Electrophoresis of proteins across a transverse sodium dodecyl sulfate gradient.

We describe a new protocol for denaturant gradient gel electrophoresis, namely the migration of proteins across transverse sodium dodecyl sulfate (SDS)-gradients. We show how such gradients may be reproducibly cast, and demonstrate their stability with time once an appropriate SDS reservoir is arranged at the cathode. SDS affects both size and surface charge of the molecules, and influences the secondary and tertiary structure to a variable extent, even in opposite directions. Hence, distinct, sometimes complex, denaturation patterns may be observed for different proteins.

Denaturation and self-association of apolipoprotein A-I investigated by electrophoretic techniques.

Purified human apolipoprotein A-I (apoA-I), when run across a transverse urea gradient at alkaline pH, gives a complex pattern characterized by a number of parallel sigmoidal curves, in which the transition between high- and low-mobility forms, i.e. from folded to unfolded structure, occurs between 1.1 and 3.2 M urea. Size differences appear to be the major cause of this isomerism. When migrated across a wide pH range in the presence of varying amounts of urea to display its titration curve, apoA-I is resolved into two pairs of bands, running parallel in the neutral to basic pH region while merging at acidic pH; such a finding does not correlate with a differential exposure of His residues, as shown by diethyl pyrocarbonate titration. Ferguson plot analysis, confirmed by cross-linking experiments, demonstrates a gradual shift from higher to lower mass aggregates as the urea concentration is raised; the monomeric form undergoes denaturation by swelling to an approximately 50% larger hydrodynamic volume than in its native state. At alkaline pH, where apoA-I exists as aggregated species, disaggregation and unfolding appear to happen at once, the larger aggregates being less stable than the smaller ones. At acidic pH, apoA-I does not form aggregates and has little secondary structure; unfolding is then a progressive rather than a cooperative process.