Untitled

Abstract

The glycosaminoglycan microenvironment of testicular hyaluronidase was simulated by multipoint covalent attachment of the enzyme to glycans as a result of benzoquinone activation. The efficiency of their binding was assessed using gel chromatography, ultrafiltration, titration of surface amino groups of the enzyme, electrophoresis, as well as judging by the value of residual endoglycosidase activity and its inhibition with heparin. Copolymer glycosaminoglycans, such as dermatan sulfate and heparin, inactivated the endoglycosidase activity as a result the C-5 epimerization of hexuronic acid. It was shown that glucuronic acid and, to a lesser extent, N-acetylglucosamine determine the specificity of hyaluronidase. The chondroitin-sulfate microenvironment made the enzyme resistant to heparin inhibition because the equatorial orientation of the OH groups is similar to that in hyaluronic acid. Model experiments with dextran and dextran sulfate showed that sulfation of the glycan chain increased its rigidity, thus hampering the stabilizing effect on hyaluronidase. The effect of chondroitin sulfate on the endoglycosidase activity of hyaluronidase had additive character and did not directly affect the small fragment of the active site of the enzyme located at the bottom of a groove. The glycosaminoglycan microenvironment of hyaluronidase, containing an iduronic acid residue, the alpha1-3 and alpha1-4 glycosidic bond, inactivated the hyaluronidase activity of the enzyme, whereas simple polymers (such as gluco- and galactoaminoglycans) potentiated it due to a similar way of linking--beta(1e-4e) and beta(1e-3e). To understand the nature of these interactions in detail, the effect of oligomeric glycosaminoglycan fragments and their derivatives on hyaluronidase should be studied.

Electrophoresis studies on the contaminating glycosaminoglycans in commercially available hyaluronic acid products

“…It has been reported that GAGs having iduronic acid residues in their disaccharide units such as heparin or DS (Fig. 1B) can be a potent inhibitor of HAases from testis and snake venom [19,20]. Therefore, we concluded that HA samples that were not digested with testicular HAase were due to the presence of contaminated DS, although we did not perform any studies using an antibody specific to DS.…”

Improved pharmacokinetics and stability properties of catalase by chemical glycosidation with end‐group activated dextran

“…16,17 In the present work, dextran was functionalized at its reducing end-group D-glucose residue with a reactive carboxylate group by treatment with a molar excess of e-aminocaproic acid in the presence of sodium cyanoborohydride. The activated dextran was further attached to the free amino groups located at the protein surface of catalase by using a water-soluble carbodiimide as coupling agent.…”

Do electrostatic interactions determine glycation of hyaluronidase derivatives with N-acetylhexosamines?

“…The first approach was realized by stabilization of proteins against glycation by creation of polysaccharide envi ronment around them. This was achieved by multiple covalent attachment of such glycosaminoglycan as chondroitin sulfate (CS) to a known carbohydrase hyaluronidase (HU) [9]. Resultant HU CS derivatives exhibited higher stability than native HU during glyca * To whom correspondence should be addressed.…”

“…Hyaluronidase chondroitin sulfate conjugate (HU CS) was prepared as described earlier [9]. Remaining endoglycosidase activity of HU CS repre sented 76⎯78% of initial enzyme activity and the modification degree of amino groups was 84⎯86%.…”

“…Protein content in native and modified HU preparations was determined by the method of Bradford [19]. Titration of surface amino groups of HU derivatives with trinitrobenzene sulfonic acid was performed as recommended in [9][10][11]20].…”