From: Jo Butler <pjgb@mrc-lmb.cam.ac.uk>
  To  : Chin, Christopher <cchin@utmb.edu>
  Date: Wed, 13 Sep 2000 11:51:23 +0100

Re: RNA vbar

Dear Christopher,

My question was really semi-rhetorical - to raise the point in your mind. 
I know perfectly well what I would prefer to do myself and that is to 
measure the apparent partial specific volume via the density increment.  We

have, and use, a Paar density meter here.  While I have never needed an 
accurate v-bar of RNA (or DNA for that matter) and have got away with 
assumed values when I have wanted an approximate one, this is certainly how

I would proceed should I need a good value.
We do routinely use a Paar instrument to measure apparent partial specific 
volumes (phi') and these have included detergent-solubilised membrane 
proteins etc.
Incidentally, I would be particularly dubious of values calculated, whether

with SEDNTERP or otherwise, for nucleic acids, unless I could be certain 
that they would allow for the counter-ion binding.  Clearly polyanions, 
such as nucleic acids, are going to be special cases where the solvation 
will be particularly important.

With specific reference to your comment about glycoproteins, we have 
published work on the Limulus CRP (Tennent et al., 1993, Molecular 
characterization of Limulus polyphemus C-reactive protein.  I. Subunit 
composition. Eur. J. Biochem. 214, 91-97) where we found phi' =
0.722±0.003 
ml/g.  The concentration was measured by refractometry and so is the total 
glycoprotein, not merely the amino acid component, so (Casassa & Eisenberg,

1964, Thermodynamic analysis of multicomponent solutions. Adv. Prot. Chem. 
19, 287-395) the molecular mass will be that of the whole glycoprotein.

Yours,

Jo Butler


--On Tuesday, September 12, 2000 10:21 am -0500 "Chin, Christopher" 
<cchin@utmb.edu> wrote:

> Jonathan,
>
> In reference to your previous question about RNA v-bar (4/6/00), I have
> not 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=c0*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
> -------------------------------------
>



P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 2QH, UK.
Tel. +44 (0)1223 402296