To assess the role of charge density on polyelectrolyte mobility, both gel and free solution electrophoresis experiments are performed on poly(acrylic acid) and acrylic acid/acrylamide copolymers. Control of charge density for poly-(acrylic acid) is achieved through solution pH, while control for acrylic acid/ acrylamide copolymers is obtained through chain composition. In either approach, the effective fraction of charged repeat units can be varied from 0 to 100% without a major interruption of solvent quality. Polyelectrolyte mobility in the presence of a monovalent counterion is observed to rise linearly with charge density when this density is low. A transition to charge density independence then occurs over a surprisingly narrow window of charge density. For vinyl polymers of the sort examined here, the transition occurs when 35-40% of the repeat units are charged. These observations are qualitatively consistent with the free solution electrophoresis model proposed by Manning and several previous data sets. An unexpected overlap of normalized gel and free solution data reveals that the charge density exerts a comparable influence in either environment. Results from the present study help define the experimental conditions in which electrophoresis can provide polymer separation by charge density and those in which the method can provide polymer separation by molecular weight.

The favorable bile acid binding characteristics of sevelamer provide a compelling explanation for its ability to lower LDL cholesterol in hemodialysis patients and in healthy volunteers.

Movement of macromolecules through low concentration agarose gels was investigated with linear poly(styrenesulfonate), linear DNA, star-shaped poly(styrenesulfonate), and circular DNA. Mobilities of weakly entangled flexible macromolecules were independent of molecular radius; within a homologous chemical sequence, electrophoretic separation at low field strengths depended solely on the degree of polymerization. These observations cannot be explained either by sieving or by reptation mechanisms; transport was apparently controlled by spatial variations of chain configurational entropy. Only when the chain was highly entangled did chain topology affect mobility. Evidence for entropically regulated transport clarifies how gel electrophoresis separates flexible macromolecules.

The in vitro stability and metabolism of GEM[91, a 25mer phosphorothioate antisense oligonucleotide complementary to the gag mRNA region of HIV-1, was investigated using capillary electrophoresis (CE). The in vitro degradation of the parent compound at 37 degrees C was followed over the course of 120 h in human plasma. A CE method using laser-induced fluorescence detection was able to detect 5'-end intact metabolites including the parent compound extracted from biological fluids. Because the primary metabolic pathway is believed to be via 3'-exonuclease activity, the results of this study were compared with the stability of the compound in a solution containing 3'-exonuclease. The numerical solution of sequential first-order reactions was used to obtain kinetic parameters. Exonuclease digestion of the parent compound, as measured using an automated CE-UV instrument, yielded striking similarities between the two in vitro systems as well as between in vitro and in vivo systems.

For the enzymatic digestion of a 25-mer phosphorothioate (PS) oligonucleotide, the reaction kinetics was previously determined to be the sum of two parallel processes: a fast and a very slow phase of digestion suggesting a two-exponential model. A characteristic metabolite profile was observed both in vitro and in vivo. This behavior is shown to be the result of the stereoselective cleavage of chiral R-configuration and S-configuration PS internucleotide linkages by 3'-exonucleases. The stereoselective nature of 3'-exonuclease action was analyzed by reverse-phase HPLC. The separation of eight diastereomers of the tetramer TTCT (5'-3') was used to follow the stereoselective course of exonuclease hydrolysis of PS internucleotide linkages. Degradation of the 25-mer parent compound having a 3' S-terminal internucleotide linkage was calculated to be more than 300 times slower than an analog with a 3'-terminal R-configuration. These results support an approach for protecting antisense oligonucleotides based on the chirality of only the 3'-end internucleotide linkage.