Enzymatic reconstruction of a hybrid glycosaminoglycan containing 6-sulfated, 4-sulfated, and unsulfated N-acetylgalactosamine.

Using the transglycosylation reaction of testicular hyaluronidase, reconstructions of hybrid glycosaminoglycans (GAGs) containing 6-sulfated (GalNAc6S), 4-sulfated (GalNAcS) and unsulfated N-acetylgalactosamine (GalNAc) were investigated. First, chondroitin 4-sulfate (Ch4S) as a donor containing GalNAc4S and the pyridylaminated (PA) chondroitin 6-sulfate (Ch6S) hexasaccharide as an acceptor containing GalNAc6S were subjected to transglycosylation reaction. Second, when the resulting PA-Ch6S(hexa-)-Ch4S(di-)octasaccharide and chondroitin (Ch) were used as an acceptor and as a donor containing GalNAc, respectively, a new decasaccharide having a hybrid structure composed of disaccharide units derived from Ch6S, Ch4S and Ch was reconstructed. Using a systematic combination of each donor and acceptor molecule, it was possible to reconstruct various types of hybrid GAGs.     

Enzymatic reconstruction of dermatan sulfate

We investigated the enzymatic reconstruction of dermatan sulfate (DS) using the transglycosylation reaction of testicular hyaluronidase. First, in order to insert the IdoA-GalNAc disaccharide unit into chondroitin sulfate chains consisting of GlcA-GalNAc disaccharide units, desulfated DS as a donor and pyridylaminated (PA) chondroitin 6-sulfate (Ch6S) hexasaccharide as an acceptor were subjected to a transglycosylation reaction using testicular hyaluronidase. The products were analyzed by HPLC, mass spectrometry, and enzymatic digestions, and the results indicated that one of the products was IdoA-GalNAc-(GlcA-GalNAc6S)(3)-PA. Next, when the resulting PA-Ch6S (hexa-)desulfated DS (di-)octasaccharide was used as an acceptor and chondroitin as a new donor, a decasaccharide having a GlcA-GalNAc-IdoA-GalNAc-(GlcA-GalNAc6S)(3) sequence was reconstructed. Using suitable combinations of donors and acceptors, it was possible to custom synthesize DS having any IdoA sequence as its uronic acid component. It is likely that application of this system would facilitate artificial reconstruction of variant DS having different specific functions.     

Carriers for enzymatic attachment of glycosaminoglycan chains to peptide

In the previous study, we have found that the endo-beta-xylosidase from Patinopecten had the attachment activities of glycosaminoglycan (GAG) chains to peptide. As artificial carrier substrates for this reaction, synthesis of various GAG chains having the linkage region tetrasaccharide, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl, between GAG chain and core protein of proteoglycan was investigated. Hyaluronic acid (HA), chondroitin (Ch), chondroitin 4-sulfate (Ch4S), chondroitin 6-sulfate (Ch6S), and desulfated dermatan sulfate (desulfated DS) as donors and the 4-metylumbelliferone (MU)-labeled hexasaccharide having the linkage region tetrasaccharide at its reducing terminals (MU-hexasaccharide) as an acceptor were subjected to a transglycosylation reaction of testicular hyaluronidase. The products were analyzed by high-performance liquid chromatography and enzyme digestion, and the results indicated that HA, Ch, Ch4S, Ch6S, and desulfated DS chains elongated by the addition of disaccharide units to the nonreducing terminal of MU-hexasaccharide. It was possible to custom-synthesize various GAG chains having the linkage region tetrasaccharide as carrier substrates for enzymatic attachment of GAG chains to peptide.     

