Index:
[thread]
[date]
[subject]
[author]
From: Arthur Rowe <arthur.rowe@nottingham.ac.uk>
To : bgyjpaw@leeds.ac.uk
Date: Thu, 14 Sep 2000 12:12:48 +0000
Re: RNA vbar
> THIS MESSAGE IS IN MIME FORMAT. Since your mail reader does not understand
this format, some or all of this message may not be legible.
--MS_Mac_OE_3051778368_512312_MIME_Part
Content-type: text/plain; charset="ISO-8859-1"
Content-transfer-encoding: quoted-printable
Jonathan -
Jo Butler is absolutely right - the nature of the counter-ion can make a
very large difference to vbar values for nucleic acids. I have seen values
reported ranging from 0.46 to 0.58 ml/g, and that sort of uncertainly cause=
s
real problems in doing sedimentation equilibrium work.
Just to give a few examples, taken from Pearce, Rowe & Turnock (J Mol Biol
97 193-205, 1975):
16S Ribosomal RNA
Sodium salt 0.524 =B1 0.008 0.521 =B1 0.007 ml/g (independent
determinations)
Potassium salt 0.580 =B1 0.004 ml/g
Homopolynucleotides (not stated, but Na salt as I recall)
poly(U) 0.527 =B1 0.015 ml/g
poly(A) 0.531 =B1 0.003
poly(U).poly(A) 0.529 =B1 0.002
I do not think there is enough data around to be able to draw the conclusio=
n
(which would be nice to believe) that RNA (and perhaps nucleic acid in
general ?) has a vbar which is a function of the solvent rather than of the
base composition. The figures above provide some very minimal basis for
such a supposition.
If this idea holds up, one could buy in any RNA of known molecular mass, do
an equilibrium run in your local solvent, then back-calculate the vbar valu=
e
needed to return the known mass, as Christopher Chin suggests.
Best wishes
Arthur Rowe
*****************************************************
Arthur J Rowe
Professor of Biomolecular Technology
University of Nottingham
School of Biological Sciences
Sutton Bonington
Leicestershire LE12 5RD UK
Phone/voicemail +44 (0)115 951 6156
Phone/fax +44 (0)115 951 6156/7
email arthur.rowe@nottingham.ac.uk
arthur.rowe@connectfree.co.uk
Web http://www.nottingham.ac.uk/ncmh/business
*****************************************************
----------
>From: "Chin, Christopher" <cchin@utmb.edu>
>To: "'bgyjpaw@leeds.ac.uk'" <bgyjpaw@leeds.ac.uk>
>Subject: RNA vbar
>Date: Tue, Sep 12, 2000, 3:21 pm
>
>Jonathan,
>
>In reference to your previous question about RNA v-bar (4/6/00), I have no=
t
>seen any discussion in the Rasmb discussion board So I am giving my 2 cent
>worth of suggestion in the hope that the experts in this field will voice
>their opinions. We know that for protein vbar, with known amino acid
>sequence, SEDNTERP is the method of choice to obtain vbar. You can then
>input in this calculated vbar value into the edited Sedimentation
>Equilibrium curve (generated from the experiment) to get associated or
>non-associate mass of the protein molecule weight with fairly good
accuracy,
>provided your protein sample is very pure. But what happen if one works
>with glycoprotein or DNA, (in your case RNA) and cannot get vbar from
>SEDNTERP. I have done some preliminary work using a backdoor approach in
an
>attempt to get vbar from macromolecule. I don't know if this approach will
>work for small molecule like selenium or acetyl containing amino acids,
>unless one can generate a decent sedimentation equilibrium curve, which is
>the key for this approach, from AU. I call this approach "vbar via back
>calculation".
>I believe this "Back Calculation" method will yield a realistic vbar value
>for protein, glycoprotein, DNA. Perhaps it will work for RNA also(I have
not
>yet tried).
>Provided the following conditions are met:
>a. The sample is pure
>b. The SE experiment data is accurate
>c. The molecular weight is accurate
>I think this approach works well with bona fide monomer or n-mer, but I
>doubt if it works for monomer-dimer or monomer-nmer at equilibrium because
>under such condition one will have difficulty inputting a correct apparent
>MW to reflect the sedimentation equilibrium curve that was generated.
>
>If you are interested in applying this approach to get your RNA v-bar, I
>will be glad to send you the procedure describing how I use the following
>Lamm's equation to extract v-bar from the sedimentation equilibrium curve.
I
>can also share my preliminary tabulated data (v-bar overview) with anyone
>who would like to explore using this approach to obtain hard-to-get v-bar
>values.
