Chondroitin methods

Herring, G. M. Methods for the study of the glycoproteins and proteoglycans of bone using bacterial collagenase. Determination of bone sialoprotein and chondroitin sulphate. Calc. Tiss. Res. 24, 29 (1977)... 

K. Nagasawa, Y. Inoue, and T. Tokuyasu, An improved method for the preparation of chondroitin by solvolytic desulfation of chondroitin sulfates,./. Biochem., 86 (1979) 1323—1329. 

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). 

Rienits later studied the zone-electrophoretic behavior of hyaluronic acid, chondroitin hydrogen sulfate, and heparin on paper strips in nonborate buffers he found that, whereas hyaluronic acid can be separated from chondroitin hydrogen sulfate and heparin, the latter two mucopolysaccharides cannot be separated. The mucopolysaccharides could be located by the use of a method similar to that of Gardell, Gordon and Aqvist, or by staining the pherogram with Toluidine Blue. The mobility of hyalu-... 

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... 

The proof of this sequence, and of the nature of the carbohydrate-peptide bond, was based on enzymic erosion to a glycopeptide fragment that was then studied by adaptations of classical methods. A similar linkage-region probably occurs in heparin,86 heparitin sulfate,87 dermatan sulfate88 and chondroitin 6-sulfate.88a It is possible that the polypeptide core itself is branched.89... 

In order to shed more light on the Interaction of water with these mucopolysaccharides, we employ the DSC method to follow the melting behavior of aqueous solutions of the polysaccharides cooled to -50 C. The polymers chosen here are chondroitin sulfate A (Chn S-A), chondroitin sulfate C (Chn S-C), chondroitin (Chn), heparin (Hpn), and hyaluronic acid (HyA). Their chemical structures are shown in Figure 1. The DSC curves allow us to determine the amount of the non-freezing water In highly concentrated solutions, since any endothermic peak Is not observed for such solutions over a wide temperature range (17. 18). This paper will also describe the presence of more than one endothermic peak In the DSC curves for solutions of relatively low polymer concentrations. 

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