Determination of Molecular Weight of Glycoproteins by Analytical Ultracentrifugation
By: Beckman Coulter Published: June 25 2016
Analytical ultracentrifugation is a classical technique that has played a critical role in laying the foundations for modern molecular biology. Among its achievements is the demonstration that proteins are macromolecules rather than complexes of smaller units, and direct support for the semiconservative replication mechanism of DNA as proposed by Watson and Crick. Unlike techniques such as SDS PAGE or gel permeation chromatography, analytical ultracentrifugation can be used to determine absolute molecular weights without the use of molecular weight standards or interference from the sieving matrix used for separation.
With the advent of recombinant DNA technology and the explosion of protein drugs being developed, there has been a renewed interest in analytical ultracentrifugation. Many of the proteins being developed as pharmaceuticals exist as glycosylated proteins. It is unclear how the composition or content of carbohydrate affects behavior on typical gel permeation chromatography media. Often the standards used to calibrate the chromatography are globular proteins and are inadequate to obtain an accurate molecular weight of the glycosylated protein in solution. The apparent molecular weights determined may be sufficiently in error to result in the conclusion that the protein exists in an associated state in solution. The experiments presented here show two examples using glycoproteins produced at Genentech, Inc.: the recombinant DNA-derived envelope glycoprotein, rgp120 IIIB, of human immunodeficiency virus type 1, and sTNF-R1, the extracellular domain of human tumor necrosis factor (TNF-α) type 1 receptor. Gel permeation chromatography suggests that these molecules exist as dimers in solution. Molecular weights, determined by analytical sedimentation equilibrium experiments, showed these conclusions to be incorrect.
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