[RASMB] glycosylation in sigma value

[Jacob Lebowitz]

Satinder,

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: Lewis, M. S., and 
R. P. Junghans. 2000. Ultracentrifugal analysis of molecular mass of 
glycoproteins of unknown or ill-defined carbohydrate composition. Methods 
Enzymol 321:136-49. 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.

Jack Lebowitz



At 07:53 PM 4/1/2004 -0500, Satinder Singh 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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