[RASMB] glycosylation in sigma value

[pjgb at mrc-lmb.cam.ac.uk]

Dear Satinder,

Yes you can usually get a reasonable estimate by calculating in the 
contribution of the carbohydrate provided you do know the amount (and 
averages will simply smear things a little).  Of course, back on my hobby 
horse, the best way is to measure (d-rho/d-c) for the protein at constant 
chemical potential.
We did this for the C-reactive protein from Limulus polyphemus over 10 
years ago and got a very good result.
(Tennent, G. A., Butler, P. J. G., Hutton, T., Woolfitt, A. R., Harvey, D. 
J., Rademacher, T. W. & Pepys, M. B. (1993). Molecular characterization of 
Limulus polyphemus C-reactive protein.  I. Subunit composition. Eur. J. 
Biochem. 214, 91-97.)

Yours,

Jo

--On Thursday, April 1, 2004 7:53 pm -0500 Satinder  Singh 
<ss2337 at columbia.edu> wrote:

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>
> Hello,
>
> I am dealing with a glycosylated protein (N-linked). The monomer molecular
> weights determined from amino acid sequence and MALDI-TOF are 45,890 and
> 48,170, respectively, so I am guessing that 2280 Da (or 5% of the
> protein's MW) is due to glycosylation.
>
> I calculated the partial specific volume with Sednterp (using the
> sequence and a temp of 4C; Vbar = 0.7266) and then adjusted the value
> using an estimated carbohydrate Vbar of 0.63 ml/g
> The resulting value was 0.7220 ml/g.
>
> I am wondering if this is the proper way to calculate Vbar of a
> glycosylated protein if the precise carbohydrate content is unknown.
>
> Another question: with an unglycosylated protein, I am had been using
> Sednterp to calculate sigma from the amino acid composition. Since I do
> not know the carbohydrate composition of the present protein, I obviously
> cannot use a calculated sigma value. I had thought about directly
> calculating it from the mass spec molecular weight using the equation
> sigma = {MW*(1-[Vbar*solventdensity])w2}/RT, but again I am not sure if
> this correct.
>
> Could someone shed some light onto this?  (I will be using WinNONLIN for
> fitting).
>
> Thanks.
>
> Satinder
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P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 2QH, UK.
Tel. +44 (0)1223 402296