Hyaluronidase, assay

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... 

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). 

Hyaluronidase is involved in bacterial and fungal infections because of virulence factors evoked by tissue degradation and mediates host-pathogen interactions [47]. Since hyaluronic acid (HA) is a major component of the extracellular matrix involved in joint lubrication, a sensitive hyaluronidase assay is important. Current hyaluronidase assays rely on turbidimetric techniques that require high levels of the enz3Tne and are relatively inaccurate [47]. HA was previously shown to bind cyanines [48,49[. The detection scheme designed for CMA, CMC, and amylase enzyme described earlier was also applicable to HA and hyaluronidase activity [19]. Scaffold Destruction ... 

Apart from a report of the rather surprising use of indoxyl acetate as a fiuorogenic substrate (Guilbault et al., 1967 Rhodes el al., 1970), conventional hyaluronidase assays employ giycosaminoglycans for this purpose. Procedures have also been described for the visualization (Abrahamson and Friedman, 1967) and histochemical location (Davost, 1965) of hyaluronidases. 

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. 

An international standard for hyaluionidase was accepted in 1957 by the World Health Organization (WHO) [131]. This was of gnat historical value, since hyaluronidase is one of the fust biological international standard reference preparations ever made. The International Unit (IU) was and is still defined as the activity of 0.1 mg of the International Standard Preparation. It is recommended that the standard be employed for the assay of hyaluronidase from testicular origin (RC.3.2.1.35). According to the WHO, the standard should be valid for all methods of comparison, including turbidimetric and visco si metric assays. The first international WHO standard 1955 hyaluionidase is still available in tablet form with an activity of approximately 200 IU per tablet... 

These assays are used to confirm the presence of hyahuonidase in a preparation. Quantification can only be achieved by using a standard hyaluronidase prepara-... 

These assays make use of probes that hmd specifically to hyalurooan, but not to the digestion products after degradation by hyaluronidase. Hie remaining binding... 

B. Delpech, P. Bertrand, and C. Chanty. An indirect aizymeimmunologicsl assay for hyaluronidase. X lmntunol hfctk. JM223 (1967). 

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). 

B. Steiner and D. Grace. A lymographic assay for detection of hyaluronidase activity on polyacrylamide gels and its application to enzymatic activity found in bacteria. ajua mwewm. uu huj (im/. 

The activity of hyaluronidase is determined by comparing the rate at which it hydrolyzes sodium hvahironate BRP with the rate obtained with the International Standard or a reference preparation calibrated in I.U. using a slope-ratio assay. 

Frost, G.I. and Stern, R., A microtiter-based assay for hyaluronidase activity not requiring specialized reagents, Anal. Biochem., 251, 263, 1997. 

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... 

Werthessen, N Bergman, S., Greenberg, B and Gargill, S. (1945). A technique for the assay of hyaluronidase in human semen and its correlation with sperm concentration. J. Urol. 22,540-555. 

The FIP and the European Pharmacopeia have a new BRP hyaluronidase standard power with an activity of 330 IU/mg. The stability and activity of this preparation is tested regularly by interlaboratory collaborative assays. International standards for the standardization of hyaluronidases of nontesticular origin are not available. 

This assay is used to investigate hyaluronidase activities from a variety of sources or to estimate the inhibitory capacities of compounds on the enzyme. The assay is based on the determination of the liberated /V-acetyl-D-glucosamine end groups from hyaluronan after digestion by hyaluronidase [60]. A comparison has been made between the Moigan-Elson and neocuproine assays and of the influence of inhibitors [138]. 

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. 

For this assay plates or petri dishes consisting of hyaluronan dispersed in agar and buffered to the appropriate pH are used. Cylindrical holes are punched in the gel and the samples (unknowns or standards) are added. The plates are incubated at 37°C for several hours. After incubation, a solution of a quaternary ammonium compound is poured over the gel, and after reaction the areas of digested hyaluronan become visible as clear rings. This technique is a simple assay for hyaluronidase activity in biological samples and has been used to estimate hyaluronidase activity in hymenoptera venoms [143], in semen, and in seminal plasma [146],... 

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