Chimeric glycosaminoglycan oligosaccharides synthesized by enzymatic reconstruction and their use in substrate specificity determination of Streptococcus hyaluronidase

A method was developed for the reconstruction of glycosaminoglycan (GAG) oligosaccharides using the transglycosylation reaction of an endo-beta-N-acetylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repetition of the transglycosylation using suitable combinations of various GAGs as acceptors and donors made it possible to custom-synthesize GAG oligosaccharides. Thus we prepared a library of chimeric GAG oligosaccharides with hybrid structures composed of disaccharide units such as GlcA-GlcNAc (from hyaluronic acid), GlcA-GalNAc (from chondroitin), GlcA-GalNAc4S (from chondroitin 4-sulfate), GlcA-GalNAc6S (from chondroitin 6-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc4,6-diS (from chondroitin sulfate E). The specificity of the hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specificity of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disaccharide unit (GlcA-GlcNAc) is necessary for the enzymatic action of hyaluronidase SD; (ii) cleavage is inhibited by sulfation of the N-acetylgalactosamine; (iii) hyaluronidase SD releases GlcA-GalNAc and IdoA-GalNAc units as well as GlcA-GlcNAc. At the reducing terminal side of the cleavage site, the sulfated residues on the N-acetylgalactosamines in the disaccharide units were found to have no influence on the cleavage. Additionally, we found that hyaluronidase SD can specifically and endolytically cleave the internal unsulfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG oligosaccharides will open a new avenue in GAG glycotechnology.     

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.     

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.Abbreviations HA hyaluronic acid - Ch6S chondroitin 6-sulfate - GAG glycosaminoglycan - GlcA d-glucuronic acid - GlcNAc 2-acetamido-2-deoxy-d-glucose - GalNAc 2-acetamido-2-deoxy-d-galactose.     

The role of hyaluronic acid in intercellular adhesion of cultured mouse cells

Aggregation of cultured mouse cells was measured by the rate of disappearance of particles from a suspension of single cells. Treatment with several enzymes which degrade hyaluronic acid (testicular hyaluronidase, streptomyces hyaluronidase, streptococcal hyaluronidase and chondroitinase ABC) inhibited the aggregation of SV-3T3 and several other cell types. Since streptomyces and streptococcal hyaluronidases are specific for hyaluronic acid, it is suggested that hyaluronic acid is involved in the observed aggregation. Hyaluronidase-induced inhibition of aggregation was complete in the absence of divalent cations, but only partial in their presence. This finding is consistent with the hypothesis that two separate mechanisms are responsible for aggregation; one dependent upon and the other independent of calcium and magnesium. Aggregation was also inhibited by high levels of hyaluronic acid. A similar effect was obtained with fragments of hyaluronic acid consisting of six sugar residues or more. Chondroitin (desulfated chondroitin 6-sulfate) and to a lesser extent desulfated dermatan sulfate also inhibited aggregation. Other glycosaminoglycans (chondroitin 4-sulfate, chondroitin 6-sulfate, heparin and heparan sulfate) had little or no effect on aggregation. It is suggested that the hyaluronic acid inhibits aggregation by competing with endogenous hyaluronic acid for cell surface binding sites.     

Kinetics of Hyal-1 and PH-20 hyaluronidases: comparison of minimal substrates and analysis of the transglycosylation reaction

The availability of recombinant expression systems for the production of purified human hyaluronidases PH-20 and Hyal-1 facilitated the first detailed analysis of the enzymatic reaction products. The human recombinant enzymes, both expressed by Drosophila Schneider-2 (DS-2) cells, were compared to bovine testicular hyaluronidase (BTH), a commercially available hyaluronidase preparation, which has long been considered a prototype of mammalian hyaluronidases. The conversion of low molecular weight hyaluronic acid (HA) fragments was detected by a capillary zone electrophoresis (CZE) method. Surprisingly, the HA hexasaccharide, which is generally accepted to be the minimum substrate of BTH, was not a substrate of recombinant human PH-20 and Hyal-1. However, HA octasaccharide was converted efficiently by both enzymes, thus representing the minimum substrate for human PH-20 and Hyal-1. Additionally, BTH was shown to catabolize the HA hexasaccharide at pH 4.0 mainly by hydrolysis, while at pH 6.0 transglycosylation prevailed. Human PH-20 was found to catalyze both hydrolysis and transglycosylation of the HA octasaccharide. On the contrary, human Hyal-1 converted the HA octasaccharide mainly by hydrolysis with transglycosylation products occurring only at high substrate concentrations (> or = 500 microM). The differences between the hyaluronidase subtypes and isoenzymes were much more prominent than expected. Obviously, the different hyaluronidase subtypes have evolved into very specialized enzymes with respect to their catalytic mechanism of action.     

