The N-linked oligosaccharides of bovine skin proteodermatan sulphate.

Abstract

activity was collected and rotary evaporated to dryness. The residue was extracted ( x 5) with 10 ml of methanol, redissolving the dried residue in water and drying before each extraction. The pooled methanol extracts were dried under vacuum, redissolved in 1 ml of methanol/chloroform/2.5 Mammonium hydroxide (40:60:8; by vol.) and run onto a silica gel column (50 cm x 1 cm) equilibrated with the same solvent at 0.5 ml/min. Fractions (10 ml) were collected and inhibitor was shown t o be eluted between fractions 3 and 1 0 . Fractions 4-7 inclusivc were pooled, rotary evaporated to dryness then redissolved in ammonium bicarbonate (10 mM). The sample was finally purified by h.p.1.c. using a C , , (Spherisorb ODS. 25 cm x 4.6 mm) column equilibrated in 10 mM-ammonium bicarbonate and run at 1.5 ml/min. After loading of the sample, a linear gradient from 10% to 50% methanol was applied over a 25-min period. The inhibitor eluted between I 1 and 13 min and gave a single spot ( H , = 0.22) t.1.c. silica plates in methanol/chloroform/2.5 M-ammonium bicarbonate (40:60:8, by vol.). when developed with 50% (v/v) sulphuric acid. Development with phenol sulphuric acid gave a brown spot and carbazole sulphuric acid gave a violet spot, indicating the presence of a sugar. Positive fast atom bombardment mass spectroscopy on the purified material gave an M , of 323. Further study using chemical ionization of the trimethylsilylated derivative and analysis by nuclear magnetic resonance has provided the proposed structure shown in Fig. 1 . Methanolic extracts of the algae Mesotueriiitm culduriorum and Mougeotiu sp. have been shown to inhibit a-glucosidase. For both extracts the active components have been extensively purified by chromatography on Sephadex LH 2 0 (41 c m x 1.6 cm) in 95% (v/v) methanol/S% (w/v) ammonium bicarbonate/100 mM-&mercaptoethanol, followed by chromatography on silica in water containing 100 mM-~-mcrcaptocthanol for Mesotueriiitm culdurioriim and 80%) (v /v) water/20% (v/v) methanol/ 100 mM-pmercaptoethanol for Moitgeotiu sp. The active fractions were collected and each showed a single spot on silica t.1.c. (butanol/acetic acid/water, 2.8: 1 : 1, by vol.) when developed with SO‘% (v/v) sulphuric acid. However, in both cases, attempts at further analysis by h.p.1.c. and mass spectroscopy were unsuccessful due to the consistent loss of activity on concentration or drying of the sample. Concurrent with the loss of activity was the appearance of a reddish brown precipitate in each sample. Such changes are symptomatic of the polymerization o f tannins, which can be caused by air oxidation [ 41, a not uncommon problem associated with the purification o f tannins. Use of excess P-mercaptoethanol did not