[RASMB] Absorbance Scan Question

[John J. Correia]

Peter



Just did exactly the same measurement for a small protein (54 kD dimer).  In this case we compared area under a g(s) as well so it was the full signal from the same samples. This approach and comparing an equil scan may also be influenced by scattering at lower wavelength, so the trend you see is higher near the base I presume.



Another factor to consider is propagation of the error in ext into a free energy scale (if its self-associating).  The absolute error in deltaG goes as the relative error in K (deltaK*dLnK/dK )times RT, so 50% errors in K are 0.5*RT or ~.3 kcal/mol errors in deltaG.



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Please note my email has changed to jcorreia at umc.edu<mailto:jcorreia at umc.edu>

Dr. John J. "Jack" Correia
Department of Biochemistry
University of Mississippi Medical Center
2500 North State Street
Jackson, MS  39216
(601) 984-1522
fax (601) 984-1501
email address: jcorreia at umc.edu<mailto:jcorreia at umc.edu>
homepage location: http://biochemistry.umc.edu/correia.html
dept homepage location:    http://biochemistry.umc.edu/
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________________________________
From: rasmb-bounces at rasmb.bbri.org [rasmb-bounces at rasmb.bbri.org] on behalf of Peter Edward Prevelige Jr [prevelig at uab.edu]
Sent: Tuesday, January 31, 2012 10:32 AM
To: rasmb at rasmb.bbri.org
Subject: [RASMB] Absorbance Scan Question

Hi All –

I’m doing an equilibrium run on a  ~3 KDa synthetic peptide lacking aromatics in 20% TFE. I  thought I might use multiple wavelengths to extend the concentration range that I could follow.

I thought I’d be clever and obtain the ratio of extinction coefficients by dividing the absorbance at one wavelength (236 nm) by the other  (240 nm) across the cell (rather than using the spectrum).

I calculated the ratio using only the points that were at the same radial distance and plotted them vs radial distance. It is pretty apparent that the ratio is changing across the cell.

My question is whether this is likely to be an optical artifact or whether it most likely represent heterogeneity in the sample. It is probably worth noting that the ratio from the spectrum is 1.43, intermediate between the extremes seen across the cell which to me suggests heterogeneity.

Thanks

Peter

Peter E. Prevelige Jr.
Professor
Dept. of Microbiology, BBRB 416/6
Univ. of Alabama @ Birmingham
845 19th St. South
Birmingham AL. 35294-2170
Phone 205 975-5327
FAX 205 975-5479
prevelig at uab.edu<mailto:prevelig at uab.edu>
http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm



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