Mitsuo Majima

Ion-spray mass spectrometric analysis of glycosaminoglycan oligosaccharides

Oligosaccharides from hyaluronic acid and chondroitin 6-sulfate were prepared by digestion with testicular hyaluronidase and separated according to their degree of polymerization by gel-permeation chromatography. These materials were successively analyzed by negative-mode ion-spray mass spectrometry with an atmospheric-pressure ion source. An ion-spray interface was used to produce ions via the ion evaporation process, producing mass spectra containing a series of molecular species carrying multiple charges. Using two adjacent multiply charged molecular ions, the exact molecular weights up to the tetradecasaccharide were calculated with a precision of ±1 dalton. This type of mass spectrometry was also demonstrated to be feasible for the analysis of mixtures of oligosaccharides, including tetra-, hexa-, octa- and decasaccharides, from hyaluronic acid or chondroitin 6-sulfate without separation. Ion-spray mass spectrometry was thus shown to be applicable to the structural analysis of oligosaccharides from glycosaminoglycans.

Hyaluronidase assay using fluorogenic hyaluronate as a substrate

The reducing terminal of hyaluronate was labeled with a fluorogenic reagent, 2-aminopyridine. The pyridylaminohyaluronate was incubated with testicular hyaluronidase for 1 h. After incubation, 4 vol of ethanol was added to the incubation mixture, followed by centrifugation. The fluorescence of the supernatant containing the degradation products of hyaluronidase digestion was then determined by fluorospectrophotometry (excitation wavelength, 320 nm; emission wavelength, 400 nm). It was found that the increase of the pyridylamino products was linearly correlated with enzyme concentration (up to 0.1 national formulary unit), incubation time (up to 60 min), and substrate concentration (up to 2.5 microM). The fluorogenic substrate was also applicable for the determination of crude hyaluronidase. This simple, rapid, and sensitive hyaluronidase assay was made possible by the use of pyridylaminohyaluronate as a substrate.

Effect of proteoglycan on experimental colitis

Abstract The effect of proteoglycan (PG) on colitis was examined in animal experiments using mice. The PG used was extracted from nasal cartilage of salmon head with 4% acetic acid and prepared by precipitation with ethanol followed by dialysis. The PG contained about 7% protein, and had a molecular mass of 344 kDa on SDS/PAGE. The glycosaminoglycan (GAG) sugar chains of the PG were composed of hexosamine, uronic acid and sulfate at a molar ratio of 1.0:1.0:0.7. The mice were divided into a control group and an administration group. The control group was given free access to drinking water containing dextran sulfate sodium salt (DSS) to induce colitis. On the other hand, the administration group was given free access to drinking water containing DSS and PG. Then, the time course of survival rates in both groups were measured. In the administration group, the survival rate increased significantly in comparison with that of the control group. The difference in the survival rates indicated that the onset of mouse colitis induced by DSS was inhibited by administration of the PG.

Demonstration of chondroitin sulfates degrading endo-β-glucuronidase activity in rabbit liver

Reduced chondroitin sulfate was incubated with rabbit liver extracts followed by reduction once more with sodium [3H]borohydride, and then passed through a Sephadex G‐100 column. Chondroitin sulfate obtained from the incubation medium at pH 4 was only slightly depolymerized and was highly radioactive. Paper Chromatographic analyses showed that glucuronic acid residues became exposed at the reducing terminal of chondroitin sulfate after incubation with the liver extracts. These results suggest that endo‐β‐glucuronidase activity which degrades chondroitin sulfate is present in the rabbit liver.

Improved analytical method for the hexuronic acids at the reducing terminals of glycosaminoglycans

When endo-uronidases act on glycosaminoglycans, the reaction products have hexuronic acid residues at the reducing terminals. An analytical method for hexuronic acids at the reducing terminals was devised for hyaluronate oligosaccharides having hexuronic acid residues at the reducing terminals. The procedure is as follows: Hexuronic acid residues at the reducing terminals of hyaluronate oligosaccharides were tritiated with reduction using NaB[3H]4 and the products were hydrolyzed with trifluoroacetic acid and nitrous acid. As a result, the tritiated and reduced hexuronic acid residues, that is aldonic acids, were liberated from the reducing terminals. After passing them through anion and cation ion-exchange resins, the aldonic acids were lactonized. The lactones were developed on paper chromatography, and their radioactivities determined on the paper. The method is also useful for discrimination between glucuronic acid and iduronic acid at the reducing terminals of glycosaminoglycans.

The application of the Milner-Avigad method for the quantitative determination of endouronidase activities.

The Milner-Avigad method (Milner, Y. and Avigad, G. (1967) Carbohydr. Res. 4, 359-361) has been shown to have high specificity for measuring the reducing powers of free hexuronic acids; its applicability in the quantitative determination of endouronidase activity was the purpose of this investigation. Among the constituent monosaccharides of glycosaminoglycans, hexuronic acids showed high color yield by this method, while xylose, galactose and N-acetylhexosamine recorded negligible color yield. Among the monosaccharide residues at the reducing terminals of oligosaccharides, only hexuronic acids exhibited color yield. However, the color yield was less than that of free hexuronic acids. Gel filtration chromatography of reaction products revealed that the cleavage of the oligosaccharide chains and the resultant exposure of new reducing terminals were not caused by the reaction procedures involved in this method. These data indicate that the Milner-Avigad method is useful for determining the presence of hexuronic acid residues preferentially at reducing terminals of glycosaminoglycan moieties. Thus, it supported the conclusion that the Milner-Avigad method is applicable for the quantitative determination of endouronidase activity using glycosaminoglycan as a substrate.

Demonstration of N-acetylchondrosine-degrading β-glucuronidase in rabbit liver

N‐Acetylchondrosine was incubated at pH 4.0 with a rabbit‐liver crude enzyme extract. Gel filtration of the reaction products on Sephadex G‐15 revealed the presence of monosaccharide liberated from the disaccharide. The monosaccharide fraction was analyzed by gas‐liquid chromatography, and identified as a mixture of glucuronic acid and N‐acetylgalactosamine. These results indicate the presence of β‐glucuronidase, which degrades N‐acetylchondrosine, in rabbit liver. The discovery of the presence of this enzyme may help to establish the complete degradation process of chondroitin sulfates.

Effects of divalent cations on bovine testicular hyaluronidase catalyzed transglycosylation of chondroitin sulfates

Glycosaminoglycans were prepared as salts of different divalent cations and tested as donors in bovine testicular hyaluronidase catalyzed transglycosylation reactions. All of the metal cations examined had similar binding efficiency of divalent cations to hyaluronan. However, cations bound with different efficiencies to chondroitin sulfate species and the differences were marked in the case of chondroitin 6-sulfate; the numbers of cations bound per disaccharide unit were estimated to be 0.075 for Mn, 1.231 for Ba, 0.144 for Zn, and 0.395 for Cu. While barium salt of chondroitin sulfates enhanced transglycosylation, the zinc salt of chondroitin sulfates inhibited transglycosylation. Therefore, by selecting the proper divalent cation salt of chondroitin sulfates as a donor in the transglycosylation reaction it is possible to improve the yields of the products.