[RASMB] Re: vbar of DM +OG

[john.e.harlan at abbott.com]

It has been a while since I did these measurements, but if I recall 
correctly, the literature I was following suggested that the relavent vbar 
was that of the detergent above the CMC.  I am pretty sure that all the 
measurements that I did were above the CMC.  As you suggest Fumio, the 
concentrations were all from dry weight into solution (gm/mL).  It is 
likely I made a more concentrated stock (10gm/100mL?) and diluted from 
there for the measurements.  Not only did the graphs from the density vs. 
concentration measurements give a straight line, they were quite flat. 
While the graphs were flat, I did use a vbar value extrapolated to the CMC 
for each detergent I did this for.

You could probably get the concentration from the number of fringes, but 
to do this I think you would need to know the refractive index increment 
for the detergents in micelles.  I don't have a good idea on how to get 
this value.

John




Fumio Arisaka <farisaka at bio.titech.ac.jp>
Sent by: rasmb-admin at rasmb-email.bbri.org
11/24/2003 01:42 AM

 
        To:     rasmb at rasmb-email.bbri.org
        cc: 
        Subject:        [RASMB] Re: vbar of DM +OG



Dear John and Ariel,

Thank you very much for the information from both of you.

Actually, I have a question.  Ariel, why are the values obtained by Anton 
Paar density
meter designated as "not hydrated"?  It may be an elementary question, but 
both states in 
the density meter and ultracentrifuge cell are hydrated, arn't they? 

Another question to both of you is that as Ariel pointed out, the micelle 
formation
of OG and DM may make the measurement difficult.  In your measurement, as 
the
concentration of the solute increases, the fraction of OG or DM in the 
micelle would 
increase with respect to the free ones.  Does the graph of rho vs. c still 
give a straight line?
(Actually, I do not know the cmc of these reagents and the range of 
concentration
that you used.)  We have recently purchased an Anton Paar DMA5000.  Could 
you give
any advice concerning precaution of the measurement if we measure it 
ourselves? 
I suppose the concentrations are estimated from the dry weight of OG or DM 
or
can you get the concentration from the number of fringe?
 
Thank you in advance.  Fumio

--------------------------------------------------------------------
Dear Fumio and dear colleagues,
the Vbar of  OG is 0.867 not hydrated  and hydrated  mesured in sucrose or 
similar
densifiers  0.924 (mesured in the model E)
That of DM is 0.813 (measured in the A. Paar desity-meter) and the 
hydrated
one is 0.885 (measured in  model E).In my next  ? paper  will be the 
reference?

OG reference under Nー15 of Lustig A, Engel A, Tsiotis G, Landau EM, 
Baschong W.
" Molecular weight
determination of membrane proteins by sedimentation equilibrium at the
sucrose or nycodenz-adjusted density of the hydrated detergent micelle."
Biochim Biophys Acta. 2000 Apr 5;1464(2):199-206.

Yours ....ariel

--------------------------------------------------------------------
Fumio- 

Partial specific volume of octylglucoside is 0.859 (Reynolds & McCaslin 
(1985), Methods in Enzymology, 117:41-53) 

Partial specific volume of decylmaltoside is 0.795.  I determined this for 
my thesis work using density measurments at several detergent 
concentrations using a Mettler/Paar density meter (DMA 01 C). 

Regards, 

John Harlan 
---------------------------------------------------------------------


At 15:10 03/11/22 +0100, Lustig wrote:

Dear John Harlan , Fumio  and colleagues,
I'm not astonished  that  you =John report on a bit different  Vbar values 
as I have done.
The deviation of detergent  micelles densities  stem from several reasons.
We have to understand that a detergent solution is mixture  of underand 
over CMC.
This equilibrium of particles may  differ  by concentrationand  used 
buffer/water.
In density-meters you  need  to  know  the  realconcentration of micelles 
and this
is  quasi impossible for the Vbar determination, where you have tocross
the under CMC range.
In AUC you can determine the micelles density  with schlieren and 
interference
in densifiers between sedimentation and floatation, but  thisdensities are 
hydrated once.
with a water shell so they are 40-50% off.
Different producers of detergents reports on  different subunits fora 
micelle so
also the density variegates.
The reference of Shire (15 of the BBA paper I sent to Fumio) gives 
alsodifferent values
as the Meth.of Enzymology.if not to trust in my own measurements.

                     Yours....ariel
***********************************************************
Fumio Arisaka, Ph.D.
Department of Biomolecular Engineering
Graduate School of Bioscience and Biotechnology
Tokyo Institute of Technology
5249 Nagatsuta, Midori-ku
Yokohama 226-8501  JAPAN
Tel/Fax:+81-45-924-5713
E-mail: farisaka at bio.titech.ac.jp
URL  http://www.farisaka.bio.titech.ac.jp/　　
　　　 http://edpex104.bcasj.or.jp/pricps2004/
***********************************************************

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20031124/3e8d99a3/attachment.htm>