<HTML><HEAD>
<META http-equiv=Content-Type content="text/html; charset=iso-8859-1">
<META content="MSHTML 6.00.2900.3086" name=GENERATOR></HEAD>
<BODY style="MARGIN: 4px 4px 1px; FONT: 10pt Tahoma">
<DIV><FONT face=Arial size=3>Mitra</FONT></DIV>
<DIV><FONT face=Arial size=3></FONT> </DIV>
<DIV><FONT face=Arial size=3>Detection of aggregation or heterogeneity was to my knowledge 1st dealt with quantitatively in a book chapter: </FONT></DIV>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt"><FONT size=3><SPAN style="FONT-SIZE: 11pt; FONT-FAMILY: Arial; mso-bidi-font-size: 10.0pt; mso-bidi-font-family: 'Times New Roman'">DA Yphantis, J.J. CORREIA, M.L. Johnson and G.-M. Wu (1978).<SPAN style="mso-spacerun: yes"> </SPAN>"Detection of<SPAN style="mso-spacerun: yes"> </SPAN>Heterogeneity in Self-Associating Systems," in "Physical Aspects of Protein Interactions," </SPAN><?xml:namespace prefix = st1 ns = "urn:schemas-microsoft-com:office:smarttags" /><st1:place><SPAN style="FONT-SIZE: 11pt; FONT-FAMILY: Arial; mso-bidi-font-size: 10.0pt; mso-bidi-font-family: 'Times New Roman'">N. Catsimpoolas</SPAN></st1:place><SPAN style="FONT-SIZE: 11pt; FONT-FAMILY: Arial; mso-bidi-font-size: 10.0pt; mso-bidi-font-family: 'Times New Roman'">, ed., Elsevier, pp. 275-303.<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" /><o:p></o:p></SPAN></FONT></P>
<DIV><FONT face=Arial size=3></FONT> </DIV>
<DIV><FONT face=Arial size=3>Recently Yujia Xu published a graphical method for indicating the presence of heterogeneity and extracting K's from Kapp values. <SPAN class=bf>Characterization of macromolecular heterogeneity by equilibrium sedimentation techniques, Biophys Chem 108, </SPAN><I>141-163, 2004.</I></FONT></DIV>
<DIV><FONT face=Arial size=3></FONT> </DIV>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 11pt; FONT-FAMILY: Arial; mso-bidi-font-family: 'Times New Roman'">In practice it has been noted for sometime now that fitting individual loading concentrations will give a trend of increasing Kapp with decreasing concentration - this applies to equilibrium data, a feature implemented in earlier versions of Nonlin, and more recently to velocity data. see: J. J. CORREIA, C. A. Sontag, W. F. Stafford and P. J. Sherwood, (2005) “Models for Direct Boundary Fitting of Indefinite Ligand-Linked Self-Association,” <SPAN style="mso-spacerun: yes"> </SPAN>in <B style="mso-bidi-font-weight: normal">Analytical Ultracentrifugation: Techniques and Methods.</B> (Ed. D. Scott, S. Harding and A. Rowe) pp 51-63.</SPAN></P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 11pt; FONT-FAMILY: Arial; mso-bidi-font-family: 'Times New Roman'"></SPAN> </P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 11pt; FONT-FAMILY: Arial; mso-bidi-font-family: 'Times New Roman'">As David said, when the aggregates pellet there is no problem, just use the species in equilibrium at the plateau concentrations. The problem arises when the heterogeneity is aggregated dimer or inactive monomer. Careful analysis and direct boundary fitting can reveal these features while also extracting the reversible component of the reactions. Aggregation during the run is more difficult and favors velocity methods or changing conditions like temperature, pH, salt or reducing agents to induce stability.</SPAN></P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 11pt; FONT-FAMILY: Arial; mso-bidi-font-family: 'Times New Roman'"></SPAN> </P>
<DIV> </DIV>
<DIV> </DIV>
<DIV>-------------------------------------------------------------------<BR> Dr. John J. "Jack" Correia<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@biochem.umsmed.edu">jcorreia@biochem.umsmed.edu</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> <BR><BR><BR>>>> <mitrana@mail.utexas.edu> 05/29/07 3:04 PM >>><BR></DIV>
<DIV style="COLOR: #000000">Hi Folks<BR>I have a question (s) - how does the aggregation (and maybe the subsequent loss)<BR>of a protein sample during SV affect the Kd of the monomer-dimer equilibrium?<BR>Either by direct fitting of the boundary or considering the average<BR>sedimentation coefficient. Is there any way around this?<BR><BR>Mitra<BR>-- <BR>Mitra S. Rana<BR>Graduate Student<BR>Institute for Cellular and Molecular Biology<BR>2500 Speedway, UT-Austin<BR>Austin, TX-78712<BR>_______________________________________________<BR>RASMB mailing list<BR>RASMB@rasmb.bbri.org<BR><A href="http://rasmb.bbri.org/mailman/listinfo/rasmb">http://rasmb.bbri.org/mailman/listinfo/rasmb</A><BR></DIV></BODY></HTML>