News
Entropic trapping of macromolecules The amount of time it takes for macromolecules, such as polymers or DNA, to be transported through a porous gel matrix depends on the size of the molecule relative to the pore radius of the gel. Smaller molecules tend to diffuse through the matrix in a spherical conformation, whereas larger molecules move in a snakelike fashion. Molecules with sizes that fall somewhere in between, however, tend to get trapped in the largest pores of the matrix to maximize their conformational entropy. Sanford A. Asher and co-workers at the University of Pittsburgh provide direct experimental evidence that this "entropic trapping" phenomenon increases the dependence of diffusion rate on molecular size The authors describe a fabrication approach that could be used to design new separation media for retarding trapping and separating macromolecules A hydrogel with a cubic array of spherical water voids was fabricated by allowing monodisperse silica spheres to electrostatically self-assemble into a crystalline colloidal ar-
Fabrication of a polymerized CCA of spherical water voids. (1) Monodisperse silica spheres electrostatically self-assemble into a CCA. (2) CCA is immobilized in a polyacrylamide hydrogen network. (3) Silica spheres are etched with hydrofluoric acid, yielding water voids. (Adapted with permission. Copyright 1999 Macmillan Magazines.)
ray (CCA). The CCA was then immobilized in a polyacrylamide hydrogen network, forming a polymerized CCA Finally, the silica spheres were etched with hydrofluoric acid, yielding a polymerized CCA of ~100-nm-diameter water voids. The film was soaked in sodium polystyrene sulfonate (NaPSS) solutions of varying molecular masses until equilibrium was established. The chemical composition in the voids and the gel was probed by analyzing diffracted wavelength and intensity changes, both of which depend on the refractive indices. Higher concentrations of NaPSS in
Monitoring protein unfolding
is a promising (but not yet common) technique for examining protein unfolding. A protein's stability can be assessed by Max L. Deinzer and co-workers at Oreexamining the transitions it goes through gon State University studied the thermal while unfolding. Most single-domain prodenaturation of Escherichia coli thioredoxin teins approximate a two-state unfolding (TRX) with H/D exchange and electromechanism, which means that only the spray ionization (ESI) MS. The experifolded and unfolded states are populated ments were performed in a capillary assemat equilibrium. The combination of hydro- bled in a conventional injection valve and gen/deuterium (H/D) exchange and MS directly connected to the infusion capillary of the ESI source. The continuous-flow design allowed the direct monitoring of the unfolding transition. Mass spectra were recorded as a function of temperature over the range of 20-80 °C. MS has been shown to distinguish between different H/D exchange mechanisms during folding transitions. When conditions favor the native state, the mass spectra show a single peak that shifts to higher masses with increasing exchange-in time. If interInstrumentation used for the H/D exchange-in experiments conversion between the monitored online by ESI-MS.
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Analytical Chemistry News & Features, April 1, 1999
the voids than in the gel decrease the refractive index difference between the voids and the gel, decreasing the overall diffraction intensity. Uptake of NaPSS also slightly red-shifts the diffracted wavelength. The NaPSS partition coefficient KnG (NaPSS concentrations in the voids/ NaPSS concentrations in the gel) is shown to increase with increasing NaPSS molecular mass; however, it levels off at the highest molecular masses. KHG is also shown to depend on the NaPSS concentration in the reservoir solution. (Nature 1999,397, 141-44)
folded and unfolded conformations is much faster than the H/D exchange time, two distinct mass peaks develop after a short exchange-in time. For TRX, at temperatures
