One can make measuremets of the
flourescene polerization (anisotrophy) to measure rotational motions of
proteins in dilute physiological aqueous solutions. With this method, one
can solve for molecular weights.
see ref: Taylor,waggoner et al (1986) "applications of Flourescence in
Biomedical Sciences". Liss,New York
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