Hyaluronidase, polymerization

The enzymatic polymerization to provide synthetic Ch and its derivatives catalyzed by hyaluronidase has been reported by Kobayashi et al. (113). Synthetic oxazoline monomers were recognized and catalyzed by ovine testicular HAase (OTH) to produce Ch and the polymerization behaviors greatly depend on the reaction conditions. The Mn value of synthetic Ch reached 4600, which corresponds to that of naturally occurring Ch. 

Kobayashi and coworkers also showed that hyaluronidase is able to catalyze the enzymatic polymerization of chondroitin [229], chondroitin sulfate [230], their... 

Another class of polymers where hydrolases are used successfully comprises polysaccharides. This approach has been pioneered by Kobayashi et al (3c,36) and is exemplified by their article (18) where hyaluronan and chondroitin were prepared via hyaluronidase-catalyzed polymerizations. A slightly different approach is to use glycosidases to prepare oligosaccharides (17,37). 

The present paper focuses on precision synthesis of natural HA (46) and Ch (47) with well-defined structures of biological importance, via hyaluronidase-catalyzed polymerization of sugar oxazoline derivatives (Scheme 1). Similarly, unnatural Chs (6) were prepared using the same enzyme (47) (Scheme 2). These reactions provide a facile and efficient approach to synthesis of GAGs with well-defined structures. 

The quantitative determination of hyaluronidase is based on its activity which is customarily expressed in empirically defined units. The activity is measured by the resulting transformation of the substrate, hyaluronic acid. In vitro, this is accomplished by estimating the changes in the high polymeric properties of the substrate, by determining the amount of remaining unreacted substrate, or by measuring the products of hydrolysis. 

Hyaluronidase activity was originally determined biol( cally by the intradermal spread of colored indicator solutions and chemically by the reductometric method. The purification of hyaluronidase and the study of its mechanism of action depended on the development of assay methods more accurate than the spreading effect and less time consuming than the determination of reducing substances. New methods were based on certain high polymeric properties of hyaluronic acid (a) the viscosity of aqueous solutions of the polysaccharide, and (b) the formation of mucin clots with acidified proteins. 

Ragan and Meyer (354) have recently reported hyaluronic acid contents and relative viscosities of synovial fluids from the knee joints of 35 patients with rheumatoid arthritis and 11 patients without joint diseases. They found with very few exceptions that the apparent degree of polymerization of the hyaluronic acid was lower in the arthritic patients, although the total amount of the mucopolysaccharide present was greater. Hyaluronidase was not detected in joint fluid or in synovial or periarticular tissue and, in any event, its presence would not explain the increase in total hyaluronic acid. Therefore, it was concluded that the defect lies not in an enzymatic hydrolysis or nonspecific depolymerization but in abnormal synthesis of this polysaccharide, involving an increased production of incompletely polymerized hyaluronic acid. 

Polymeric phosphates of diethylstilbestrol, dienestrol, hexesterol, and benzesterol are reported by Diczfaluzy et al. (1959) to be potent inhibitors of j3-gIucuronidase as well as of phosphatase and hyaluronidase. 

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