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From: Geoff=Howlett%BioChem%UNIMELB@muwaye.unimelb.EDU.AU
To : tml@christa.unh.edu
Date: Wed, 18 May 94 9:40:17 +1000
re: Re: Differential sedimentation.
Open letter to Tom Laue
My experience with differential sedimentation velocity is from a while back
and is as follows:
There are basically two methods.
method one: Use of wedge windows and schlieren optics. This method accounts
for the overwelming proportion of delta S experiments. The main reason is
that it is not as sensitive to small amounts of convection in the plateau
region. Aspartate transcarbamylase undergoes a conformational change on the
binding of substrate analogues (about 3%) and this can be titrated with
accuracies of 0.1%. The secret here is to have good centerpieces (The ones
from Beckman have improved out of sight in recent years (Personal
communication , HK Schachman). The other point is to align the cell in the
rotor accurately. My understanding is this is presently done on a trial and
error basis where the line underneath the cell is calibrated with respect to
the line on the rotor. The ultimate test is to get a zero delta s for
identical solutions. Small corrections have to be made to the measured
delta S for the effects of added ligands on density and viscosity. These
corrections are standard and not large, at least in the case of high affinity
ligands. Method one does not require absolute matching of menisci.
method 2: This method was originally described for interference optics but
has I believe been adapted for the XLA optical system. It involves filling
the reference sector with the protein sample and the sample sector with the
protein plus ligand sample. It is not necessary to exactly match menisci but
they shold be relatively close together because the data appears as a peak
due initially to the mismatch of menisci. This peak then gets either bigger
or smaller depending on the relative velocities of the sedimenting protein in
the two sectors. This method is much more sensitive to convection in the
upper plateau but could well be preferred with the XLA.
One of the interesting adaptions which needs to be made for method two in the
XLA is to disable the auto gain capacity of the optical system. I believe
the XLA system adjusts the gain (voltage) on the photo multiplier due to
changes in the OD in the reference sector at different radii. You don't want
this to happen in delta S experiments.
How to disable this function? I don't know.
As you correctly point out the delta S method is an extaordinarily sensitive
method for picking up global conformational changes and is potentially a
powerful tool to studies the effects of ligand binding and single amino acid
changes on overall protein structure.
I'm not sure how we got here from sloping baselines.
Cheers
Geoff H.
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