Selective hydrolysis of chondroitin sulfates by hyaluronidase

Chondroitin 4-sulfate and chondroitin 6-sulfate were incubated with testicular hyaluronidase in the presence of excess beta-glucuronidase. The beta-glucuronidase caused rapid removal of the nonreducing terminal beta-D-glucuronosyl residues from the oligosaccharides formed by the action of the hyaluronidase, destroying the oligosaccharide acceptors required for the transglycosylation activity of hyaluronidase and releasing free D-glucuronic acid at a rate that was equal to the rate of the hyaluronidase-catalyzed hydrolysis. When hyaluronidase was assayed at 37 degrees C in the presence of 0.05 M NaCl, 0.05 M Na2SO4, and 0.1 M sodium acetate at pH 5, chondroitin 4-sulfate was hydrolyzed at 1.5 times the rate found for chondroitin 6-sulfate. When hyaluronidase was assayed at 45 degrees C in 0.06 M sodium acetate at pH 6, chondroitin 4-sulfate was hydrolyzed at 8 times the rate observed for chondroitin 6-sulfate. Under the pH5 conditions, the chondroitin 4-sulfate was converted to a mixture of tri- and pentasaccharides, while the chondroitin 6-sulfate was converted primarily to a mixture of penta- and heptasaccharides, with only a small amount of trisaccharide. Under the pH 6 conditions, the chondroitin 4-sulfate was converted to a mixture of penta- and heptasaccharides, with only a small amount of trisaccharide, but the products from chondroitin 6-sulfate were a mixture of oligosaccharides ranging in degree of polymerization from 7 to 25 monosaccharides per oligosaccharide. End-group analyses of the products formed at pH 6 showed that both substrates were cleaved preferentially at the glycosidic bonds of the 4-sulfated disaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)     

The major human urinary trypsin inhibitor is a proteoglycan

The major urinary trypsin inhibitor (Mr 44 000), isolated from human urine, contains 35% carbohydrate. In addition to N-acetylglucosamine and neutral sugars (primarily mannose and galactose), the carbohydrate moiety contains hexuronic acid and N-acetylgalactosamine and corresponds to a glycosaminoglycan. This carbohydrate chain is an integral component of the inhibitor: it does not dissociate from the inhibitor when using dissociative conditions such as sodium dodecyl sulfate, guanidinium chloride, or by increasing ionic strength or mixing with cetylpyridinium chloride. This glycosaminoglycan chain is sensitive to chondroitinase ABC or testicular hyaluronidase digestion and corresponds to slightly sulfated chondroitin 4-sulfate or 6-sulfate. After treatment by these enzymes, the urinary inhibitor has a lower molecular mass (Mr 26 000) but still inhibits trypsin.     

Glycosaminoglycan synthesis in the chick gastrula

Explanted definitive primitive streak to four somite chick embryos were labeled with [H³]glucosamine or S³⁵O₄ and the glycosaminoglycans were isolated and characterized. On the basis of susceptibility to Streptomyces hyaluronidase, which specifically degrades hyaluronic acid, hyaluronic acid is the major glycosaminoglycan produced by these embryos (at least 84%). On the basis of electrophoretic mobility, about 10% of the [H³]glucosamine-labeled glycoaminoglycan is sulfated. At least 55% of the sulfate-labeled glycosaminoglycan is sensitive to testicular hyaluronidase, and 36–39% is resistant to testicular hyaluronidase, but sensitive to nitrous acid treatment. About 94% of the labeled glycosaminoglycans can be accounted for in ratios of 22:1:5:1 as hyaluronic acid:chondroitin sulfate:heparan sulfate. No stage-related changes were observed. It is suggested that hyaluronic acid synthesis at this time might be related to the appearance of extensive cell-free spaces.     

