Detection and characterization of oligosaccharides in column effluents using surface plasmon resonance.

Surface plasmon resonance was used to detect oligosaccharides derived from glycoproteins. No sample derivatization or labeling is required for this technique. Sensor surfaces were prepared by immobilizing lectins. In the case of Sambucus nigra agglutinin reactive to terminal sialic acid or Ricinus communis agglutinin preferring terminal beta-linked galactose, femtomole levels of oligosaccharides could be detected. Using this affinity detector system, oligosaccharides and glycopeptides from a chromatographic separation on a gel filtration column were detected either by on-line monitoring or by analyzing the fractions individually.

Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis.

A system for real-time biospecific interaction analysis using biosensor technology based on the optical phenomenon surface plasmon resonance is described. The biospecific interface is a sensor chip covered with a hydrogel matrix. One component of the interaction to be studied is immobilized covalently to the hydrogel and other interactants are passed over the chip in solution. The mass change at the sensor surface, reflecting the progress of the interaction studied, is monitored in real time. The technique, which does not require molecular labels for detection, can measure mass changes down to 10 pg/mm2. Repeated analyses can be performed on the same sensor chip. Applications shown include kinetic measurements, binding site analysis and concentration determination.

Detection of antigen-antibody interactions by surface plasmon resonance. Application to epitope mapping.

Surface plasmon resonance (SPR) detection requires no labeling of antigen or antibodies and allows quantification of two or more interacting molecular species. The automated SPR instrument used here consists of an optical detection unit, an integrated liquid handling unit, and an autosampler. A first molecule is immobilized to the dextran modified surface of the sensor chip. By sequential introduction, the stepwise formation of multimolecular complexes can then be monitored. A two-site binding assay which allows characterization of MoAb epitope specificities is described. A polyclonal rabbit anti-mouse IgG1 (RAMG1) immobilized to the dextran surface is used to capture the first MoAb from unprocessed hybridoma culture supernatants. After introducing the antigen, the ability of a second MoAb to bind to the antigen is tested. The analysis cycle which is fully automated can be performed more than 100 times using the same RAMG1 surface. Since the detection principle allows monitoring of each reactant in the consecutive formation of a multimolecular complex, multi-site binding experiments can be performed. Five MoAbs recognizing different epitopes on an antigen were shown to bind sequentially, forming a hexamolecular complex. MoAbs were further characterized by inhibition analysis using synthetic peptides derived from the primary structure of their antigen. As a model system MoAbs against recombinant HIV-1 core protein p24 were used in all experiments.

Fractionation of human red cell membrane proteins by ion-exchange chromatography in detergent on Mono Q, with special reference to the glucose transporter.

The anion exchanger Mono Q has been used for rapid and efficient fractionation of human red cell membrane proteins in the easily removable detergents n-octyl-beta-D-glucopyranoside or nonanoyl-N-methylglucamide. In practice the chromatographic resolution of membrane proteins was lower than for water-soluble proteins, perhaps due to protein-protein interactions and microheterogeneity, but several components, or groups of components, separated well upon salt gradient elution. The glucose transporter (or transportase) was eluted early, glycophorins later, and the anion transporter still later. The detergents Berol 185 and the zwitter-ionic derivatives of cholate, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonat e and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propan esulphonate, gave similar chromatographic results but differed in solubilization selectivity. A relatively pure material was also fractionated; viz., a glucose transportase which had been prepared by DEAE-cellulose chromatography. Mono Q, in the presence of octyl glucoside, afforded additional purification, which made automatic sequence determination possible for eighteen amino acid residues. The results indicate that two polypeptides were present in about equimolar amounts.

Hb-Linköping (beta 36 Pro----Thr): a new hemoglobin mutant characterized by reversed-phase high-performance liquid chromatography.

Preparative separation of alpha- and beta-chains from a newly identified abnormal hemoglobin has been performed on a new C1/C8 column. Tryptic peptides from the abnormal beta-chain were subjected to peptide mapping on a new C2/C18 column with a reversed-phase system including potassium dihydrogen phosphate (pH 2.9) as a hydrophilic ion-pairing reagent. A hydrophobic substitution, beta 36 proline-threonine, was evident after amino acid analysis and Edman degradation of the isolated mutant peptide. Replacement of proline as the second amino acid in the C-helix represents an important structural change in the alpha 1 beta 2-contact.

Fast chromatofocusing of human serum proteins with special reference to alpha 1-antitrypsin and Gc-globulin.

A new chromatofocusing medium, MonoP, was used for fast (60 min or less) separations of human serum proteins. Separations in the broad pH interval 6.0-3.8 were analysed by fused rocket immunoelectrophoresis to identify a number of proteins, and by gradient gel electrophoresis to determine the molecular weight distribution of the eluted material. To illustrate further the high resolving power of chromatofocusing, narrow pH intervals of about 0.5 pH units were used to study the microheterogeneity of alpha 1-antitrypsin and Gc-globulin. Due to its high resolving power and preparative capacity, chromatofocusing is attractive as the first dimension in two-dimensional techniques for the resolution of complex protein mixtures.

A 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414. Purification, characterization and preparation of an immunoadsorbent for the enzyme.

A 1,4-beta-glucan glucanohydrolase (EC 3.2.1.4) was isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by molecular-sieve chromatography on Bio-Gel P-30, ion-exchange chromatography on DEAE-Sephadex A-50 and isoelectric focusing in a density gradient. Polyacrylamide-gel electrophoresis at two different pH values, analytical isoelectric focusing in a polyacrylamide-gel slab and molecular-sieve chromatography of the reduced and alkylated enzyme in a denaturing medium indicated a homogeneous protein. The enzyme has a mol.wt. of 51,000 and is not a glycoprotein. The pI was found to be 4.66 at 23 degrees C. Antiserum against the purified enzyme was prepared and the amount of enzyme in the original filtrate was determined by rocket immunoelectrophoresis to be about 50mg/liter. An immunoadsorbent made from CNBr-activated sepharose 4B and antiserum affords a rapid and highly specific purification of the enzyme.