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Abstract

Properties of native and aldehyde dextran-modified hyaluronidase (with surface amino group modification about 98%) were investigated. Optimal endoglycosidase activity of the native enzyme was observed at 0.15 M NaCl and pH 5.5 and electrostatic interactions influenced the enzyme activity. The inhibitory effect of heparin on hyaluronidase activity slightly differed at pH 5.5 (1.5-fold inhibition) and 7.5 (1.2-fold inhibition). Ionic strength of the reaction medium only slightly influenced the effect of heparin. Modification of hyaluronidase with dextran increased hydrophobic interactions and steric hindrance. Conjugation with dextran increased the resistance of hyaluronidase activity to denaturing agents (urea, guanidinium hydrobromide) and extended the optimal conditions for maximal endoglycosidase activity (pH 4.5-6.5, the range of NaCl concentration from 0.1 to 0.3 M). The conjugation also reduced electrostatic effects on the active site of hyaluronidase and efficacy of heparin inhibition. At pH 7.5 the enzyme was almost insensitive to heparin. The resistance of dextran-modified hyaluronidase to heparin points to approaches for subsequent studies of the heparin-binding site of this enzyme and biomedical trial of the stabilized enzyme for the treatment of acute cardiovascular lesions.

Improved Pharmacokinetics Properties for Catalase by Site‐Specific Glycosidation with Aminated Dextran

“…This value agrees with the result achieved by SDS/PAGE analysis. On the other hand, in contrast to other polymer-catalase conjugates previously described in the literature, [13,21] the conjugate prepared with monoaminated dextran showed an increased catalytic activity in comparison with the native enzyme. This fact suggests that hydrophilization of the enzyme protein surface with dextran yielded a more active enzyme conformation, as well as that the catalytic active site in catalase was not blocked by the macromolecular chains of the polysaccharide.…”

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

“…Different con formation of the native and modified enzyme (curve 4 does not reach the position similar to that of curves 1 and 2, Fig. 2), their different pH dependence (differ ent shape and position of the curves on Fig 2a and 2b, respectively), multistage formation of various glyca tion products especially after prolonged incubation play evident roles in this process [9][10][11]15]. In addi tion, bovine testicular HU preparation may be con taminated with N deacetylase, an accompanying enzyme that catalyzed conversion of N acetylglu cosamine at the reducing end into glucosamine [22].…”

“…MATERIALS AND METHODS Testicular hyaluronidase (EC 3.2.1.35) from bovine testes (Immunopreparat, Ufa, Russia) was used for preparation of modified form of HU and study of N acetylhexosamine action on functioning of HU deriv atives; the commercial enzyme was further purified by gel chromatography on Sephadex G 100 (Pharmacia, Sweden) as described in [15]. The following reagents were purchased from Sigma (USA): human umbilical hyaluronic acid potassium salt (average molecular mass of 700-800 kDa), bovine high molecular weight heparin sodium salt (16)(17)(18), chondroitin 4 sul fate from bovine trachea (average molecular mass of 30-50 kDa), trinitrobenzene sulfonic acid, N acetyl glucosamine and N acetylgalactosamine.…”

“…Resistance of HU derivatives to inhibition by hep arin was evaluated by their remaining endoglycosidase activity assayed in the presence of heparin excess (the ratio of weight concentrations hyaluronidase/heparin was 1 : 100) [9,15]. Heparin (0.2 mg/mL) was added in 0.1 M phosphate buffer, pH 5.5, to the reaction mixture containing the same protein concentration (2 µg/mL) and endoglycosidase activity of HU deriv atives was assayed viscosimetrically after incubation at room temperature for 2⎯4 min.…”

Polymeric micelles for oral drug delivery: Why and how

“…Polymeric micelles formed by amphiphilic DEXs of structure similar to that of DEX10-g-PEO y -C n , but prepared from a higher molecular weight DEX (Dextran T40; MW 40 000 Da; DEX40) are larger than those formed by DEX10-g-PEO y -C n , independently of the size of the hydrophobic substituent and of the level of modification [93]. This observation can be taken as an indication of the steric hindrance induced by the carbohydrate chains, which are expected to take place over a larger volume for the polymer of higher molecular weight [94,95]. It has been shown that the uptake of particles within the intestine and the extent of drug absorption increase with decreasing particle size and increasing specific surface area [6].…”