Hyaluronidase chondroitin

A dodecasaccharide that was obtained by hydrolysis of dermatan sulphate with testicular hyaluronidase, chondroitin lyase AC, and jS-D-glucuronidase yielded a sulphated 2-amino-2-deoxy-D-galactose derivative when treated with human serum at pH 4.5 or 7.0. No further degradation of the substrate occurred, suggesting that normal human serum possesses an cxo-jS-D-acetamidodeoxyhexosidase that is active towards dermatan sulphate. 

M sodium chloride oligosaccharides from hyaluronidase degradation of chondroitin 4-sulfate 89... 

Hyaluronidase from Flavobaaerium and Proteus vulgaris has been shown to hydrolyze chondroitin-4- and -6-sulfate and to possess weak activity toward dermatan sulfec [30]. Hyaluronidase from Streptomyces hyaiurolyacus... 

Several studies have shown that hyaluronidase is iidiibited by ft1, Fe Cu +, Ag+, Hg2+, Zn2+, Cd2+, and Pb2+ salts [26,51,58,77,78]. Substrate analogs like chondroitin sulfate tit desulfeted chondroitin sulfate B, dermatan sulfate, keratan sulfate, heparitin sulfate, and heparin were shown to be competitive inhibitors of hyaluronidase [14,54]. 

Kobayashi S, Fujikawa S, Ohmae M. Enzymatic synthesis of chondroitin and its derivatives catalyzed by hyaluronidase. J. Am. Chem. Soc. 2003 125 14357-14369. 

Histochemical methods will not differentiate between the isomeric chondroitin sulfates, and identification with any degree of certainty requires isolation of the mucopolysaccharide. A method has been reported (M8) that differentiates mucopolysaccharides sulfated at C-6 from other sulfated mucopolysaccharides, and depends on application of the Mor-gan-Elson reaction to the oligosaccharides released by the action of testicular hyaluronidase. It is well known that substitution at C-6 does not interfere with the Morgan-Elson determination of A -acetylhexo-samines, whereas the chromogens are not formed in significant amount if C-4 is substituted. 

Dermatan sulfate may be distinguished from chondroitin 4- and 6-sulfates in that it is not degraded by testicular hyaluronidase and, furthermore, the desulfated mucopolysaccharide is unattacked by testicular and bacterial hyaluronidases (M17). Further diflFerentiation of dermatan sulfate from hyaluronic acid and the foregoing chondroitin sulfates is readily made on the basis of color reactions given by the different uronic acid components. Dermatan sulfate shows equimolar ratios of uronic acid ihexosamine sulfate when the uronic acid content is determined by the orcinol (K7) or decarboxylation (T4) methods, whereas significantly lower values are obtained by the carbazole method (D8). 

This mucopolysaccharide, possessing a strueture similar to those of chondroitin 4- and 6-sulfates but with a small content of sulfate, was isolated from bovine cornea (M16). Chondroitin resembles hyaluronic acid in its rate of hydrolysis by testicular and bacterial hyaluronidases, but was differentiated from hyaluronic acid ([a]n —65° to — 78°) by its optical rotation ( [o]d — 21°). Its structural similarity to chondroitin 4- and 6-sulfates was indicated by the fact that chondrosine was released in high yield on controlled, acidic hydrolysis (D3). The isolation of this mucopolysaccharide is of particular interest since it may be a precursor in the biosynthesis of chondroitin 4- and 6-sulfates. 

Commercial preparations usually contain this type of hyaluronidase. The enzyme is found in testicular tissue of most mammals and is located in the acrosomal cap of spermatozoa [6], Testicular hyaluronidase degrades hyaluronan, chondroitin, chondroitin-4- and -6-sulfate to oligosaccharides, mainly tetrasaccharides [1]. Partial degradation of dermatan sulfate has been described [7]. Testicular hyaluronidase had a broad pH range of activity [5]. 

