From: Dr A.J. Rowe <ajr@leicester.ac.uk>
  To  : rasmb@bbri.harvard.edu
  Date: Wed, 4 Jun 1997 17:48:52 +0100 (BST)

buffers in the far uv

USE OF BUFFERS IN THE FAR UV

For some purposes - including of course AUC of dilute solutions - it is
desirable to be able to study one*s protein/whatever in the far uv, where the
sensitivity is at least 5x greater than at 280 nm. 

We have been doing this on the XL-A with a number of systems, including
defining four new species of ciliary dynein (Tharia et al, JMRCM in press)
available only in very small quantity.

It is useful to be able to know whether samples received in various buffered
solutions can or can not be investigated in this way. To give ourselves a quick
guide as to what might at least be worth a try, we have measure up the uv
spectrum of a range of common buffers. The lists appended below are classified
as follows:

List A: very low absorabance at 220 nm, rising to only a modest absorbance at
200 nm (i.e. the best of the bunch)

List B: absorbance less than 0.3 at 220 nm, rising more steeply towards 200 nm
(i.e. could be usable with care, if 220+ nm is acceptable, and if the problems
of working on a *shoulder* can be accepted)

List C:  forget them !  Too much absorption to be of use in the far uv.

Some comments on other factors which might be added are also appended. Hope
these may be of some use to the RASMB fraternity. 

Arthur Rowe

Far uv absorbance of commonly used buffer systems
(all at 10 mM concentration)

			   Absorbance at
Buffer			220 nm	200 nm

List A
CHES			.02		.01
Glycine			.08		.56
MES			.05		.24
MOPS			.09		.26
phosphate		.01		.44
TAPS			.03		.22
TES			.10		.47

List B
acetate			.19		1.34
bicine			.15		0.96
cacodylate		.00		1.36
glutamate		.26		1.59
pyrophosphate		.28		1.20
tris/various		.05		1.52

List C - avoid totally !
bis-tris propane
acetamide
butylamide
ACES
tartrate
cysteine
citrate
PIPES
LEEDS
CHAPS
HEPES
TEA
ethan-di-ol
maleate

notes:

(1) addition of neutral salt makes the absorbance higher (e.g. glycine is
totally unusable in the presence of 0.5 M NaCl)

(2) addition of significant (mM) concentrations of compounds such as EDTA,
CyDTA, DTT is likely to render an otherwise acceptable buffer system unusable

(3) GuHCl produces a blackout, but (pure) urea is usable with care (at usual
sort of levels)

***************************************************
Dr Arthur J Rowe
Director
UK National Centre for Macromolecular Hydrodynamics
Leicester Laboratory
Adrian Building
University of Leicester
Leicester LE1 7RH    UK

Tel: +44 (0)116 252 3448
Fax: +44 (0)116 252 5602
ajr@leicester.ac.uk
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