(424g) The Charge of Green Fluorescent Protein, and Its Effect On Advanced Electrophoretic Separations

The charge of proteins as they vary with pH determines in large part how one protein may be separated from another.  The isoelectric point of many proteins is known, and this information can be used to guide development of an advanced electrophoretic separations method.  The isoelectric point alone, however, gives no information on how the protein charge changes.  Often simple group contribution methods that sum the pKa of the amino acid residues are used to estimate the charge of a protein as pH is varied.  It is recognized that the group contribution approach has many limitations, though it is rare that they are explored in detail for any particular protein.

In this presentation the charge of Green Fluorescent Protein, a widely-used fluorescent reporter, is presented as a function of pH over the range of 3 to 10.  The charge measured by experiment differs drastically from what would be expected from its amino acid residues.  Probable reasons for the difference, and its effect on electrophoretic separations, is discussed.