Bollet et al. [11] demonstrated the presence of hyaluronidase activity in various mammalian tissues. They showed that this type of hyaluronidase differed from the testicular type concerning pH optimum and pH range of activity. Subsequent studies revealed that the enzyme was present in the lysosomal fraction of the tissues [12]. The liver is an especially rich source [13]. Degradation of hyaluronan leads to the same end products as testicular hyaluronidase [11]. Lysosomal hyaluronidase from rat liver degrades chondroitin-4- and -6-sulfate, but not dermatan sulfate, desulfated dermatan sulfate, heparan sulfate, keratan sulfate, or heparin [14], Lysosomal hyaluronidase has an acid pH optimum and a narrow pH range of activity [14]. This difference in pH profile of activity has commonly been used to differentiate between testicular and lysosomal hyaluronidase. A similar acid-active hyaluronidase is present in human serum [15]. 

Animal venoms usually possess hyaluronidase activity [17]. The enzymatic properties, including hyaluronidase, of snake venoms have been extensively studied by Tan et al. [18]. Snake hyaluronidase acts on hyaluronan, chondroitin, and chondroitin-4- and -6-sulfate, producing various oligosaccharides, mainly tetrasaccharides [1]. 

Hyaluronidase from bee venom has about the same substrate specificity as snake hyaluronidase [19]. Venom of social wasps was found to contain high levels of hyaluronidase activity, whereas venom from ants contains low levels of activity [20]. Lizard venom contains a hyaluronidase that acts almost specific on hyaluronan, i.e., it has no activity toward chondroitin-6-sulfate, dermatan sulfate, or heparin and only weak activity toward chondroitin-4-sulfate [21]. 

A hyaluronidase has been purified and characterized from stonefish (Synanceja horrida) venom. It acts specifically on hyaluronan, producing tetra-, hexa-, octa-, and decasaccharides, but does not act on chondroitin sulfate or dermatan sulfate [22,23]. 

In this chapter we describe some methods used to determine the kinetics of the action of hyaluronidase. Table 2 presents a survey of the Michaelis-Menten constants (Km) of the action of hyaluronidase on hyaluronan and chondroitin sulfate obtained using different methods. These assays usually make use of hyaluronan as a substrate for hyaluronidase. Various sources of hyaluronan are employed, but these substrates have different physicochemical properties (molecular weight, intrinsic viscosity). Payan et al. [130] investigated the action of Streptomyces hyaluronidase on hyaluronan from several sources. 

The current method for the hyaluronidase assay described in the United States Pharmacopeia (USP) [132] is based on the inability of hydrolyzed potassium hyaluronate to form a complex precipitate with proteins from added serum, reflected in a decreased turbidity of the reaction mixture (measured after 30 min). The method is, from the enzymological point of view, not well defined since it does not actually evaluate the kinetics of the hydrolysis of the substrate. An assay with end-point determination is only valid if the reaction rate does not change during this reaction time. We found that only with the two lowest test concentrations (0.15 and 0.3 IU) was this condition fulfilled, while with the three higher test concentrations the reaction is not linear. Commercially available hyaluronates can be contaminated with chondroitin sulfates. They are more acidic than hyaluronic acid itself and hence can form better protein complexes and influence the turbidity. In a suitability test of the USP [133], the substrate must pass both an inhibitor content test and a turbidity-production test. The assumption is made that... 

P. Hoffman, K. Meyer, and A. Linker. Transglycosylation during the mixed digestion of hyaluronic acid and chondroitin sulfate by testicular hyaluronidase. J. Biol. Chem. 279 653 (1956). 

Furthermore Kobayashi et al. were able to show that hyaluronidase is able to catalyze the in vitro synthesis of hyaluronan [228], chondroitin [229], chondroitin sulfate [230], their derivatives [229, 231] and of non-natural GAGs [232]. 

Hyaluronidase (HAase systematic name hyaluronate 4-glycanohydrolase EC 3.2.1.35) is a glycoside hydrolase that cleaves (l-4)-(i-N-acetylhexosaminide linkages in hyaluronan, chondroitin, chondroitin sulfate. 

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