From: Jack Lebowitz <Jack_Lebowitz@micro.microbio.uab.edu>
  To  : Arturo J. Morales <art@scripps.edu>
  Date: Tue, 5 Jan 1999 11:26:26 -0600 

RE: Interesting problem...

We have faced a similiar problem with a protein-DNA interaction and decided
to use the Edelstein-Schachman (Methods of Enzymol. vol.27) approach of
simultaneously measuring v bar and molecular weight in an equilibrium
experiment with the nucleic acid-protein complex in aqueous buffer and in
D2O buffer. With absorbance measurements in the UV the tRNA absorance should
overwhelm the protein absorbance at 260nm for your Kd and you should be able
to obtain the exponential distrubtion for a fully complexed tRNA following
the tRNA signal. We do a global analysis at 3 speeds to get the best reduced
molecular weight in H2O and D2O and then solve for v bar and then molecular
weight.  We assume full deuteration of the protein and use the deuteration
constant given by E-S.  When I was on sabbatical at NIH in Marc Lewis' lab I
measured the density of 99.9% D2O to be 1.105455. If you have a Anton Parr
density meter you can measure the density of D2Obuffer and the H2O buffers
or you can increment the density changes using the Sednterp values. The
above appears to have worked for us. If you have any questions let me know.

Jack Lebowitz
Dept. of Microbiology
520 CHSB
Jack Lebowitz
University of Alabama at Birmingham
Birmingham AL 36294-2041
(205) 934-9475
FAX (205) 975-4621

> -----Original Message-----
> From:	Arturo J. Morales [SMTP:art@scripps.edu]
> Sent:	Monday, January 04, 1999 6:36 PM
> To:	rasmb@alpha.bbri.org
> Subject:	Interesting problem...
> 
> 
> Hello all,
> 
> I have an interesting problem and I hope that anyone out there with more
> expertise than me in AU can provide some insight... this list has been
> great so far at helping me learn how to use the XL-I mostly on my own
> (since I wasnt the one going to the Beckman training in our lab :)
> 
> Ok, here's the problem:
> 
> I have a protein-RNA interaction that I'm trying to quantify... I know it
> binds... Kd in the low nm range...
> 
> If I do a velocity run on the RNA, I get about 3S which assuming vbar of
> .53 gives me MW of ~25Kd (just about right for tRNA)... so far so good... 
> 
> If I do the same on the protein, I get about 2S, which is about 24Kd using
> a vbar of .7595 (calculated from composition).  that works well too...
> since it is expected to be a dimer (12kd monomers)
> 
> The problem arises when I mix them both... I end up getting about
> 2.5S-2.8S
> (or so, I need to refine that part).  I know this is happening because now
> the vbar for the complex is somewhere in between .53 and .75, of course,
> depending what vbar I use, I can get any MW I want... which is not really
> that useful... My main question is, how can I approach this problem?  I
> would like to get stoichoimetry (which I believe is 2:1 monomer:rna, but
> it
> could be 2:2...)  Is it acceptable to show the shift in S without
> estimating MW? Can I determine vbar directly for a complex? what is the
> best way?
> 
> I would appreciate any comments 
> 
> THANKS!
> 
> Art
> 
> ----------------------------------------------------------------------
>      Arturo J. Morales    (RPI '94)             art@scripps.edu
>    Department of Biology - Massachusetts Institute of Technology &
>   Skaggs Institute for Chemical Biology - Scripps Research Institute
>                    http://schimmel.scripps.edu/~art