Studies on glycosaminoglycans of regenerating rabbit ear cartilage

Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitiated. Normal cartilage had a $\text{Δ}\text{Di-6S}\text{Δ}\text{Di-4S}$ ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.     

Enzymatic attachment of glycosaminoglycan chain to peptide using the sugar chain transfer reaction with endo-beta-xylosidase.

Endo-beta-xylosidase from the mid-gut gland of the molluscus Patinopecten is an endo-type glycosidase that hydrolyzes the xylosyl serine linkage between a core protein and a glycosaminoglycan (GAG) chain, releasing the intact GAG chain from proteoglycan. In this study, we investigated GAG chain transfer activity of this enzyme, in order to develop a method for attaching GAG chains to peptide. Peptidochondroitin sulfate (molecular mass of sugar chain, 30 kDa) from bovine tracheal cartilage as a donor and butyloxycarbonyl-leucyl-seryl-threonyl-arginine-(4-methylcoumaryl-7-amide) as an acceptor were incubated with endo-beta-xylosidase. As a result, a reaction product with the same fluorescence as the acceptor peptide was observed. High pressure liquid chromatography analysis, cellulose acetate membrane electrophoresis, and enzymatic digestion showed that this reaction product had the chondroitin sulfate (ChS) from the donor. Furthermore, the acceptor peptide was released from this reaction product after hydrolysis by endo-beta-xylosidase. Therefore, it was confirmed that the ChS chain released from the donor was transferred to the acceptor peptide by the GAG chain transfer reaction of endo-beta-xylosidase. The optimal pH for hydrolysis by this enzyme was found to be about 4.0, whereas that for this reaction was about 3.0. Not only the ChS but also the dermatan sulfate and the heparan sulfate were transferred to the acceptor peptide by this reaction. By using this reaction, the GAG chain could be attached to the peptide in one step. The GAG chain transfer reaction of endo-beta-xylosidase should be a significant glycotechnological tool for the artificial synthesis of proteoglycan.     

Cell surface glycosaminoglycans: Identification and organization in cultured human embryo fibroblasts

A morphologically detectable cell coat, composed of glycoprotein, glycolipid, and glycosaminoglycan, is present on the external surface of most vertebrate cells. We have invetigated the composition and organization of glycosaminoglycans in the cell coat of cultured human embryo fibroblasts by labeling cells with ³H-glucosamine and Na₂³⁵SO₄ and subsequently treating cultures with specific enzymes. Components released were identified by chromatography and specific enzymatic digestion. In situ incubation with leech hyaluronidase (4 μg/ml) removed only hyaluronic acid from the cell surface whereas testicular hyaluronidase (0.5 mg/ml) removed both hyaluronic acid and chondroitin sulfate. Trypsin (0.1 mg/ml) released a large mass of glycopeptides in addition to hyaluronic acid, chondroitin sulfate, and heparan sulfate. The affinity of the cell coat for the cationic dye, ruthenium red, was reduced by leech hyaluronidase treatment. Sequential enzyme digestions of the cell surface showed that hyaluronic acid could be removed without the concomitant or subsequent release of sulfated glycosaminoglycans, suggesting that the hyaluronic acid is not a structural backbone for glycosaminoglycan complexes of the external cell surface.     