>
>
>
>f=3Dc0*exp((w*3.1416/30)^2*m*(1-v*p)*(x^2-x0^2)/(2*R*T))
>
>
>-------------------------------------
>Christopher Chin
>Manager
>Sealy Center for Structural Biology
>HBC&G, 5136 MRB. rt1055 UTMB
>cchin@utmb.edu, 409-772-1693, efax 708-585-1920
>-------------------------------------
>
--MS_Mac_OE_3051778368_512312_MIME_Part
Content-type: text/html; charset="ISO-8859-1"
Content-transfer-encoding: quoted-printable
<HTML>
<HEAD>
<TITLE>Re: RNA vbar</TITLE>
</HEAD>
<BODY BGCOLOR=3D"#FFFFFF">
<FONT SIZE=3D"2">Jonathan -<BR>
<BR>
Jo Butler is absolutely right - the nature of the counter-ion can make a ve=
ry large difference to vbar values for nucleic acids. I have seen values rep=
orted ranging from 0.46 to 0.58 ml/g, and that sort of uncertainly causes re=
al problems in doing sedimentation equilibrium work.<BR>
<BR>
Just to give a few examples, taken from Pearce, Rowe & Turnock (J Mol B=
iol 97 193-205, 1975):<BR>
</FONT><B><BR>
16S Ribosomal RNA</B><FONT SIZE=3D"2"><BR>
<BR>
Sodium salt 0.524 =B1 0.008 &=
nbsp; 0.521 =B1 0.007 ml/g (independent determinations)<B=
R>
Potassium salt 0.580 =B1 0.004 ml/g<BR>
<BR>
</FONT><B>Homopolynucleotides</B><FONT SIZE=3D"2"> (not stated, but Na salt a=
s I recall)<BR>
<BR>
poly(U) =
; 0.527 =B1 0.015 ml/g<BR>
poly(A) =
; 0.531 =B1 0.003<BR>
poly(U).poly(A) 0.529 =B1 0.002<BR>
<BR>
I do not think there is enough data around to be able to draw the conclusio=
n (which would be nice to believe) that RNA (and perhaps nucleic acid in gen=
eral ?) has a vbar which is a function of the </FONT><U>solvent</U><FONT SIZ=
E=3D"2"> rather than of the base composition. The figures above provide some =
very minimal basis for such a supposition. <BR>
<BR>
If this idea holds up, one could buy in any RNA of known molecular mass, do=
an equilibrium run in your local solvent, then back-calculate the vbar valu=
e needed to return the known mass, as Christopher Chin suggests.<BR>
<BR>
Best wishes<BR>
<BR>
Arthur Rowe<BR>
*****************************************************<BR>
Arthur J Rowe<BR>
Professor of Biomolecular Technology<BR>
University of Nottingham<BR>
School of Biological Sciences<BR>
Sutton Bonington<BR>
Leicestershire LE12 5RD UK<BR>
<BR>
Phone/voicemail +44 (0)115 951 6156<BR>
Phone/fax +44 (0)115 951 6156/7<BR>
email arthur.rowe@nottingham.ac.uk<BR>
arthur.rowe@connectfree.co.uk<BR>
Web http://www.nottingham.ac.uk/ncmh/business<BR>
*****************************************************<BR>
<BR>
----------<BR>
>From: "Chin, Christopher" <cchin@utmb.edu><BR>
>To: "'bgyjpaw@leeds.ac.uk'" <bgyjpaw@leeds.ac.uk><BR>
>Subject: RNA vbar<BR>
>Date: Tue, Sep 12, 2000, 3:21 pm<BR>
><BR>
<BR>
>Jonathan,<BR>
><BR>
>In reference to your previous question about RNA v-bar (4/6/00), I have=
not<BR>
>seen any discussion in the Rasmb discussion board So I am giving my 2 c=
ent<BR>
>worth of suggestion in the hope that the experts in this field will voi=
ce<BR>
>their opinions. We know that for protein vbar, with known amino acid<B=
R>
>sequence, SEDNTERP is the method of choice to obtain vbar. You can then=
<BR>
>input in this calculated vbar value into the edited Sedimentation<BR>
>Equilibrium curve (generated from the experiment) to get associated or<=
BR>
>non-associate mass of the protein molecule weight with fairly good accu=
racy,<BR>
>provided your protein sample is very pure. But what happen if one work=
s<BR>
>with glycoprotein or DNA, (in your case RNA) and cannot get vbar from<B=
R>
>SEDNTERP. I have done some preliminary work using a backdoor approach =
in an<BR>
>attempt to get vbar from macromolecule. I don't know if this approach w=
ill<BR>
>work for small molecule like selenium or acetyl containing amino acids,=
<BR>
>unless one can generate a decent sedimentation equilibrium curve, which=
is<BR>
>the key for this approach, from AU. I call this approach "vbar vi=
a back<BR>
>calculation".<BR>
>I believe this "Back Calculation" method will yield a realist=
ic vbar value<BR>
>for protein, glycoprotein, DNA. Perhaps it will work for RNA also(I hav=
e not<BR>
>yet tried).<BR>
>Provided the following conditions are met:<BR>
>a. The sample is pure<BR>
>b. The SE experiment data is accurate<BR>
>c. The molecular weight is accurate<BR>
>I think this approach works well with bona fide monomer or n-mer, but I=
<BR>
>doubt if it works for monomer-dimer or monomer-nmer at equilibrium beca=
use<BR>
>under such condition one will have difficulty inputting a correct appar=
ent<BR>
>MW to reflect the sedimentation equilibrium curve that was generated.<B=
R>
><BR>
>If you are interested in applying this approach to get your RNA v-bar, =
I<BR>
>will be glad to send you the procedure describing how I use the followi=
ng<BR>
>Lamm's equation to extract v-bar from the sedimentation equilibrium cur=
ve. I<BR>
>can also share my preliminary tabulated data (v-bar overview) with anyo=
ne<BR>
>who would like to explore using this approach to obtain hard-to-get v-b=
ar<BR>
>values.<BR>
><BR>
><BR>
><BR>
>f=3Dc0*exp((w*3.1416/30)^2*m*(1-v*p)*(x^2-x0^2)/(2*R*T))<BR>
><BR>
><BR>
>-------------------------------------<BR>
>Christopher Chin<BR>
>Manager<BR>
>Sealy Center for Structural Biology<BR>
>HBC&G, 5136 MRB. rt1055 UTMB<BR>
>cchin@utmb.edu, 409-772-1693, efax 708-585-1920<BR>
>-------------------------------------<BR>
><BR>
<BR>
<BR>
</FONT>
</BODY>
</HTML>
--MS_Mac_OE_3051778368_512312_MIME_Part--
Index:
[thread]
[date]
[subject]
[author]