ABSTRACT
A methods for the simultaneous separation and determination of amino acids, amino sugars and neutral carbohydrates is described. Stepwise elution systems with sodium citrate and borate buffers have developed for the ion-exchange liquid chromatographic separation of amino acids and sugars, using 8-micrometer particle size resins and the Stein and Moore and orcinol colorimetric method for detection. With the aid of this system, the direct quantitative comparison of sugars and amino acids by liquid chromatography becomes possible for the first time.
Subject(s)
Amino Acids/analysis , Amino Sugars/analysis , Carbohydrates/analysis , Glycoproteins , Autoanalysis , Chromatography, High Pressure Liquid/instrumentation , Chromatography, High Pressure Liquid/methods , OvalbuminABSTRACT
The i.r. spectra of disaccharides differing in monosaccharide composition and in the position and configuration of the glycosidic linkage, and also those of raffinose and model saccharides, were studied in the region 1,000-40 cm-1. Two ranges may be of interest for structural analysis. The first, called "the anomeric region=, is suitable for the determination of the configuration of the glycosidic linkage. The spectra of the oligosaccharides in the second region, called "the region of crystallinity", depend upon the packing of the molecules in the solid. The reasons for the present impossibility of using the far-infrared region of the i.r. spectra of lower oligosaccharides for the determination of the position of the glycosidic linkage are considered.
Subject(s)
Oligosaccharides , Binding Sites , Crystallization , Molecular Conformation , Spectrophotometry, InfraredABSTRACT
beta-D-Glucopyranosyl-(1S and 1R)-epoxyethanes (I and II), 1-(beta-D-glucopyranosyl)-(2R and 2S)-2,3-epoxypropanes (III and IV), beta-D-glucopyranosyl isothiocyanate (V) and beta-D-galactopyranosylepoxyethane (VI) are active-site-directed irreversible inhibitors of sweet-almond beta-glucosidase B (beta-D-Glucoside glucohydrolase, EC 3.2.1.21). Formation of the covalent bond is preceded by the binding of these inhibitors in the active site of the enzyme. This is testitified by the competitive character of inhibition of beta-glucosidase component B by compounds I-VI at the early period and by the protection of the enzyme from inactivation by its competitive inhibitors D-glucose and 1,5-D-gluconolactone. Epoxides I-IV are bound covalently with componet B at a molar ratio 1 : 1 as shown with the aid of 14C-labelled inhibitors. The release of the label from modified enzyme (E-I covalent) by treatment with hydroxylamine suggests the formation of an ester bond between inhibitors I-IV and the carboxyl group of the enzyme active site. The pH dependence curve of the inactivation rate of beta-glucosidase B is of a bell-shaped form for V and of a sigmoid character for I-IV and points to the involvement of the active site groups with pKa 5.6-5.9 and 4.2-4.4.
