[RASMB] Help with Small Self-Associating Peptide

[John Philo]

Peter, I have successfully used both SE and SV for peptides in that size
range at concentrations up to ~15 mg/mL, with absorbance detection. If this
peptide is really associating up to something like a hexamer you should be
able to use 6-channel equilibrium cells, at least for the higher
concentrations, but for samples at low concentrations which are mostly
monomer you'll probably need to do equilibrium in velocity cells so you can
go to 60K. The rotor speed limitations mean you can't really get to very
high sigma values, and that's exactly why you don't want to use interference
optics. However the low sigmas are okay for absorbance data if (and only if)
you don't float the baseline offsets. For the higher concentrations I think
I was using wavelengths somewhere out near 300 nm.
 
I really did the velocity dilution series mostly to check that the
association was fully reversible, but to my surprise I was able to get a
pretty good global fit of the 3 concentrations to a reversible association
model, and the stoichiometry was quite clear (and not what I expected). I
can't recall exactly how high I went in concentration but I think it was > 5
mg/mL (using 3 mm centerpieces, which is quite important).
 
I will warn you though that the non-ideality effects may be much stronger
than you expect (they certainly were for the peptides I ran). The apparent
MW from SE reached a maximum as the concentration went up and then turned
strongly downward, and that maximum MW never really approached the
stoichiometry implied by the best-fit model. In my experience the strong
non-ideality was the biggest drawback to the velocity approach. If you
really need to go higher than 10 mg/mL, the complications of hydrodynamic
non-ideality may be a killer.
 
Good luck and best regards,
 
John
  _____  

From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Peter Edward Prevelige Jr
Sent: Friday, December 09, 2011 2:57 PM
To: rasmb at rasmb.bbri.org
Subject: [RASMB] Help with Small Self-Associating Peptide



Hi All -

 

Can you give me some advice on the best way to analyze a small (~2800 Da)
peptide that we think is self associating. The CD shows increasing helicity
with concentration which continues to increase even at 10 mg/ml meaning we
will likely have to record data at a less than ideal wavelength (240 nm) Our
interest is really in determining the stoichiometry of the species formed at
high concentration  (we suspect a hexamer), we care less about Kd. Looking
through the archives I've seen conflicting suggestions, (ie. sed velocity vs
sed equilibrium).  I was thinking about mimicking the approach in the
Braswell/Holtzer paper but the peptide is somewhat valuable and limited so I
wanted to ascertain whether or not this was the optimal way to proceed. 

 

Thanks!

 

Peter

 

 

Peter E. Prevelige Jr.

Professor

Dept. of Microbiology, BBRB 416/6

Univ. of Alabama @ Birmingham

845 19th St. South

Birmingham AL. 35294-2170

Phone 205 975-5327

FAX 205 975-5479

prevelig at uab.edu

http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm

 

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