No abstract

1H n.m.r. spectra in [2H6]dimethyl sulphoxide of dodecyltrimethylammonium salts of chondroitin sulphates and hyaluronate, or sodium salts of oligomers from hyaluronate, showed unambiguous NH signals. The acetamido NH occurs in two different environments: environment I ('normal') in simple sugars, and environment II (hydrogen-bonded NH) appearing in tri- or tetrasaccharides, indicating a secondary structure in hyaluronate (and some chondroitin sulphates) involving a hydrogen-bonded acetamido NH.

The growth of the mould Trichoderma viride on a defined medium containing either potassium D-glucose 6-0-sulphate or potassium D-galactose 6-0-sulphate as sole sources of both carbon and sulphur is marked by the production of an enzyme system capable of liberating inorganic SO42-ions from either of the sulphate esters. The enzyme is not produced when the organism is grown with glucose (or galactose) and potassium sulphate or with glucose and methionine as sole sources of carbon and sulphur. Experimental conditions are described whereby inorganic S042-ions liberated from potassium glucose 6-0-sulphate by the growing mould appear in the culture medium after a constant lag period of 21-24hr. The enzyme has been shown to be a simple glycosulphatase that is active towards the 6-0-sulphate esters of D-glucose and D-galactose but not towards potassium glucose 3-0-sulphate. The properties of the crude glycosulphatase show the enzyme to be appreciably different from analogous molluscan enzymes that can degrade monosaccharide sulphate esters.

Studies on the effect of pH and ionic strength upon the activity of purified bovine testicular hyaluronidase have shown that the pH optimum for the hydrolysis of hyaluronic acid occurs at 5.2 in the presence of, and at 6.0 in the absence of NaCl. Hydrolytic activity towards various mucopolysaccharide and hyaluronate octasaccharide substrates was dependent upon the presence of strong electrolyte (LiCl, NaCl, KCl, CsCl, NaNO3 and Na2SO4), maximum activity being obtained at electrolyte strengths of 0.2. Identical weights of sulphated and unsulphated mucopolysaccharides were hydrolysed at similar rates under optimal conditions, except that double chains of chondroitin 4-sulphate were hydrolysed at twice the rate of the other polysaccharides. Hydrolytic activity towards hyaluronate hexasaccharide was favoured at pH values below 5.2 whereas transglycosylation activity was favoured at higher pH. Hyaluronate tetrasaccharide was neither a substrate for the hydrolytic or transglycosylation activity, nor was it an inhibitor of the enzymic hydrolysis of hyaluronic acid. No conformational change in hyaluronic acid was detected by CD-spectroscopy in the presence of varying concentrations of salt and the collective results suggest that the salt effect is exerted on the enzyme rather than on the substrate.

'H NMR spectra were obtained in fully deuterated dimethyl sulphoxide of sodium chondroitin 4-sulphate and of the tetrasaccharide, hexasaccharide and octasaccharide prepared from it. Two types of N H resonance were observed, at 7.6ppm and 8.8ppm. The latter was the only one in the polymer spectrum, whereas both were present in the oligomer spectra : N-acetylchondrosinate produced only the 7.6-ppm signal. The upfield resonance (7.6 ppm) is characteristic of 'normal', monomeric acetamido sugars and disaccharides, while the downfield resonance (8.8 ppm) suggests a hydrogen-bonded NH. The elongation of disaccharide to tetrasaccharide produces a new environment, involving a hydrogen bond at the non-reducing disaccharide N H . A structure, with three hydrogen bonds per tetrasaccharide unit, is proposed which accounts for the quantitative and qualitative aspects of the N M R spectra of polymers and oligomers. A laser light-scattering study showed that sodium chondroitin 4-sulphate has the same molecular weight in water as in dimethyl sulphoxide. The observed hydrogen bonds are therefore intramolecular, as required by the proposed structure, and probably exist in water as well as in dimethyl sulphoxide.Chondroitin 4-sulphate and 6-sulphate are glycosaniinoglycuronans of molecular weight 10000 -40000, occurring as side chains in proteoglycans of molecular weight about 10" and characteristically present in the extracellular matrix [I]. Together with hyaluronate, they appear in a space-filling capacity, e.g. as a pressure-resistant 'filling' responsible for the elasticity of cartilage [2], or to keep collagen fibrils apart, e.g. to preserve the transparency of cornea or, as in young tendon, to inhibit the growth and fusion of the fibrils [3]. These functions contrast with those of isomeric dermatan sulphate, which, as a proteoglycan, appears always to be bound to collagen fibrils at a specific locus [4]. Although the polypeptide part of the proteoglycan may be important in determining the specificity of the interaction with collagen [4], the properties of the glycosaminoglycuronan side chains must be decisive in the space-filling role. It is therefore relevant that hyaluronate and the chondroitin sulphates probably have preferred configurations in solution, stabilised by a hydrogen-bonding system which cannot form in dermatan sulphate [5]. As a result, the chains must be stiffened, helping their space-filling function. Their interaction with other molecules (e.g. collagen) by secondary valencies is correspondingly diminished, since much oftheir capacity is satisfied by intramolecular bonds [6]. In this paper we present evidence, obtained on oligosaccharides from chondroitin sulphate, that the previously proposed hydrogenbonding system, involving the acetamido NH, is operative in dimethyl sulphoxide solution. We also investigate by a laser light-scattering method the state of aggregation of sodiumAbhreviarion. (C2H,)2S0, fully deuterated dimethyl sulphoxide, (*H,)diinethyl sulphoxide.Enzymes. Chondroitinase ABC or chonduoiti...