<html dir="ltr">
<head>
<meta http-equiv="Content-Type" content="text/html; charset=Windows-1252">
<style>@font-face {
font-family: Cambria Math;
}
@font-face {
font-family: Calibri;
}
@font-face {
font-family: Consolas;
}
@page WordSection1 {margin: 1.0in 1.0in 1.0in 1.0in; }
P.MsoNormal {
MARGIN: 0in 0in 0pt; FONT-FAMILY: "Calibri","sans-serif"; FONT-SIZE: 11pt
}
LI.MsoNormal {
MARGIN: 0in 0in 0pt; FONT-FAMILY: "Calibri","sans-serif"; FONT-SIZE: 11pt
}
DIV.MsoNormal {
MARGIN: 0in 0in 0pt; FONT-FAMILY: "Calibri","sans-serif"; FONT-SIZE: 11pt
}
A:link {
COLOR: blue; TEXT-DECORATION: underline
}
SPAN.MsoHyperlink {
COLOR: blue; TEXT-DECORATION: underline
}
A:visited {
COLOR: purple; TEXT-DECORATION: underline
}
SPAN.MsoHyperlinkFollowed {
COLOR: purple; TEXT-DECORATION: underline
}
SPAN.EmailStyle17 {
FONT-FAMILY: "Calibri","sans-serif"; COLOR: windowtext
}
</style><style id="owaParaStyle">P {
MARGIN-TOP: 0px; MARGIN-BOTTOM: 0px
}
</style>
</head>
<body lang="EN-US" link="blue" vlink="purple" fPStyle="1" ocsi="0">
<div style="direction: ltr;font-family: Tahoma;color: #000000;font-size: 10pt;">
<p>Peter</p>
<p> </p>
<p>Just did exactly the same measurement for a small protein (54 kD<a></a> dimer<a></a>). 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<a></a> scan may also be influenced
by scattering at lower wavelength, so the trend you see is higher near the base I presume.</p>
<p> </p>
<p>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<a></a> goes as the relative error in K (deltaK<a></a>*dLnK<a></a>/dK<a></a> )times RT<a></a>, so 50% errors in
K are 0.5*RT<a></a> or ~.3 kcal/mol errors in deltaG. </p>
<div>
<p> </p>
<div><font size="2" face="Tahoma">
<div>-------------------------------------------------------------------</div>
<div><font face="tahoma">Please note my email has changed to <a href="mailto:jcorreia@umc.edu">
jcorreia@umc.edu</a><a></a></font></div>
<div><font face="tahoma"></font> </div>
<div>Dr. John J. "Jack" Correia<a></a><br>
Department of Biochemistry<br>
University of Mississippi Medical Center<br>
2500 North State Street<br>
Jackson, MS 39216<br>
(601) 984-1522 <br>
fax (601) 984-1501 <br>
email address: <a href="mailto:jcorreia@umc.edu">jcorreia@umc.edu</a><a></a> <br>
homepage location: <a href="http://biochemistry.umc.edu/correia.html">http://biochemistry.umc.edu/correia.html</a><br>
dept homepage location: <a href="http://biochemistry.umc.edu/">http://biochemistry.umc.edu/</a><br>
-------------------------------------------------------------------<br>
<br>
</div>
</font></div>
</div>
<div style="FONT-FAMILY: Times New Roman; COLOR: #000000; FONT-SIZE: 16px">
<hr tabindex="-1">
<div style="DIRECTION: ltr" id="divRpF466773"><font color="#000000" size="2" face="Tahoma"><b>From:</b> rasmb-bounces@rasmb.bbri.org [rasmb-bounces@rasmb.bbri.org] on behalf of Peter Edward Prevelige Jr [prevelig@uab.edu]<br>
<b>Sent:</b> Tuesday, January 31, 2012 10:32 AM<br>
<b>To:</b> rasmb@rasmb.bbri.org<br>
<b>Subject:</b> [RASMB] Absorbance Scan Question<br>
</font><br>
</div>
<div></div>
<div>
<div class="WordSection1">
<p class="MsoNormal">Hi All –</p>
<p class="MsoNormal"> </p>
<p class="MsoNormal">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.
</p>
<p class="MsoNormal"> </p>
<p class="MsoNormal">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).
</p>
<p class="MsoNormal"> </p>
<p class="MsoNormal">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.</p>
<p class="MsoNormal"> </p>
<p class="MsoNormal">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. </p>
<p class="MsoNormal"> </p>
<p class="MsoNormal">Thanks</p>
<p class="MsoNormal"> </p>
<p class="MsoNormal">Peter</p>
<p class="MsoNormal"> </p>
<p class="MsoNormal"><span style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">Peter E. Prevelige Jr.</span></p>
<p class="MsoNormal"><span style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">Professor</span></p>
<p class="MsoNormal"><span style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">Dept. of Microbiology, BBRB 416/6</span></p>
<p class="MsoNormal"><span style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">Univ. of Alabama @ Birmingham</span></p>
<p class="MsoNormal"><span style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">845 19th St. South</span></p>
<p class="MsoNormal"><span style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">Birmingham AL. 35294-2170</span></p>
<p class="MsoNormal"><span style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">Phone 205 975-5327</span></p>
<p class="MsoNormal"><span style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">FAX 205 975-5479</span></p>
<p class="MsoNormal"><span style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt"><a href="mailto:prevelig@uab.edu" target="_blank">prevelig@uab.edu</a></span></p>
<p class="MsoNormal"><span style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt"><a href="http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm" target="_blank">http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm</a></span></p>
<p class="MsoNormal"> </p>
</div>
</div>
</div>
</div>
<br clear=all> Individuals who have received this information in error or are not authorized to receive it must promptly return or dispose of the information and notify the sender. Those individuals are hereby notified that they are strictly prohibited from reviewing, forwarding, printing, copying, distributing or using this information in any way.
</body>
</html>