[RASMB] back to reality ... and overspeeding

[Walter Stafford]

All:

Ariel's point is well taken. It reminded me of 
two other studies on overspeeding:

One very detailed theoretical study from David 
teller's group in the 1960's. (cf page 78: D.C. 
Teller and T.A. Horbett, "Ultracentrifuge Studies 
with Rayleigh Interference Optics. III ...", 
Annals NY Academy Sciences pp 66-101.) gives over 
speeding protocols. It also warns of the serious 
dangers of over - overspeeding. In 1976 Dennis 
Roark published a handy little formula for 
calculating overspeed times based on the 
calculations done by the Teller Lab. The 
overspeeding procedures cited can - if properly 
applied (and properly is the key word here) - 
reduce the time to equilibrium to about 1/6 th 
the time not using overspeeding. However, if you 
look at the figure on page 78, you'll see that 
the choice of overspeeding time is quite 
critical. Use of overspeeding with unknown mixed 
systems could lead to undesirable results since 
each species has a very narrow useable 
overspeeding time.



Here is the reference (and abstract) to Denny's paper.

Roark, D. E. (1976). "Sedimentation equilibrium 
techniques: multiple speed analyses and an 
overspeed procedure." Biophysical Chemistry 
5(1-2): 185-96.
	The use of a single high-speed 
sedimentation equilibrium experiment to analyze 
mixed associating systems is inadequate to 
determine the association mode even if the 
molecular weight of one species is known. 
Simultaneous analysis of the concentration 
distributions at three equilibrium speeds greatly 
reduces the lack of uniqueness. Linear 
least-squares multi-speed fits discriminate 
between association models in which the molecular 
weights are assumed. Experiments at a series of 
initial concentrations as well as rotor speeds 
further increases the discrimination. An 
overspeed procedure is proposed. The overspeed 
time depends only slightly on the sedimenting 
species molecular weight, but significantly on 
the frictional ratio. A minimal overspeed time 
may be estimated.


Walter

p.s. Also a must read from David Teller's Lab: 
Teller, DC, Characterization of proteins by 
sedimentation equilibrium in the analytical 
ultracentrifuge. Methods Enzymol. 1973;27:346-441.



At 5:58 PM +0200 9/29/04, Ariel Lustig wrote:
>Dear colleauges, lets come back to reality
>At  least for new-comers , but not only for 
>them, the subject of predicting or better , 
>calculating the time to achieve equilibrium 
>makes no / or only very little sense , since  as 
>I belive that every  one of us use the
>"overspeed procedure" discribed  by Hexner, 
>Radford and Beams PNAS (1961), 47,1848 that 
>reduces time to achieve equilibrium to  50% or 
>more. If so, I ask myself what means time to 
>equilibrium ? from the time  rotor speed is 
>achieved and not yet a concentration gradient is 
>established, or  a steep graient at both 
>solution  column ends? all non -sense ! I agree 
>that for polydispersed or selfassociating 
>systems
>using an overspeed procedure is difficult (and 
>dangrous especially it some parts are not 
>reversible), but not less difficult as to 
>calculate time to equilibrium as you suggested 
>of a non monodispersed systeme.
>In  FRACTION N° 1 1967 issued  by  Beckman 
>Instruments ( may be based on the same  equation
>that Walter  Stafford mentioned recently) K.E 
>Van Holde discribed  a formula where the time 
>depends on Diffusion ! and here again to 
>new-comers. If at all making the exercise to 
>calculate time to  equilibrium.
>It is to use Dobs and not D20w that is 
>correected  for Viscosity  of  water. There may 
>be a huge  difference
>between the two Diffusion  values if  per 
>example you use sucrose in the  buffer as I do 
>measuring
>membrane  proteins. The density has no influence 
>for  Diffusion  only the  viscosity of the 
>solvent.
>yours....ariel
>ariel.lustig at bluewin.ch


-- 
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Walter Stafford
mailto:stafford at bbri.org
direct dial:    617-658-7808
receptionist: 617-658-7700
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