[RASMB] Sv and Se for V

[Ariel Lustig]

Dear Colleauges,
I wanted to leave the discussion (see Walter  Staffords last e-mail 7 June 08) for the Newcastle AUC Symposium in  September 08, but it developed already now, so  I shall  give here the  abstract of the  my 
contribution about  this  theme and   a new approach to solve it.:

The use of an elder mode to determine the density of vesicles by S0 versus ? is a good approach that can be used also to determine protein/RNA or protein/DNA complexes. 

First of all we have to bear in our mind that the molecular masses achieved from centrifugation, based on the buoyant term (the partial specific volume, V the particle), here in this discussed case Vpot.DNA or Vprot.RNA. Usually we invest a big effort on sophisticated methods to calculate molecular weights Mw, but the lack of precise V values is like a weak link of a chain. In such mentioned case we are able to calculate   the V from the protein amino acids and nucleic acids but the exact ratio of both components is usually unknown. 

The classical density determination by pycnometry even with digital precise instruments needs the knowledge of the total particle concentration of the solution that can be only estimated.

The use of S.J. Edelstein and  H.K.Schachman's mode, (JBC 196, vol.247 , 306-317) to make  parallel  runs  in H2O and D2O buffer as mentioned also in the  RSC published  book >> Analytical Ultracentrifugation, Techniques and  Methods<< page 197, edited by  D. Scott et al, can be employed only if the samples are pure, monodispersed and  reveal  with sedimentation equilibrium SE straight  slopes and single Mw's. The reality  in case of  biological  samples is, that  the slopes  tends  to self-association , nonideality or both effects and even  show partly aggregation, so that  the deviated lines from them  the V is  calculated  are not single and  straight.

Using the SEGAL computer program when the slopes of 2 parts are calculated, it is easy to realize this discrepancy.

Using the S0 relative versus ? mode developed by C.H: Huang and J Charlton (JBC 

1971 Vol. 216) that was used to study the density of vesicles, it is possible to apply it also for high molecular complexes. The advantage in this case is, that even the sample sediments with a boundary that contains  partly  a  faster  or a slower component (as it  happens often with biological samples) it can be used  to achieve  S°0 (at a D2O -H2O buffer density where S' comes  at  rest) that represents the V of  the particle. Important is to use the whole series of S0 relative at the same particle concentration to avoid S' differences that stem from concentration dependence of S'.

All our measurements were done with absorption optics using the  XLA at the same  wavelength WL since the used samples do not show a distinct  WL maximum at 260 nm or 280 nm.

...............yours......ariel





 

 
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20080608/9a43eb7f/attachment.htm>