Heparinase-II-catalyzed degradation of N-propionylated heparin.

Currently there is great interest in the preparation of modified heparins and heparin-like polymers that possess specific and useful bioactivities. This paper demonstrates the potential of a particularly versatile endopolysaccharide lyase (heparinase II) as an analytical tool with which to assess both the chemical modification occurring during synthesis of such polymers and the actual primary structure of the final product of the enzyme activity. Additionally, the work widens our knowledge of the specificity range of this enzyme. The study involved a novel derivative of heparin containing the unnatural N-propionyl group, which was prepared from de-N-sulfated heparin. The extent of the chemical modification was followed throughout the preparation process by incubating samples with heparinase II and analyzing, with HPLC, the products of degradation catalyzed by the enzyme.

Zn(2+)-heparin interaction studied by potentiometric titration.

Measurement of the decrease in pH that accompanies the addition of Zn2+ to heparin in solution provided an indirect method of examining cation-polyanion interaction. Construction of plots analogous to isothermal saturation binding plots revealed the existence, for defined conditions of interaction, of a [heparin]-independent direct proportionality between the fraction of the maximal pH change occurring and the [Zn2+]/[heparin disaccharide] ratio. This accords with results from polarimetric examination of Ca(2+)- and Cu(2+)-heparin interactions. It suggests that, under the conditions used, cation-heparin interaction may result in the formation of a complex that exists in a colloid-like phase, between which and the aqueous phase, exchange of cations does not follow simple solution-phase reversible equilibrium thermodynamic behaviour. The results suggest that the putative Zn(2+)-containing complex is less stable in the presence of NaCl than is the corresponding Ca(2+)-containing complex. Addition of Zn2+ to low concentrations of heparins is accompanied by the usual decrease in pH, followed by a removal of H+ from solution as the [Zn2+]/[heparin disaccharide] ratio increases, suggesting dissolution of the putative complex. This reversal of the initial pH change was not seen for most other cation-heparin interactions under the conditions studied.

Inhibition by heparin of Fe(II)-catalysed free-radical peroxidation of linolenic acid.

Heparin, in a concentration-dependent manner, inhibited the generation of conjugated dienes and thiobarbituric acid-positive substances when incubated with Fe2+ and gamma-linolenic acid. In the conjugated diene assay, other glycosaminoglycans, on a molar basis calculated with respect to their respective hydrated disaccharide repeat units, were less effective than heparin. Heparin which had been re-N-sulphonated after removal of both N-sulphonates and O-sulphates, and heparin in which iduronate residues had been reduced to idose residues, were largely unaffected in their activity. Removal of both N-sulphonates and O-sulphates greatly reduced the effectiveness of the heparin. Analysis of the effects of heparin fragments generated by heparinase I treatment of heparin showed that depolymerization decreased the effectiveness of the heparin. It is possible that heparins and related strongly acidic polysaccharides may function as endogenous antioxidants, and that sequestration by them, or harmless oxidation by them, of ions such as Fe2+, contributes to their effectiveness.

A potentiometric titration study of the interaction of heparin with metal cations.

Potentiometric titrations, at ionic strengths (I) ranging from 0.0057 to 0.336, suggest that Ca2+, Cu2+, Li+ and Na+ bind to heparin in a manner that depends on cation identity. These interactions were less affected by the value of I than those of heparin with Mg2+, which binds weakly below I0.050 and with K+, which binds weakly at I0.0057. Of the interactions studied, that of heparin with Cu2+ was the least readily reversible.

A putative role for colloidal silicates in primitive evolution deduced in part from their relevance to modern pathological afflictions.

Biogenic and bioactive silicas have structural subtleties that are only now beginning to be appreciated. Their ability to interact with particular biological systems is related to these structures. Some of these interactions result in pathological effects. It is suggested that such interactions mirror events occurring in a prebiotic environment.

Degenerative and inflammatory diseases may result from defects in antimineralization mechanisms afforded by glycosaminolglycans.

Many human cellular and tissue compartments are supersaturated with respect to calcium oxyanion salts. In order to prevent the formation of injurious crystals efficient anti-crystallization protective mechanisms must be necessary. We suggest that depletion of such systems, particularly in ageing organisms and under conditions of oxidative stress, plays an important role in degenerative and inflammatory diseases, including cancer.

Cu(2+)-heparin interaction studied by polarimetry.

Plots analogous to isothermal binding curves were derived from polarimetric studies of solutions of Na(+)-heparin to which Cu2+ was added. The shape of the plots suggests that discrete Cu(2+)-heparin complexes may form, the nature and stoichiometry of which depend upon the conditions under which the interaction occurs. Initially, complexes were formed with a stoichiometry of about 1 Cu2+ ion per heparin tetrasaccharide unit. Under these conditions, the complex may exist as a colloid-like phase that is not subject to simple solution equilibrium processes, and that is maintained by forces in addition to electrostatic ones. Further addition of Cu2+ resulted in the formation of a different complex with a stoichiometry of about 1 Cu2+ ion per disaccharide unit.

A study of Ca(2+)-heparin complex-formation by polarimetry.

Possible inflexions in isothermal binding plots derived from equilibrium-dialysis measurements and in equivalent plots obtained by polarimetric measurements accord with the possibility that discrete Ca(2+)-heparin-water complexes/phases may exist, the nature and proportions of which depend on the conditions under which the interaction occurs. Analysis of the plots obtained by polarimetric study of chemically modified heparins suggests, for individual substituents groups, an order of importance of carboxylate greater than N-sulphonate greater than N-acetyl greater than O-sulphate for the Ca(2+)-heparin interaction occurring at [Ca2+]/[heparin disaccharide] ratios of less than 0.5. At higher ratios, transitions occur that eventually lead to the formation of a complex in which the stoichiometry of association is 1 Ca2+ ion/heparin disaccharide unit.