[RASMB] Sedimentation coefficient of human serum albumin monomer and good protein standard with well established s value

[Mattia Rocco]

Hi - As an electronic supplement to our May 2005 Structure paper (Rai et 
al., 13:723-734), we have published a detailed analysis of the literature 
hydrodynamic data of BPTI, RNase A and lysozyme. Although they are 
relatively small, and thus not the best standards for sedimentation 
velocity, it seems to me that both RNase and lysozyme could be used, as 
they can be obtained commercially in large quantities and relatively pure. 
For lysozyme, be careful to use it at pH<5, as above it dimerizes. Needless 
to say, if you want to properly calibrate, you will need to run a 
concentration series to extrapolate the sed. coeff. value reported in the 
literature. Anyway, I'll be happy to send the supplement to anyone that 
cannot download it from the Structure website.

Regarding BSA (why use HSA while BSA is much cheaper, and surely much more 
studied?), it can be purified by size-exclusion chromatography using a 
modern HPLC (or FPLC) apparatus and suitable columns. For instance, we use 
two TSK PWXL G3000 analytical columns connected in series. In our hands, 
the dimer and trimer peaks can be separated almost to baseline, so if you 
take the peak fractions you'll have quite pure material, and in enough 
quantity for AUC work. However, I don't know if you can find a "good" BSA 
sedimentation coefficient value in the literature, i.e. one determined 
after those purification steps have been taken, I have never examined this 
issue.

Cheers - Mattia
________________________________________________

Dr. Mattia Rocco
Proteomica
Istituto Nazionale per la Ricerca sul Cancro (IST)
c/o Centro per le Biotecnologie Avanzate (CBA)
IST c/o CBA, Largo Rosanna Benzi 10
I-16132 Genova, Italy

Phone: +39-0105737-310
Fax: +39-0105737-325
e-mail: mattia.rocco at istge.it

At 15:58 17/12/07 -0500, Tom Laue wrote:
>Hi all-
>Be careful with the Tanford data. The D20,w for RNAaseA is off (low as I 
>recall) by 20%.
>Serum albumin may not be a good standard due to its tendency to form 
>dimers, and possible contamination by trace quantities of IgG.
>I'll be interested in other's suggestions of possible standards... I've 
>never met a protein that was well behaved, except under a limited range of 
>conditions.
>Best wishes,
>Tom
>
>John Burgner wrote:
>>Yiming,
>>On page 381 (Table 22-1) of Charlie Tanford's book "Physical Chemistry of
>>Macromolecules" there is a list proteins with s20,w, D20,w, and vbar for a
>>number of different macromolecules with s values from 1.6 to 170S.
>>
>>John Burgner
>>
>>
>>
>>-----Original Message-----
>>From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
>>Behalf Of Yi-Ming_Li at hgsi.com
>>Sent: Monday, December 17, 2007 1:50 PM
>>To: rasmb at rasmb.bbri.org
>>Subject: [RASMB] Sedimentation coefficient of human serum albumin monomer
>>and good protein standard with well established s value
>>
>>
>>Hi! Everyone
>>We are looking for some "protein standard" to qualify our sedimentation
>>velocity method. We think that HSA may be a good candidate for the
>>standard. Does anyone know the sedimentation coefficient of human serum
>>albumin monomer (corrected for buffer density and viscosity as well as
>>vbar)? Some literatures said HSA has a sedimentation coefficient of 4.6 s,
>>this is significantly higher than the value of the monomer sedimentation
>>coefficient of HSA I got by analyzing the SV data of Sigma HSA using C(s)
>>model. The C(s) profile of this Sigma HSA showed that the product contains
>>about 80% monomer, 17% dimer and 3% trimer. So I guess the 4.6 s value
>>reported in some literatures is the weight average apparent s value of the
>>HSA monomer, dimer and trimer (or even some oligomers) in the sample
>>instead of the apparent s value of HSA monomer. Therefore it will be very
>>helpful for me if anyone can tell me the accurate s value of HSA monomer.
>>Also the suggestions for better protein standard (better commercial
>>available) with a well established s value will be very helpful.
>>
>>Thanks!
>>
>>Yiming
>>
>>
>>_______________________________________________
>>RASMB mailing list
>>RASMB at rasmb.bbri.org
>>http://rasmb.bbri.org/mailman/listinfo/rasmb
>>
>>_______________________________________________
>>RASMB mailing list
>>RASMB at rasmb.bbri.org
>>http://rasmb.bbri.org/mailman/listinfo/rasmb
>>
>>
>
>--
>Department of Biochemistry and Molecular Biology
>University of New Hampshire
>Rudman Hall 379
>46 College Road
>Durham, NH 03824-3544
>Phone: 603-862-2459
>FAX:   603-862-0031
>www.camis.unh.edu
>www.bitc.unh.edu
>
>
>_______________________________________________
>RASMB mailing list
>RASMB at rasmb.bbri.org
>http://rasmb.bbri.org/mailman/listinfo/rasmb