[RASMB] Use of O-18 heavy water for SE of protein-adduct complexes

[Arthur Rowe]

Greetings, everyone

This may seem like a shameless plug for a recent paper, but never mind.
There is one aspect of the said paper (reference below) that could be of
interest to those not involved in protein-protein interaction and/or STEM
Cells and their regulation.

I guess most out there are familiar with the use of high density solvent to
'match out' the detergent part of a micellar complex in which a protein
moiety is included within a micelle (the original Tanford-Reynolds method,
fully reviewed with subsequent advances recently by Karen Fleming, ref
below). This renders the detergent or other adduct part of the complex
'invisible' in terms of any contribution to the solute sedimentation
potential in an SE experiment.

Traditionally deuterium oxide ('heavy water') has been used to increase the
density level so that matching is achieved, either fully or by
extrapolation. However deuterium oxide has two drawbacks: (1) the protein
becomes deuterated to an extent which is not exactly known, thereby
undergoing changes in its partial specific volume; and (2) it is far from
biologically inert, indeed it is toxic to humans, and some protein systems
can be de-stabilised by its presence.

An obvious alternative candidate to deuterium oxide would be 'heavy oxygen'
- i.e O-18 water. This produces much the same level of density increment,
without any 'side effects', since the labelled oxygen does not interchange
with O atoms within a protein to any significant extent. I know that this
has long been appreciated - I was told recently that Howard Schachman tried
it out once in the 60's - but the use of O-18 water had one obvious
drawback: enormous cost!

However, in present times the cost has come down a long way, no doubt
because of its importance in relation to PET Scanners. A gram of O-18 water
will suffice for one SE experiment, maybe two if one uses deuterium oxide
for the reference channel. At present prices that is around £30 (~$US50) per
run, which is much less than the full economic cost of doing an SE
experiment. And it works nicely - see Mullin et al (2008).

The paper is: Mullin et al (2008) The plutipotency rheostat Nanog functions
as a dimer. Biochem J 4 227-231

Karen Fleming's review is Chapter 19 in Analytical Utracentrifugation -
Techniques & Methods (2005) eds D J Scott, S E Harding & A J Rowe.

Regards to all

Arthur


--
*******************************************************
Arthur J Rowe
Professor of Biomolecular Technology
NCMH Business Centre
University of Nottingham
School of Biosciences
Sutton Bonington
Leicestershire LE12 5RD   UK

Tel:        +44 (0)115 951 6156
           +44 (0)116 271 4502
Fax:        +44 (0)115 951 6157
email:      arthur.rowe at nottingham.ac.uk
Web:        www.nottingham.ac.uk/ncmh/business
*******************************************************



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