Hyaluronidase substrates

Daoust R. Histochemical localization of enzyme activities by substrate film methods Ribonucleases, proteases, amalyses and hyaluronidase. Int Rev Cytol 1965 18 191-221. 

Lysosomal hyaluronidase possesses transglycosylation properties using octo-saccharides as a substrate, rather than hexasaccharides like testicular hyaluronidase [14, lti]. 

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]. 

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

T. Hirsyama, T. Hasegawa, and M. Hiioi. The measurement of hyaluronidase activity in human spermatozoa by substrate slide assay and its clinical application. Fen Stcr. 51 330(1989). 

Hyal-1, an acid-active lysosomal enzyme, was the first somatic hyaluronidase to be isolated and characterized.191,192 It is a 57 kDa single polypeptide glycoprotein that also occurs in a processed 45 kDa form, the result of two endoprotease reactions. The resulting two chains are bound by disulfide bonds. This is not a zymogen-active enzyme relationship, since the two isoforms have similar specific activities. Why two forms should occur is unknown. Only the larger form is present in the circulation, while both isoforms occur in urine,193 in tissue extracts, and in cultured cells. Why an acid-active hyaluronidase should occur in plasma is not clear. Some species do not have detectable enzymatic activity in their circulation,194 but an inactive 70 kDa precursor form of the enzyme is present in such sera, detectable by Western blot (L. Shifrin, M. Neeman, and R. Stern, unpubl. data). Hyal-1 is able to utilize HA of any size as substrate, and generates predominantly tetrasaccharides. 

The catabolic scheme, formulated only recently [173,174], proposes that the high molar mass HA polymer is cleaved in a series of controlled enzymatic scissions, in which the product of one reaction becomes the substrate for the subsequent reaction. These successive enzymatic reactions generate oligomers of ever-decreasing sizes. It may be reasonable to assume that these very enzymes are responsible for the supply and maintenance of size-specific oligomers. However, there is no evidence to date that the hyaluronidases perform such a function. 

The vertebrate hyaluronidases, previously neglected enzymes [185], are all endo-P-A-acetylhexosaminidases employing substrate hydrolysis as their mechanism of action. These hydrolases have, until recently, been difficult to assay. Turbidometric and... 

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]. 

Hyaluronidase has an absolute requirement for cations, as dialyzed preparations show no activity toward dialyzed substrates. K+, Na+, Ca2+, Mg2+, and Mn2+ all have stimulating properties toward hyaluronidase. The monovalent cations show the strongest stimulating effects and act over a broader range of concentration than divalent cations [50]. Testicular, serum, and lysosomal hyaluronidase seem to have different sensitivities toward NaCl [54,61]. A noncompetitive mechanism was proposed to explain the activation of hyaluronidase by Na+ [59,62]. 

Table 2 Michaelis-Menten (Km) Constants of the Action of Hyaluronidase from Different Sources on Different Substrates...

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... 

The European Pharmacopoeia and HP standard viscosimetric method is described in Chapter IS. In the proposed method, both hyaluronidase and hyaluronan are available as well-defined stable standard preparations, eliminating variations in test results due to the absence of a well-defined substrate. With slight modifications the method can be used for the determination of hyaluronidase in serum [137]. 

The response (a decrease of viscosity) is a direct consequence of the action of the enzyme on its substrate, since the splitting of the glycosidic bonds gives a decrease in the viscosimetric average molecular weight and hydrodynamic volume of the hyaluronan chains and hence a decrease in the intrinsic and relative viscosities. With this method the rate at different concentrations cannot be compared because the initial viscosities are different and rheological measurements do not coincide. To eliminate these problems, a kinetic dilution methodology for the viscosimetric study of the substrate concentration dependence of the action of hyaluronidase was proposed [135,136]. We were able to determine the rate of reaction, expressed as the number of moles of bonds broken per unit of time, from viscosimetric data [136]. 

The substrate for this assay consists of hyaluronan with the reducing terminals labeled with the fluorogenic reagent 2-aminopyridine [144], It should be noted that the reaction products by hyaluronidase also contain oligosaccharides that do not contain the fluorogenic end group. As these products are not detectable by this method, not all scissions occurring in the substrate are detected. 

J. Demeester, O. M. Awad, M. Bracke, and A. Lauwers. Kinetic dilution methodology for the viscosimetric study of the substrate concentration dependence of hyaluronidase action. Fres. Z Anal Chem. 330 366 (1988). 

T. Nakamura, M. Majima, K. Kubo, K. Takagaki, S. Tamura, and M. Endo. Hyaluronidase assay using fluorogenic hyaluronate as a substrate. Anal Biochem. 797 21 (1990). 

Heparin, de-esterified nitrated hyaluronic acid, the related sulphonic acid dyes (e.g. chlorazol fast pink) and a non-specific inhibitor of rabbit and human serum, all inhibit various hyaluronidases from different sources but there are great differences, depending on substrate, enzyme and inhibitor. As inhibitors, cellulose trisulphuric acid and chitin disulphuric acid are much stronger, while treburon is similar to heparin. 

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