Size fractionation of viral structural proteins

Once pure virus is obtained, gentle disruption of the virions with mild detergents or appropriate salt treatments can lead to disruption of the particle and solubilization of the components. Proteins and nucleic acids can be separated from each other by a variety of extraction or differential degradation regimens. For example, small amounts of nuclease could be used to digest nucleic acid into nucleotides, or proteases could be used to digest proteins. These macromo-lecular components then can be separated according to size or charge, or a combination of both.

It can be shown using physical chemical analysis that the sedimentation rate of a macromol-ecule is a function of its molecular size and its hydrodynamic volume. Thus, a globular mac-romolecule (such as most proteins) will migrate at a different rate than an extended (linear) macromolecule of the same size. Further, the same parameters apply to the rate of migration of a similarly charged macromolecule of equivalent shape when subjected to an electrical field, provided the molecules are suspended in a medium of high viscosity that discourages diffusion, such as an acrylamide gel. This is the principle of gel electrophoresis.

In electrophoresis, the rate of migration is inversely proportional to sedimentation rate (s value). Two macromolecules of equivalent hydrodynamic shape and unit charge will migrate so that the molecule with the larger molecular size will migrate more slowly than the smaller molecule.

These principles are incorporated into a very powerful technique for the size fractionation of proteins. It involves mild denaturation (disruption) of protein structure with the detergent sodium dodecyl sulfate (SDS), which associates with denatured protein to give it a uniform net negative charge. Such proteins can then be size fractionated by electrophoresis on acrylamide gels where the larger proteins move more slowly through the gel network, and the smaller proteins migrate more rapidly. If the procedure is properly done, such a gel provides good fractionation of viral structural proteins according to size.

Such gels can be stained with color reagents that provide a quantitative measure of the amount of protein of each size, as the color reaction is based on reactions with amino acids in the proteins. A small protein has fewer amino acids per polypeptide than a large one; therefore a sample of, say, 1000 small protein molecules will stain less intensely than will a sample of 1000 larger protein molecules.

A hypothetical example of protein size fractionation and a method of estimating molar ratios is shown in Fig. 11.3 where the fractionation of protein mixtures in a denaturing SDS-containing gel is represented. In this experiment, a solution of an equimolar mixture of four proteins of significantly different sizes (i.e., different number of amino acids in the peptide chain) was fractionated in lane 2. Another sample of three proteins of different sizes in variable amounts (with the smallest protein being present in higher molar concentration than the midsized one, and both present in higher concentration than the largest) was fractionated in lane 1. The staining pattern of the gel is shown in lanes 3 and 4 where the staining intensity is represented by band thickness.

The pattern of staining intensity shown in lane 3 makes it clear that the proteins are not present in equimolar amounts. Since staining intensity of the most rapidly migrating band is greater than that of the mid-sized and large bands, there must be more amino acids in the band of small protein. This can only happen if there are more copies of the small protein chains. The staining pattern of lane 4 shows a monotonically decreasing intensity of staining with size.

Anode 1

Length of peptide is proportional to its MW and number of amino acids

Band intensity (function of total mass of protein present in band)

Length of peptide is proportional to its MW and number of amino acids

Anode 1

  1. 11.3 Denaturing gel electrophoresis of proteins. If proteins are gently denatured in a detergent solution such as sodium dodecyl sulfate (SDS), they will assume globular shapes and a net negative charge due to interaction with the detergent molecules. The proteins then can be fractionated by size on acrylamide gels. The proteins migrate in specific bands, and the amount of mass in each band can be determined with a color reaction that measures protein mass. The intensity of banding is a function of the total amount of amino acids (a direct correlate with the total mass) in the band, not the number of protein molecules per se. MW = molecular weight.
  2. 11.3 Denaturing gel electrophoresis of proteins. If proteins are gently denatured in a detergent solution such as sodium dodecyl sulfate (SDS), they will assume globular shapes and a net negative charge due to interaction with the detergent molecules. The proteins then can be fractionated by size on acrylamide gels. The proteins migrate in specific bands, and the amount of mass in each band can be determined with a color reaction that measures protein mass. The intensity of banding is a function of the total amount of amino acids (a direct correlate with the total mass) in the band, not the number of protein molecules per se. MW = molecular weight.

Although a precise measure would be required, the band intensity appears to be (roughly, at least) proportional to protein size. This is the result expected for an equimolar mixture of proteins of different size, as one small protein polypeptide chain will have fewer amino acids than a single peptide chain of a larger protein.

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