From: Olwyn Byron <o.byron@bio.gla.ac.uk>
  To  : rasmb@bbri.harvard.edu
  Date: Fri, 19 Dec 1997 18:03:17 +0100

PEG-tastic

Hello again RASMBers

Further to yesterday's request for PEG data I'm now appending to this
e-mail a summary of the tremendously useful responses that I received.

Thanks very much to those who contributed:

Sue Bethell
Jo Butler
Eckhard Goernitz
Yatin Gorkan
Tom Laue
John Philo
German Rivas
Walter Stafford
Gay-May Wu


Again, Happy Christmas!
Olwyn Byron.

_____________________________________________________________________________

The original request for help:

Hello RASMBers

I've got two quick questions to ask concerning polyethylene glycol (PEG).

We're trying to characterise a protein-PEG system and are having real
trouble measuring a vbar for the conjugated molecule. However, we know the
mass fraction of PEG and so conceivably we could estimate a vbar if we knew
the vbar of the PEG we're using.

So, does anyone have any vbar data for PEG?
In particular we're after vbar for PEG 5,000, 10,000, 20,000 and 40,000 (Da)
(I don't know that vbar for these will necessarily be comparable).

The second question concerns labelling PEG: does anyone know of an easy way
to covalently (or non-covalently) label PEG (so that we can try AUC studies
using absorbance optics)?

Thanks in advance for your responses to these questions.
All the very best for Christmas and the New Year-

Olwyn Byron.


_____________________________________________________________________________

A summary of the replies:

1. vbar:

The source of PEG (i.e. Fluka, Sigma etc) makes a big difference to some
studies. This could be because of the different distribution of MW in the
PEGs
coming from different sources and /or some of the impurities which have
been detected.

However some sources are better than others: Shearwater Polymers supply PEG
with accurate molecular weight cut-offs. Yatin Gokarn used PEG 5000 and
measured the mol. weight for one batch as 4880 (accounting for
non-ideality).

Data for shorter PEGs (200, 400, 600, 1000) can be found in
Arakawa & Timasheff (1985) Biochemistry 24, 6756-6762.

Yatin Gokarn reported a measured vbar of 0.832 +/- 0.002 ml/g for PEG 5,000
in pure water and 0.825 +/- 0.002 ml/g in 50 mM Tris, 100 mM NaCl pH 7.4.
Lechner et al. (in Polymers) report a value of 0.838 ml/g for large PEGs.

Gay-May Wu measured the vbar of 12K, and 20K PEG in water, the values are
0.83973 and 0.83778 ml/g respectively. David Yphantis did a calculation of
vbar for 25K PEG, his estimate gave vbar = 0.84 ml/g.

Eckhard Goernitz found some unpublished v-bar data measured many years ago
by him and Karl Linow for PEG in water at 20°C:

M(PEG) (g/mol)          v-bar (ml/g)

  200                           0.862
  400                           0.845
  600                           0.841
 1500                           0.838
 4000                           0.836
10000                           0.834
20000                           0.832

These are in general accordance with the results of:
L.Lepori and V.Mollica, J. Polymer Sci. Polym. Phys. Ed., Vol. 16 (1978)
1123-1134


2. Labelling:

The only reactive groups on PEG are the hydroxyls at the ends, otherwise it
is chemically a poly-ether and not readily reactive. Hydroxyl groups can be
labelled with many dyes, e.g. the Procion dyes, or with a fluorescent
label. The latter are not much use in the XLA, as there is really no room
to include an emission filter between the cell and the scanning slit and
also the geometry is awful for collecting the light. The problem with a dye
is that most of them (at least in the usual visible range) have extinction
coefficients only in the range around 30,000 M-1 cm-1, i.e. for Mr 100k one
is looking for ~1mg/ml for an OD of 0.3.

***********

In principle, it should be possible to label PEG with a reagent for
modifying alcohols. The Molecular Probes catalog (chapter 3, section 3.1)
is a possible source (email:tech@probes.com or eurotech@probes.nl or
www.probes.com).
A probe with a good extinction coefficient in the visible region is best.

***********

PEG-aldehyde can be used to make protein conjugates. This can then be more
easily labelled, eg with lucifer yellow (available form Sigma).

***********

PEG 20,000 (and perhaps others) has a phenolic group already attached
(possibly as a polymerisation initiation group). It (PEG 20,000) has an
absorption spectrum exactly like tyrosine.




_____________________________________________________________________
Olwyn Byron
Division of Infection and Immunity
Institute of Biomedical and Life Sciences
Joseph Black Building, University of Glasgow
Glasgow, G12 8QQ, Scotland.

tel: +44 (0)141 330 3752  email: o.byron@bio.gla.ac.uk
fax: +44 (0)141 330 4600  web:   www.gla.ac.uk/Acad/IBLS/II/ob/ob.htm
_____________________________________________________________________