Proteolytic cleavage of soybean Bowman-Birk inhibitor monitored by means of high-performance capillary electrophoresis. Implications for the mechanism of proteinase inhibitors.

The hydrolysis of the soybean Bowman-Birk inhibitor in the presence of catalytic amounts of bovine trypsin and the formation of the non-covalent enzyme-inhibitor complex with an equimolar amount of enzyme are monitored by means of high-performance capillary electrophoresis (HPCE). The inhibitor is cleaved in the trypsin-reactive and more slowly in the chymotrypsin-reactive subdomain. HPCE proves itself as the only reliable analytical tool to monitor these reactions in clear contrast to classical electrophoretic, chromatographic and enzymatic methods. The most efficient separation of the intact and the two active site cleaved forms of the inhibitor was achieved in borate buffer at pH 10.0. The pH dependence of the rate constant and the final extent of hydrolysis reveal the stability of the enzyme inhibitor complex as a central aspect of the mechanism of proteinase inhibitors.

A strategy for chromatographic and structural analysis of monosaccharide species from glycoproteins.

A general strategy for the chromatographic and structural analysis of the monosaccharide species fucose (Fuc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), galactose (Gal), glucose (Glc), mannose (Man), N-acetylneuraminic acid (NANA) present in glycoproteins is described. Qualitative and quantitative aspects for the separation of these glycoprotein monosaccharides (monosaccharide species) using ligand-exchange chromatography (LEC) and high pH anion-exchange chromatography (HPAEC) in combination with pulsed-amperometric detection (PAD), refractive index (RI) and ultraviolet (UV) monitoring are discussed in detail. The conditions for the acidic hydrolysis of glycoproteins and for the liquid chromatographic analyses of glycoprotein monosaccharides using HPAEC and LEC technique were optimised. Furthermore, the characterisation of glycoproteins according to their purity and molecular mass connected with a comparison to biomolecules that are not glycosylated or whose extent of glycosylation is low was carried out by means of matrix-assisted laser-desorption ionisation mass spectrometry (MALDI-MS). The identification of glycoprotein monosaccharides using an on-line coupling liquid chromatography mass spectrometry (LC-MS/MS) was performed by means of their characteristic "quasi molecule ions" such as (M + NH(4))(+) and (2M + NH(4))(+). The different chromatographic and structural methods used in combination with each other were applied to characterise and determine the monosaccharide species of fetuin and a membrane glycoprotein fraction.

HPLC analysis of carbohydrates on POLYSPHERCH(R)OH columns using pulsed amperometric detection (PAD) with sodium hydroxide as post column detection reagent.

Carbohydrates have been separated on POLYSPHER(R)CH OH columns using pulsed amperometric detection (PAD) and UV detection (lambda =196 nm) in series and pure water as mobile phase. Nearly baseline separations have been obtained for the glycoprotein carbohydrates of sialic acid ( N-acetylneuraminic acid, NANA), N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). As carbohydrates dissolved and eluted with pure water are present in the neutral form they are not detectable with PAD in contrast to carbohydrate anions formed at high pH values. Therefore an additional NaOH post column reagent has been continuously pumped through a mixing chamber into the mobile phase to form carbohydrate anions resulting in improved detection limits. Monosaccharides as well as glycoprotein carbohydrates could be detected in the microg/ml-range. This method has been applied successfully to the analysis of sugars in fruit juice. With only 2 microl of juice per 50 ml water, the determination of the main constituents, sucrose, glucose and fructose, was possible in a few minutes without sample preparation.

Calculation of the molecular masses of two newly synthesized thermostable enzymes isolated from thermophilic microorganisms.

Two thermostable enzymes synthesized by thermophilic microorganisms were isolated and purified. A thermostable beta-galactosidase was produced in a continuous fermentation process by Bacillus stearothermophilus TP 32 as an intracellular enzyme. After applying different concentration procedures the raw extract enzyme was prepurified on a Sephadex G-200 size exclusion column. The isolated beta-galactosidase fraction was then separated with HPLC on a TSK G 3000 SW size exclusion column to determine the molecular mass based on calibration curves of standard proteins. The other enzyme, a thermostable protease, was synthesized by Bacillus stearothermophilus TP 26 as an extracellular enzyme. After its concentration, the enzyme was purified on a classical size exclusion column (Sephacryl S-200) and on a HPLC size exclusion column (BIO-SIL TSK-250). The micropreparatively isolated fraction was separated again on this HPLC column to determine its molecular mass. The optimum temperature of both enzymes was approximately 75 degrees C.

[Results valuation of adhesive bridges--3-year study]. / Erfolgsbewertung von Adhäsivbrücken--eine 3-Jahresstudie.

139 of 169 adhesive-bridges, incorporated from 1984 bis 1987, were reexaminated in 1988. The failure rate was 37.5% with a mean functional period of 27.3 months. The patients were in average 40.5 years old. Rate of success is discribed depending on the localization and pillar-link-relation. The peaks of loss were after 300 and 900 days incorporation period. Conclusions for indication, prognosis and methodical references are given.

[Chromatographic separation of mono- and oligomeric carbohydrates from biological materials with the use of HPLC glass columns]. / Chromatographische Trennungen mono- und oligomerer Kohlenhydrate aus biologischen Materialien mit Hilfe von HPLC-Glassäulen.

By means of HPLC mono- and oligomeric carbohydrates are separated on silica, modified chemically with aminopropyl groups or impregnated in situ with an amine modifier (piperazine). Fundamentals of this sugar analysis are self-packed glass columns. The advantages of these columns are high efficiencies and handy column plugs. They are chemically inert and can be packed in different length with different HPLC packings. Applications of HPLC technique using glass columns are demonstrated by way of qualitative and quantitative determinations of carbohydrates in different food-stuffs and in polysaccharides (e.g. starch, regenerated fibre), hydrolyzed under acid and enzymatic conditions.