Biosynthesis and properties of a further member of the small chondroitin/dermatan sulfate proteoglycan family

Human osteosarcoma cells express a 78-kDa proteoglycan core protein to which an asparagine-bound oligosaccharide, O-glycosidically linked oligosaccharides and probably only a single chondroitin 6-sulfate chain of 29-kDa are bound. Prior to O-glycosylation, the N-glycosylated core protein exhibits a mass of 83 kDa. Upon digestion of the secreted proteoglycan with chondroitin ABC lyase a mature core protein with an apparent molecular mass of 106 kDa is obtained. Smaller amounts of core proteins of 101 and 115 kDa can be detected occasionally. The glycosaminoglycan composition and the relative molecular mass of the glycosaminoglycan chain distinguish this proteoglycan, tentatively named proteoglycan 100 (PG-100), from biglycan (small proteoglycan I) and decorin (small proteoglycan II) which are also expressed by osteosarcoma cells. An antiserum against PG-100 shows partial cross-reactivity with decorin, but in contrast to the latter proteoglycan it does not bind to type I collagen fibrils. PG-100 is not a unique product of osteosarcoma cells. It has also been found in the secretions of human skin fibroblasts.     

Quantitation of hyaluronic acid in tissues by ion-pair reverse-phase high-performance liquid chromatography of oligosaccharide cleavage products

A method for quantifying hyaluronic acid in biological tissues and fluids is described. The assay uses ion-pair HPLC to resolve and quantify the oligosaccharide end products of Streptomyces hyaluronidase digestion. Tissue samples were solubilized by papain, and the nondiffusate after dialysis was exhaustively digested with Streptomyces hyaluronidase. The resulting tetrasaccharide and hexasaccharide cleavage products were resolved by reverse-phase high-performance liquid chromatography in the presence of the ion-pairing agent, tetrabutylammonium phosphate. The saccharides were detected and quantified by their absorbance at 232 nm due to the alpha, beta-unsaturated carboxyl group generated by the eliminase reaction. In control experiments 93 +/- 3% of a hyaluronic acid standard so treated was reproducibly recovered as its tetra- and hexasaccharide cleavage products. As little as 0.5 microgram of the oligosaccharides could be quantified with no interference from a vast excess of chondroitin sulfate or other tissue components. The assay was applied to various types of human, bovine, and rabbit cartilage and to samples of other tissues including nucleus pulposus, annulus fibrosus, skin, aorta, cervix, cockscomb, synovial fluid, and vitreous humor. Results on human articular cartilage showed a linear increase in the content of hyaluronate from 0.1 to 0.5% of tissue dry weight between birth and 80 years of age.     

Conformational Transitions in 3D Model of Bovine Testicular Hyaluronidase during Molecular Docking with Glycosaminoglycan Ligands

In silico molecular docking of the trimer repeating unit of chondroitin sulfate (sulfated hexasaccharide) and tetramer repeating unit of heparin (sulfated octasaccharide) to the 3D model of bovine testicular hyaluronidase by the methods of computational chemistry demonstrated the presence of eight significant binding sites for these ligands (cs1–cs8). The interaction of the active site of the enzyme with the heparin ligand, which inactivates the enzyme, and the protective effect of the chondroitin sulfate ligands bound to the surface sites of the biocatalyst molecule were theoretically studied using calculation approaches. We sequentially determined binding sites for the chondroitin sulfate ligands (in positions cs2, cs4, cs7, cs8 or cs1, cs2, cs4, cs7, cs8) critical for the protein structure stabilization, whose occupancy is theoretically sufficient to prevent irreversible deformations of the enzyme molecule when the heparin ligand is introduced into the cavity of its active site. Theoretical detection of these ‘sensibility points’ on the hyaluronidase globule indicates the possibility of regulating its functioning under the binding of the glycosaminoglycan ligands that initiate the fine formation of an effective type of the surface electrostatic potential. The interaction of the glycosaminoglycan ligands with hyaluronidase is mainly determined by electrostatic forces.     

