sepharose and methyl-4-mercaptobutyrimidate

A versatile affinity matrix in which the ligand of interest is linked to the matrix through a connector arm containing a disulfide bond is described. It can be synthesized from any amino-substituted matrix by successive reaction with 2-imino-thiolane, 5,5'-dithiobis(2-nitrobenzoic acid), and a thiol derivative of the ligand of choice. The repertoire of ligands can be significantly increased by the appropriate use of avidin-biotin bridges. After adsorption of the material to be fractionated, elution can be effected by reducing the disulfide bond in the connector arm with dithiothreitol. Examples of the preparation and use of various affinity matrices based on amino-substituted Sepharose 6MB are given. One involves the immobilization of the Fab' fragment of a monoclonal antibody against Aspergillus oryzae beta-galactosidase and the specific binding of that enzyme to the resulting immunoaffinity matrix. Another involves the immobilization of N-biotinyl-2-thioethylamine followed by complex formation with avidin. The resulting avidin-substituted matrix was used for the selective adsorption and subsequent recovery of mouse hybridoma cells producing anti-avidin antibodies. By further complexing the avidin-substituted matrix with appropriate biotinylated antigens, it should be possible to fractionate cells producing antibodies against a variety of antigens.

Topics: Antibodies, Monoclonal; Aspergillus oryzae; beta-Galactosidase; Chromatography, Affinity; Disulfides; Dithionitrobenzoic Acid; Galactosidases; Imidoesters; Indicators and Reagents; Sepharose

2-Iminothiolane, a cyclic thioimidate, is known to react readily with amino groups of proteins to give amidinated derivatives containing reactive sulfhydryl groups. In this report it will be shown that 2-iminothiolane also reacts with hydroxyl and sulfhydryl groups. Such a reaction with hydroxyl groups can be used to introduce sulfhydryl groups into polysaccharides. When this reaction is carried out in the presence of 4,4'-dithiodipyridine, polysaccharides containing 4-dithiopyridyl groups can be prepared. These activated polysaccharides couple easily via intermolecular disulfide bond formation with proteins containing thiol or 4-dithiopyridyl groups. The resulting polysaccharide--protein conjugates have good stability, and they are useful reagents for different biochemical applications such as the purification of proteins containing thiol groups and affinity chromatography.

Topics: Allergens; Antigens, Plant; Chemical Phenomena; Chemistry; Chromatography, Affinity; Imidoesters; Immune Sera; Kinetics; Plant Proteins; Pollen; Polysaccharides; Proteins; Sepharose; Sulfhydryl Compounds