sepharose and sodium-chlorate

The crystallization of sodium chlorate (NaClO3) is a classic example of spontaneous chirality, since it is achiral in solution but adopts a chiral form in the solid state. While crystal growth of NaClO3 from pure aqueous solutions yields a 50:50 statistical distribution of d- and l-crystals, large enantiomeric excesses of either d- and l-crystals can be achieved by crystal growth in agarose gel, a naturally occurring chiral polysaccharide. The influence of gel density (0.1-0.75 wt %), temperature, and the diffusion of cosolvents on crystal distribution was discerned from statistical data obtained from 752 gel-mediated crystallization experiments yielding 12,384 individual crystals. These studies demonstrate that the magnitude and direction of the bias can be selectively engineered toward either d- or l-forms by changing the gelation conditions. Aqueous agarose gels infused with 48 wt % NaClO3 at 6 degrees C, favored the growth of d-NaClO3 crystals, with ee's reaching 22% at the highest gel concentrations. Crystal growth under methanol diffusion favored deposition of the opposite enantiomorph, l-NaClO3. The bias in the crystal distribution is enhanced at higher temperatures. Aqueous gels at 24 degrees C infused with methanol cosolvent favored l-NaClO3, with ee's reaching 53%. The changing magnitude and direction of the enantiomorph bias can be ascribed to differences in the agarose conformation and intermolecular interactions between the gel and crystal surfaces that inhibit the formation of the two enantiomers to different extents.

Topics: Chlorates; Crystallization; Hydrogels; Sepharose; Stereoisomerism

Glycosaminoglycans (GAG) are a group of negatively charged molecules that have been shown to bind and directly regulate the bioactivity of growth factors and cytokines such as basic fibroblast growth factor, transforming growth factor-beta, IL-7, and interferon-gamma. The ability of GAG to interact with human IL-10 (hIL-10) and the effect of these interactions on its biologic activity were analyzed. It was demonstrated by affinity chromatography that hIL-10 binds strongly to heparin-agarose at physiological pH. Biosensor-based binding kinetic analysis indicated an equilibrium dissociation constant, K(d), of 54 nmol/L for this interaction. Human IL-10 stimulated CD16 and CD64 expression on the monocyte/macrophage population within peripheral blood mononuclear cells, with optimal concentrations between 1 and 10 ng/mL. Soluble heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate were shown to inhibit the hIL-10-induced expression of CD16 and CD64 in a concentration-dependent manner. Heparin and heparan sulfate were most effective with IC(50) values of 100 to 500 microg/mL. Considerably higher concentrations of dermatan sulfate and chondroitin 4-sulfate were required with an IC(50) of 2,000 to 5,000 microg/mL, whereas chondroitin 6-sulfate was essentially inactive. The antagonistic effect of heparin on hIL-10 activity was shown to be dependent on N-sulfation, inasmuch as de-N-sulfated heparin had little or no inhibitory effect on the IL-10- induced expression of CD16, whereas the effect of de-O-sulfated heparin was comparable to that of unmodified heparin. Furthermore, the inhibition of cell-bound proteoglycan sulfation reduced the hIL-10-mediated expression of CD16 molecules on monocytes/macrophages. Taken together, these findings support the hypothesis that soluble and cell-surface GAG and, in particular, their sulfate groups are important in binding and modulation of hIL-10 activity. (Blood. 2000;96:1879-1888)

Topics: Amino Acid Sequence; Antigens, CD; Binding, Competitive; Cells, Cultured; Chlorates; Chromatography, Affinity; Dose-Response Relationship, Drug; Flow Cytometry; Glycosaminoglycans; Heparin; Heparitin Sulfate; Humans; Interleukin-10; Kinetics; Leukocytes, Mononuclear; Macrophages; Molecular Sequence Data; Monocytes; Receptors, IgG; Recombinant Proteins; Sepharose