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<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Hello, Rasmb</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>I have a question about viscosity corrections for s20,w due
to varying concentrations of protein.</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>We are using sedimentation velocity to determine the binding
constant for association of an 18.1 kDa protein with a 224kDa protein. The
concentration of the larger protein is maintained constant at 0.3 mg/mL (about
5 uM subunits for the tetrameric protein) and the concentration of the smaller
protein is varied from 0-1 mg/mL (about 50 uM). The binding stoichiometry
is known to be 1 small protein per subunit of the larger protein (4 per
tetramer). The dissociation constant is known from independent studies to
be about 5 uM.</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>We run sedimentation velocity experiments at 28,500 rpm,
using absorbance optics at 280 nm, standard aluminum double sector cells.
We have the great fortune that the smaller protein is almost entirely
transparent at 280 nm.</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>At this juncture, we analyze the data by using SVEDBERG to
obtain a single value for s20,w. We see the s20,w increase from about 11S
to about 12+S as the concentration of the smaller protein is increased 0-1
mg/mL. This increase is about what we expect. We used the same
corrections for s20,w at all concentrations of the smaller protein, although
the solution viscosity should increase with increasing concentrations of the
smaller protein. Thus, we are underestimating the s20,w at the higher
concentrations of the smaller protein. However, I have no feel for the extent
of the error.</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>We are sort of following the paper: Dam et al (2005) Biophys
J 89:619-634 from Peter Schuck, although we have not yet gotten to the elegant
analysis part. I am not sufficiently familiar with the computational
methods that are described in the paper to see whether a correction for
viscosity is there.</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>We can determine empirically the viscosity effect on
sedimentation of the larger protein by using mutants of it that we know do not
bind the smaller protein. However, I wonder whether this is sufficient or
are there better ways to correct for the viscosity differences? If so,
what are they? Does anyone on the listserv know about this issue?</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Thanks for your consideration of this lengthy email.</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoAutoSig><font size=3 face=Arial><span style='font-size:12.0pt;
font-family:Arial'>Dr. Donald W. Pettigrew</span></font></p>
<p class=MsoAutoSig><font size=3 face=Arial><span style='font-size:12.0pt;
font-family:Arial'>Department of Biochemistry & Biophysics</span></font></p>
<p class=MsoAutoSig><font size=3 face=Arial><span style='font-size:12.0pt;
font-family:Arial'>Texas</span></font><font face=Arial><span
style='font-family:Arial'> </span></font><font face=Arial><span
style='font-family:Arial'>A&M</span></font><font face=Arial><span
style='font-family:Arial'> </span></font><font face=Arial><span
style='font-family:Arial'>University</span></font></p>
<p class=MsoAutoSig><font size=3 face=Arial><span style='font-size:12.0pt;
font-family:Arial'>College Station</span></font><font face=Arial><span
style='font-family:Arial'>, </span></font><font face=Arial><span
style='font-family:Arial'>TX</span></font><font face=Arial><span
style='font-family:Arial'> </span></font><font face=Arial><span
style='font-family:Arial'>77843-2128</span></font></p>
<p class=MsoAutoSig><font size=3 face=Arial><span style='font-size:12.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoAutoSig><font size=3 face=Arial><span style='font-size:12.0pt;
font-family:Arial'>phone: 979-845-9621</span></font></p>
<p class=MsoAutoSig><font size=3 face=Arial><span style='font-size:12.0pt;
font-family:Arial'>fax: 979-845-9274</span></font></p>
<p class=MsoAutoSig><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'> </span></font></p>
<p class=MsoAutoSig><font size=3 face=Arial><span style='font-size:12.0pt;
font-family:Arial'>"Nearly all men can stand adversity, but if you
want to test a man's character, give him power"</span></font></p>
<p class=MsoAutoSig><font size=3 face=Arial><span style='font-size:12.0pt;
font-family:Arial'>A. Lincoln</span></font></p>
<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'> </span></font></p>
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