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<DIV><FONT face=Arial size=2>Dear Colleauges,</FONT></DIV>
<DIV><FONT face=Arial size=2>I wanted to leave the discussion (see Walter
Staffords last e-mail 7 June 08) for the Newcastle AUC Symposium in
September 08, but it developed already now, so I shall
give here the abstract of the my </FONT></DIV>
<DIV><FONT face=Arial size=2>contribution about this theme
and a new approach to solve it.:</FONT></DIV>
<DIV><FONT face=Arial size=2></FONT> </DIV>
<DIV>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">The use of an elder mode to
determine the density of vesicles by S</SPAN></B><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-FAMILY: 'WP Greek Courier'; mso-ansi-language: EN-GB; mso-ascii-font-family: Arial; mso-hansi-font-family: Arial; mso-bidi-font-family: Arial; mso-char-type: symbol; mso-symbol-font-family: 'WP Greek Courier'"><SPAN
style="mso-char-type: symbol; mso-symbol-font-family: 'WP Greek Courier'">0</SPAN></SPAN></B><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB"> versus ρ is a good
approach that can be used also to determine protein/RNA or protein/DNA
complexes</SPAN></B><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">. <?xml:namespace prefix =
o ns = "urn:schemas-microsoft-com:office:office" /><o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">First of all we have to
bear in our mind that the molecular masses achieved from centrifugation, based
on the buoyant term (the partial specific volume,<B
style="mso-bidi-font-weight: normal"> V </B>the particle), here in this
discussed case <B style="mso-bidi-font-weight: normal">V</B></SPAN><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-SIZE: 8pt; FONT-FAMILY: Arial; mso-ansi-language: EN-GB">pot.DNA</SPAN></B><SPAN
lang=EN-GB style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB"> or <B
style="mso-bidi-font-weight: normal">V</B></SPAN><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-SIZE: 8pt; FONT-FAMILY: Arial; mso-ansi-language: EN-GB">prot.RNA</SPAN></B><SPAN
lang=EN-GB style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">. Usually we
invest a big effort on sophisticated methods to calculate molecular weights <B
style="mso-bidi-font-weight: normal">Mw</B>, but the lack of precise <B
style="mso-bidi-font-weight: normal">V </B>values is like a weak link of a
chain. In such mentioned case we are able to calculate<SPAN
style="mso-spacerun: yes"> </SPAN>the <B
style="mso-bidi-font-weight: normal">V</B> from the protein amino acids and
nucleic acids but the exact ratio of both components is usually unknown.
<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">The classical density
determination by pycnometry even with digital precise instruments needs the
knowledge of the total particle concentration of the solution that can be only
estimated.<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">The use of S.J. Edelstein
and<SPAN style="mso-spacerun: yes"> </SPAN>H.K.Schachman’s mode, (JBC 196,
vol.247 , 306-317) to make<SPAN style="mso-spacerun: yes">
</SPAN>parallel<SPAN style="mso-spacerun: yes"> </SPAN>runs<SPAN
style="mso-spacerun: yes"> </SPAN>in <B
style="mso-bidi-font-weight: normal">H</B></SPAN><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-SIZE: 9pt; FONT-FAMILY: Arial; mso-ansi-language: EN-GB">2</SPAN></B><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">O</SPAN></B><SPAN
lang=EN-GB style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB"> and <B
style="mso-bidi-font-weight: normal">D</B></SPAN><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-SIZE: 9pt; FONT-FAMILY: Arial; mso-ansi-language: EN-GB">2</SPAN></B><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">O </SPAN></B><SPAN
lang=EN-GB style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">buffer as
mentioned also in the<SPAN style="mso-spacerun: yes"> </SPAN>RSC
published<SPAN style="mso-spacerun: yes"> </SPAN>book >> Analytical
Ultracentrifugation, Techniques and<SPAN style="mso-spacerun: yes">
</SPAN>Methods<< page 197, edited by<SPAN style="mso-spacerun: yes">
</SPAN>D. Scott et al, can be employed only if the samples are pure,
monodispersed and<SPAN style="mso-spacerun: yes"> </SPAN>reveal<SPAN
style="mso-spacerun: yes"> </SPAN>with sedimentation equilibrium SE
straight<SPAN style="mso-spacerun: yes"> </SPAN>slopes and single Mw’s.
