Inhibition Of Gelation Research Articles

Viscosity-Reducing Bulky-Salt Excipients Prevent Gelation of Protein, but Not Carbohydrate, Solutions.

The problem of gelation of concentrated protein solutions, which poses challenges for both downstream protein processing and liquid formulations of pharmaceutical proteins, is addressed herein by employing previously discovered viscosity-lowering bulky salts. Procainamide-HCl and the salt of camphor-10-sulfonic acid with L-arginine (CSA-Arg) greatly retard gelation upon heating and subsequent cooling of the model proteins gelatin and casein in water: Whereas in the absence of additives the proteins form aqueous gels within several hours at room temperature, procainamide-HCl for both proteins and also CSA-Arg for casein prevent gel formation for months under the same conditions. The inhibition of gelation by CSA-Arg stems exclusively from the CSA moiety: CSA-Na was as effective as CSA-Arg, while Arg-HCl was marginally or not effective. The tested bulky salts did not inhibit (and indeed accelerated) temperature-induced gel formation in aqueous solutions of all examined carbohydrates-starch, agarose, alginate, gellan gum, and carrageenan.

Direct visualization of changes in deacylated Na(+) gellan polymer morphology during the sol-gel transition.

Changes in gellan polymer morphology during the sol-gel transition were directly visualized by transmission electron microscopy and a model incorporating these changes and existing physical data is proposed. Our observations suggest that the most thermodynamically stable conformations of gellan polymers in solution, in the absence of added cations, are the double helix and double-helical duplexes. We have demonstrated two forms of lateral aggregation of gellan helices in the presence of Ca(2+) and K(+) ions. One type forms junction zones that lead to network formation and gelation, while the second type leads to the formation of isolated fibers of aggregated helices and inhibition of gelation. The proposed model of gellan gelation is based on these observations where thermoreversibility, gel strength, and endothermic transitions of gellan gels can be explained.

Direct visualization of changes in deacylated Na+ gellan polymer morphology during the sol-gel transition

Changes in gellan polymer morphology during the sol-gel transition were directly visualized by transmission electron microscopy and a model incorporating these changes and existing physical data is proposed. Our observations suggest that the most thermodynamically stable conformations of gellan polymers in solution, in the absence of added cations, are the double helix and double-helical duplexes. We have demonstrated two forms of lateral aggregation of gellan helices in the presence of Ca(2+) and K(+) ions. One type forms junction zones that lead to network formation and gelation, while the second type leads to the formation of isolated fibers of aggregated helices and inhibition of gelation. The proposed model of gellan gelation is based on these observations where thermoreversibility, gel strength, and endothermic transitions of gellan gels can be explained.

Potential roles of hypochlorous acid and N-chloroamines in collagen breakdown by phagocytic cells in synovitis

We have tested the effects of the neutrophil/macrophage products, hypochlorous acid (HOCl) and N-chloroamines, on the structural integrity and proteolytic susceptibility of collagen to determine if these agents could play a role in inflammatory joint destruction. Rates of HOCl reaction with collagen, and collagen gelation were monitored by spectrophotometric methods. Direct fragmentation, and degradation by collagenase were measured by the release of acid-soluble counts from 3H-collagen. Physiologically relevant concentrations of HOCl (5–50 μM) reacted rapidly and quantitatively at several sites in the collagen polypeptide chain, causing extensive protein fragmentation and preventing collagen gelation. In contrast, reaction with (5–50 μM) N-chloroalanine induced little direct collagen fragmentation. Oxidative damage by N-chloroamines was, however, evident because collagen displayed greatly increased proteolytic susceptibility following N-chloroamine treatment. Collagen degradation by collagenase increased as much as 3-fold after exposure to N-chloroamine treatment. Collagen degradation by collagenase increased as much as 3-fold after exposure to N-chloroalanine. N-chloroleucine caused a small increase in proteolytic susceptibility, but N-chlorotaurine had no effect. Collagen fragmentation by HOCl, inhibition of gelation by HOCl, and N-chloroalanine-induced proteolytic susceptibility, all increased with linear kinetics at oxidant concentrations of 5 μM to 1.0 mM. In synovitis, phagocytes expose collagen to HOCl, N-chloroamines, and collagenase. It is known that HOCl can activate neutrophil procollagenase. Based on our new findings, we propose a model of inflammatory joint destruction that also includes collagen fragmentation, and increased susceptibility of collagen to degradation by collagenase. It may also be possible that taurine exerts a protective effect against HOCL/OCL − damage by reacting to form what appears to be essentially an inert N-chloroamine. The validity of this model must now be tested.

Effect of Salts on the Thiol-dependent Gelation of Bovine Serum Albumin.

A previous report [M. Hirose, Y. Nishizawa and J. Y. Lee, J. Food Sci., 55, 915 (1990)] has shown that the thio-dependent gelation of bovine serum albumin was strongly inhibited by high concentrations of salts. The inhibition mechanism was investigated in relation to the conformation and disuliide cleavage of the protein. The effects on the thiol-dependent disulfide cleavage of the protein varied with the salt species, especially with the anion species. The circular dichroism spectra and difference UV-spectra showed that anions induced some conformational changes in the tertiary structure, but not in the secondary structure. With regard to a chaotropic anion, perchlorate, this conformational change was closely correlated with the inhibition of gelation and disulfide reduction. In contrast, a lyotropic anion such as sulfate inhibited the gelation at a higher concentration than the concentrations that induced the conformational change. In addition, this anion showed no inhibition effect on disulfide reducti...

Purification and characterization of actinogelin, a calcium-sensitive actin-accessory protein, from rat liver.

Although cell-free extracts prepared from several types of free-living cells, including Ehrlich tumor cells, macrophages and sea-urchin eggs, readily form gels under low Ca2+ conditions, no such ability to induce actin-related gel has been detected in tissue-cell extracts. Ca2+ -insensitive gelation activity was discovered, however, in several tissue-cell extracts, including liver and brain, provided that the extracts were supplemented with skeletal muscle actin. Based on sodium dodecylsulfate/polyacrylamide gel electrophoretic analysis of the gel, these extracts seem to contain both a Ca2+ -insensitive gelation factor and Ca2+ -sensitive one, actinogelin. A procedure for purification of actinogelin from rat liver was developed, and the properties of actinogelin thus purified were compared with those of Ehrlich tumor cell actinogelin. No appreciable difference was found in these two proteins, and Ca2+ sensitivity (50% inhibition of gelation at 1 microM) was very similar. Some of the molecular characteristics are described, and the importance of the presence of actinogelin in tissue cells is discussed.

Actin-related gelation of Ehrlich tumour cell extracts is reversibly inhibited by low concentrations of Ca2+

THE intracellular filament system has been shown to have an important role in several biological phenomena1–5, but the mechanisms of control of the organisation of these filaments are little understood. Temperature-induced gelation of cell-free extracts6–11 provides a convenient system for studying interaction of cytoskeletal components in cell-free conditions. Actin and a high molecular weight (MW) actin-binding protein (MW 270,000) (ABP) are concentrated in the gel formed by warming of mammalian cell extracts9. In other cell types, protein other than actin and ABP are also required for gelation, such as 58,000 MW protein in sea urchin egg extracts10 and four low molecular weight proteins (23,000 to 38,000) for amoeba extracts11. A requirement of ATP for gelation6 and inhibition of the reaction by cytochalasin B8,9 have been found recently. We now describe reversible inhibition of gelation by micromolar concentration of Ca2+ and possible involvement of this type of regulation in a virus-induced cell fusion.