Hyaluronidase kinetics

Flavonoids are well known as inhibitors of hyaluronidase. Li 1950 Rodney et al, reported the effect of several flavonoids on hyaluronidase using in vitro and in vivo methods [79], Knppusamy et al. investigated the structure-activity relationship of die flavonoids as inhibitors of testicular hyaluronidase [80] and hyaluronidase from snake, scorpion, and bee venom [81]. They showed that flavones. flavanols. and chalcones possess the general ability to inhibit hyaluronidase Kinetic investigations showed that these compounds acted as competitive inhibitors [80]. Several hyaluronidase inhibitors were shown to be able to delay the action of snake venom when injected into mice [8ll One caimnt rule out the possible interaction of the flavonoids with other components of the venom, as the inhibitory effects of flavonoids are not specific to hyaluronidase... 

The PL-catechin conjugate showed greatly amplified concentration-dependent inhibition activity against bacterial collagenase (ChC) on the basis of the catechin unit, which is considered to be due to effective multivalent interaction between ChC and the catechin unit in the conjugate. The kinetic study suggests that this conjugate is a mixed-type inhibitor for ChC. Hyaluronidase is an enzyme which catalyzes hydrolysis of hyaluronic acid and is often involved in a number... 

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. 

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 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]. 

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). 

K. P. Vercruysse, A. Lauwers, and J. Demeester. Absolute and empirical determination of the enzymic activity and kinetic investigation of the action of hyaluronidase on hyaluronan using viscosimetry. Biochem. J. 306 153 (1995). 

K. P. Vercruysse, A. R. Lauwers, and J. M. Demeester. Kinetic investigation of the degradation of hyaluronan by hyaluronidase using gel permeation chromatography. J. Chrom. Biomed. Appl. 656 179 (1994). 

Asteriou, T., Gouley, F., Deschrevel, B., and Vincent, J. C. (2002). In Influence of Substrate and Enzyme Concentrations on Hyaluronan Hydrolysis Kinetics Catalyzed by Hyaluronidase. J. F. Hyaluronan, G. O. Kennedy, and P. A. Phillips Williams (Eds.). Woodhead Publishing, Wrexham, Wales, vol. 1, pp. 249-252. 

Vincent, J. C., Asteriou, T., and Deschrevel, B. (2003). Kinetics of hyaluronan hydrolysis catalysed by hyaluronidase. Determination of the initial reaction rate and the kinetic parameters. Journal of Biological Physics and Chemistry 3, 35-44. 

Asteriou, T., Deschrevel, B., Gouley F., Vincent, J.C. (2002) Influence of substrate and enzyme concentration on hyaluronan hydrolysis kinetics catalyzed by hyaluronidase, in Hyaluronan Proceedings of an International Meeting, September 2000, North East Wales Institute, UK (eds J.F. Kennedy, G.O. Phillips, P.A. Williams, V.C. Hascall), Woodhead Publishing Ltd, Cambridge, pp. 249-252. 

Type 3. Microbial hyaluronidases (e.g. Streptococcus hyaluronidase). Microbial hyaluronidases hydrolyse p-N-acetylaminoglycoside bonds of a substrate and simultaneously dehydrate the residue of uronic acid at the non-reducing terminus of the molecule. Substrate specificity of bacterial hyaluronate lyases varies considerably in the different species of microbe producers. Hyaluronidase of Streptococcus pneumoniae has the highest substrate specificity it hydrolyses HA alone and does not destroy other glucosami-noglycans [43]. The hyaluronate lyase, when isolated from Streptococcus pneumoniae, reaches optimal activity at pH 6.0 with the Michaelis constant with respect to HA being equal to 3.8x 10" mol/l (in terms of Michaelis-Menten kinetics) [44]. The presence of Cd3 (about 10 mM) is necessary in order to show the maximum enzyme activity. 

A polymer of natural origin chitosan attracts attention of researchers because it can be used to produce bioactive protecting coatings for treatment surgical wounds and bums. In this case, the biodegradability (enzymatic destmction) of the chitosan material will be affected under the action of nonspecific enzymes of the human body (e.g., hyaluronidase which is present at the wound surface, etc.). The speed of this process will determine the life of the polymer material on the wound surface. Thus, the study of the process of enzymatic destmction of chitosan under the action of nonspecific enzyme preparations will be important, both from scientific and practical points of view. In this paper we determine the kinetic parameters of the process of enzymatic destmction of chitosan by the enzyme hyaluronidase. 

Thus, the first time to determine the kinetic characteristics of the enzyme activity of hyaluronidase for the enzymatic destruction of a CHT solution of 1% acetic acid. Certain value of the Michaelis constant 3.4 g/dL significantly higher than the K =0.03 g/dL, as defined in Ref. [1] for the carboxymethylcellulose under the cellulose system of Geotrilium candi-... 

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