The reality<SPAN style="mso-spacerun: yes"> </SPAN>in case of<SPAN
style="mso-spacerun: yes"> </SPAN>biological<SPAN
style="mso-spacerun: yes"> </SPAN>samples is, that<SPAN
style="mso-spacerun: yes"> </SPAN>the slopes<SPAN
style="mso-spacerun: yes"> </SPAN>tends<SPAN
style="mso-spacerun: yes"> </SPAN>to self-association , nonideality or
both effects and even<SPAN style="mso-spacerun: yes"> </SPAN>show partly
aggregation, so that<SPAN style="mso-spacerun: yes"> </SPAN>the deviated
lines from them<SPAN style="mso-spacerun: yes"> </SPAN>the V is<SPAN
style="mso-spacerun: yes"> </SPAN>calculated<SPAN
style="mso-spacerun: yes"> </SPAN>are not single and<SPAN
style="mso-spacerun: yes"> </SPAN>straight.<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">Using the SEGAL computer
program when the slopes of 2 parts are calculated, it is <FONT
color=#000000>easy to realize this discrepancy.<o:p></o:p></FONT></SPAN></P>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">Using the <B
style="mso-bidi-font-weight: normal">S</B></SPAN><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-FAMILY: 'WP Greek Courier'; mso-ansi-language: EN-GB; mso-ascii-font-family: Arial; mso-hansi-font-family: Arial; mso-bidi-font-family: Arial; mso-char-type: symbol; mso-symbol-font-family: 'WP Greek Courier'"><SPAN
style="mso-char-type: symbol; mso-symbol-font-family: 'WP Greek Courier'">0</SPAN></SPAN></B><SPAN
lang=EN-GB style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB"> </SPAN><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-SIZE: 9pt; FONT-FAMILY: Arial; mso-ansi-language: EN-GB">relative</SPAN></B><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB"> </SPAN></B><SPAN
lang=EN-GB style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">versus<B
style="mso-bidi-font-weight: normal"> ρ </B>mode<B
style="mso-bidi-font-weight: normal"> </B>developed by C.H: Huang and J Charlton
(JBC <o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">1971 Vol. 216) that was
used to study the density of vesicles, it is possible to apply it also for high
molecular complexes. The advantage in this case is, that even the sample
sediments with a boundary that contains<SPAN style="mso-spacerun: yes">
</SPAN>partly<SPAN style="mso-spacerun: yes"> </SPAN>a<SPAN
style="mso-spacerun: yes"> </SPAN>faster<SPAN
style="mso-spacerun: yes"> </SPAN>or a slower component (as it<SPAN
style="mso-spacerun: yes"> </SPAN>happens often with biological samples)
it can be used<SPAN style="mso-spacerun: yes"> </SPAN>to achieve <B
style="mso-bidi-font-weight: normal">S°</B></SPAN><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-FAMILY: 'WP Greek Courier'; mso-ansi-language: EN-GB; mso-ascii-font-family: Arial; mso-hansi-font-family: Arial; mso-bidi-font-family: Arial; mso-char-type: symbol; mso-symbol-font-family: 'WP Greek Courier'"><SPAN
style="mso-char-type: symbol; mso-symbol-font-family: 'WP Greek Courier'">0</SPAN></SPAN></B><SPAN
lang=EN-GB style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB"> <B
style="mso-bidi-font-weight: normal">(</B>at a D</SPAN><SPAN lang=EN-GB
style="FONT-SIZE: 9pt; FONT-FAMILY: Arial; mso-ansi-language: EN-GB">2</SPAN><SPAN
lang=EN-GB style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">O –H</SPAN><SPAN
lang=EN-GB
style="FONT-SIZE: 9pt; FONT-FAMILY: Arial; mso-ansi-language: EN-GB">2</SPAN><SPAN
lang=EN-GB style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">O buffer density
where S’ comes<SPAN style="mso-spacerun: yes"> at rest</SPAN>) that
represents the <B style="mso-bidi-font-weight: normal">V</B> of<SPAN
style="mso-spacerun: yes"> </SPAN>the particle. Important is to use the
whole series of <B style="mso-bidi-font-weight: normal">S</B></SPAN><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-FAMILY: 'WP Greek Courier'; mso-ansi-language: EN-GB; mso-ascii-font-family: Arial; mso-hansi-font-family: Arial; mso-bidi-font-family: Arial; mso-char-type: symbol; mso-symbol-font-family: 'WP Greek Courier'"><SPAN
style="mso-char-type: symbol; mso-symbol-font-family: 'WP Greek Courier'">0</SPAN></SPAN></B><SPAN
lang=EN-GB style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB"> </SPAN><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-SIZE: 9pt; FONT-FAMILY: Arial; mso-ansi-language: EN-GB">relative
</SPAN></B><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">at the same particle
concentration to avoid S’ differences that stem from concentration dependence of
S’.<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">All our measurements were
done with absorption optics using the<SPAN style="mso-spacerun: yes">
</SPAN>XLA at the same<SPAN style="mso-spacerun: yes">
</SPAN>wavelength <B style="mso-bidi-font-weight: normal">WL</B> since the used
samples do not show a distinct<SPAN style="mso-spacerun: yes"> </SPAN>WL
maximum at 260 nm or 280 nm.</SPAN></P>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB">...............yours......ariel</SPAN></P>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB"></SPAN> </P>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB"></SPAN> </P>
<P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><B
style="mso-bidi-font-weight: normal"><SPAN lang=EN-GB
style="FONT-FAMILY: Arial; mso-ansi-language: EN-GB"><o:p> </o:p></SPAN></B></P>
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align=center><SPAN lang=EN-GB
style="FONT-SIZE: 9pt; FONT-FAMILY: Arial; mso-ansi-language: EN-GB"><o:p> </o:p></SPAN></P></DIV></BODY></HTML>