<html>
Satinder,<br>
<br>
Yes, you are correct re the first question, the partial specific volume
of a conjugated protein is the sum of the weight fraction X Vbar of each
respective component. In your case the components are protein and
glycosylation. With respect to the second question you do not know
the carbohydrate composition therefore you do not know the Vbar of the
glycoprotein. I suggest you look at the following paper:
<font face="Arial, Helvetica"><b>Lewis, M. S., and R. P. Junghans.</b>
2000. Ultracentrifugal analysis of molecular mass of glycoproteins of unknown or ill-defined carbohydrate composition. Methods Enzymol <b>321:</b>136-49. They develop an equation for the Molar mass of the glycoprotein in terms of the measured buoyant molar mass, protein molar mass and values of Vbarprotein, Vbarglyc and density. The Vbarprotein is known and you can test values of Vbarglyc on the total Mw of the glycoprotein. The latter is not very sensitive to different values of Vbarglyc. Non-lin will fit for buoyant molar mass values if you set p=1.00 and vbar=0. Then you apply the Lewis and Junghans' equation. <br>
<br>
</font>Jack Lebowitz<br>
<br>
<br>
<br>
At 07:53 PM 4/1/2004 -0500, Satinder Singh wrote:<br>
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<br>
Hello,<br>
<br>
I am dealing with a glycosylated protein (N-linked). The monomer molecular<br>
weights determined from amino acid sequence and MALDI-TOF are 45,890 and 48,170,<br>
respectively, so I am guessing that 2280 Da (or 5% of the protein's MW) is<br>
due to glycosylation.<br>
<br>
I calculated the partial specific volume with Sednterp (using the<br>
sequence and a temp of 4C; Vbar = 0.7266) and then adjusted the value<br>
using an estimated carbohydrate Vbar of 0.63 ml/g<br>
The resulting value was 0.7220 ml/g.<br>
<br>
I am wondering if this is the proper way to calculate Vbar of a<br>
glycosylated protein if the precise carbohydrate content is unknown.<br>
<br>
Another question: with an unglycosylated protein, I am had been using<br>
Sednterp to calculate sigma from the amino acid composition. Since I do<br>
not know the carbohydrate composition of the present protein, I obviously<br>
cannot use a calculated sigma value. I had thought about directly<br>
calculating it from the mass spec molecular weight using the equation<br>
sigma = {MW*(1-[Vbar*solventdensity])w2}/RT, but again I am not sure if<br>
this correct.<br>
<br>
Could someone shed some light onto this? (I will be using WinNONLIN for<br>
fitting).<br>
<br>
Thanks.<br>
<br>
Satinder<br>
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