The unusual tetrasaccharide sequence GlcA{beta}-3GalNAc(4-sulfate)({beta}1-4GlcA(2-sulfate){beta}1-3GalNAc(6-sulfate) found in the hexasaccharides prepared by testicular hyaluronidase digestion of shark cartilage chondroitin sulfate D

Eight hexasaccharide fractions were isolated from commercialshark cartilage chondroitin sulfate D by means of gel nitrationchromatography and HPLC on an amine-bound silica column afterexhaustive digestion with sheep testicular hyaluronidase. Capillaryelectrophoresis of the enzymatic digests as well as one- andtwo-dimensional 500 MHz ¹H-NMR spectroscopy demonstrated thatthese hexasaccharides share the common core saccharide structureGlcAß1-3GalNAcß1-4GlcAß1-3GalNAcß1-4GlcAß1-3GalNAcwith three, four, or five sulfate groups in different combinations.Six structures had the same sulfation profiles as those of theunsaturated hexasaccharides isolated from the same source afterdigestion with chondroitinase ABC (Sugahara et al., Eur. J.Biochem., 293, 871–880, 1996) and the other two have notbeen reported so far. In the new components, a D disaccharideunit, GlcA(2-sulfate)ß1-3GalNAc(6-sulfate), characteristicof chondroitin sulfate D was arranged on the reducing side ofan A disaccharide unit, GlcAß1-3GalNAc(4-sulfate),forming an unusual A-D tetrasaccharide sequence, GlcAß1-3GalNAc(4-sulfate)-4GlcA(2-sulfate)ß1-3GaINAc(6-sulfate)which is known to be recognized by the monoclonal antibody MO225.These findings support the notion that the tetrasaccharide sequence,GlcAß1-3GalNAc(4-sulfate)ß1-4GlcAß1-3GalNAc(6-sulfate)is included in the acceptor site of a hitherto unreported 2-O-sulfotransferaseresponsible for its synthesis. The sulfated hexasaccharidesisolated in this study will be useful as authentic oligosaccharideprobes and enzyme substrates in studies of sulfated glycosaminogly-cans. sulfated hexasaccharides chondroitin sulfate D hyaluronidase ¹ H-NMR     

Change of glycosaminoglycan in pus of patients with purulent pleurisy

The glycosaminoglycan content in pus from patients with purulent pleurisy was studied. The uronic acid content rose in the first 4 hospital days, continued at a high level during hospital days 5-8, and then fell to a low level after 9 hospital days. Four glycosaminoglycans were isolated from the preparation; they were identified as hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate. Hyaluronic acid was the main component and its relative proportion increased with increasing hospital days. The relative proportions of chondroitin 4-sulfate and chondroitin 6-sulfate were low during the first 4 day and during Days 10-21, whereas they were high during Days 5-9. The proportion of dermatan sulfate was high during the early hospital days, and thereafter decreased with increasing hospital days.     

Interfacial behaviour of bovine testis hyaluronidase

The interfacial properties of bovine testicular hyaluronidase were investigated by demonstrating the association of hyaluronidase activity with membranes prepared from bovine testis. Protein adsorption to the air/water interface was investigated using surface pressure-area isotherms. In whichever way the interfacial films were obtained (protein injection or deposition), the hyaluronidase exhibited a significant affinity for the air/water interface. The isotherm obtained 180 min after protein injection into a pH 5.3 subphase was similar to the isotherm obtained after spreading the same amount of protein onto the same subphase, indicating that bovine testicular hyaluronidase molecules adopted a similar arrangement and/or conformation at the interface. Increasing the subphase pH from 5.3 to 8 resulted in changes of the protein isotherms. These modifications, which could correspond to the small pH-induced conformational changes observed by Fourier-transform IR spectroscopy, were discussed in relation to the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the interfacial properties of hyaluronidase. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed substrate binding to a preformed film of hyaluronidase at pH 5.3, the optimal pH for the enzyme activity. Such effects of hyaluronic acid were not observed when the subphase was constituted of pure water, a medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation to the modelled structure of bovine testis hyaluronidase where a hydrophobic region was proposed to be opposite of the catalytic site.