Gel Chromatography as an Analytical Tool for Characterization of Size and Molecular Mass of Proteins

Abstract

Although experience with watersoluble, globular proteins supports Stokes radius as an appropriate size parameter, governing elution position, for both classical and HPLC gel columns, extrapolation of this concept to other kinds of conformation and substances requires caution. Current evidence suggests the viscosity based Stokes radius as the best suited size parameter for many flexible macromolecules (guanidinium HCl denatured proteins, dextrans), whereas many detergent micelles and detergent solubilized membrane proteins elute somewhat earlier, and elongated (e.g. myosin) or SDS denatured proteins elute later, than indicated by their hydrodynamic behaviour. The review includes a discussion of the use of globular proteins of known size to characterize gel pore size distribution.The purpose of this review is to briefly consider the scope of gel chromatography, or size exclusion chromatography (SEC), in the determination of sizes and molecular masses of proteins. This kind of separation is performed either as classical type chromatography, using gels such as Sepharose (agarose), Sephacryl (allyl-dextran crosslinked with Ν,Ν-methylene bisacrylamide) and Superose, or by HPLC with the aid of matrices capable of withstanding high pressures such as silica based TSK SW-or polyacrylamide based TSK PW columns. We first briefly review the theoretical foundation for use of the technique in the estimation of molecular mass and size of watersoluble, globular proteins. We then proceed to consider the use of the technique to analyze other kinds of proteins (elongated or fibrous type proteins, detergent-solubilized membrane proteins) and the effect of a disordered conformation (SDS-or GuHCl denaturation). Finally, we consider the use of globular proteins in the characterization of the pore characteristics of the gels.

The oxygenase component of the2-aminobenzenesulfonate dioxygenase system from Alcaligenes sp. strain O-1 The GenBank accession number for the sequence reported in this paper is AF109074.

“…The weight to put on this deduction is limited, because the older data are based on cross-linking experiments, whereas ours are based on gel filtration, which is known to deviate from the theoretical when the protein concerned is not spherical (e.g. le Maire et al, 1996). Indeed, naphthalene dioxygenase oxygenase was long considered to have an α # β # structure and was shown to be α $ β $ only when the crystal structure became available (Kauppi et al, 1998) ; the oxygenase was found not to be spherical but mushroom-shaped.…”

Characterization of TsaR, an Oxygen-Sensitive LysR-Type Regulator for the Degradation ofp-Toluenesulfonate inComamonas testosteroniT-2

“…Gel filtration chromatography indicated that active TsaR is a monomer in solution, though le Maire et al (18) warn of errors sometimes encountered with this method. TsaR as the active monomer differs from several LysR-type regulators.…”

Interaction of Amphipols with Sarcoplasmic Reticulum Ca2+-ATPase

“…This difference could be due to a difference in the Stokes radii of the complexes, to electrostatic repulsion between the ATPase⅐A8-35 complex and negative charges on the gel pore walls (either residual charges on the matrix itself or charges due to adsorbed A8-35 molecules), or both. 2 Correct calibration of size exclusion columns in terms of Stokes radii is known to be a difficult task (22,(62)(63)(64)(65). However, another feature of the chromatograms for diluted delipidated ATPase complexes was the larger width of the main ATPase peak following trapping by amphipols.…”