A280 vs. protein assay?

Jeff R. Livingstone livingst at ncifcrf.gov
Tue Sep 20 02:20:42 EST 1994


In Article <17034B9CAS86.MESALV00 at ukcc.uky.edu>, MESALV00 at ukcc.uky.edu wrote:
>In article <tom-1509941445240001 at 131.211.54.137>
>tom at radical.chm.jhu.edu (Tom Tullius) writes:
> 
>>
>>We have encountered an unusual situation, and I was wondering if anyone
>>else has seen this. We are studying the monomer-dimer equilibrium of a
>>protein. Gel filtration chromatography of the (purified) protein sample
>>give two bands (detected at 280 nm) that have roughly equal integrals.
>>However, a Bio-Rad protein assay on each peak indicates that there is
>>approximately 20 times more protein present in the leading (high MW) peak
>>(presumably the dimer). Could dimerization somehow be "quenching" the
>>absorbance at 280 nm? Ideas or experience would be appreciated.
>>
>>--
>>Tom Tullius
>>Department of Chemistry, Johns Hopkins University
>>tom at adical.chm.jhu.edu
> 
> 
>Dear Tom,
>Perhaps your low MW peak contains a bound ligand with absorbance at 280 nm.
>The band width of some UV detectors is quite wide, wide enough to pick up
>nucleotides and other "non-280 absorbing compounds.  Try determining the
>spectra of your peak.  Good luck.
> 
>Mike Salvucci
>USDA-ARS, Lexington KY
> 
> 
Tom - 

While performing antibody domain-domain association work at Genentech, I saw
extensive quenching of interface trptophan residues upon dimerization which
led to inaccurate determinations of monomer-dimer equilibrium constants as
determined by ultracentrifugation (sed.equilibrium).  The typical assumption
in such work is the extinction coefficient of the complex is twice that of
the monomers.  This makes it easy to mathematically model the concentration
gradient  produced in the centrifugal field.  However, in this case, due to
the quencing in the interface, it was necessary to first determine how much
the extinction coefficient in the monomer dropped on association and then
model that into the fit.  This difference affected our results dramatically.

Out of curiosity, are you attempting sed. equil. or ITC on your protein to
determine K2?  Since Ernesto is in your backyard, I'd be surprised if you
weren't at least thinking about it.

- Jeff
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Jeff R. Livingstone, Ph.D.                 Net:    livingst @ ncifcrf.gov
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