David H. Bing

Purification of Subcomponents Clq, Cl[rbar] and Cl[sbar] of the First Component of Complement from Cohn Fraction I by Affinity Chromatography

Abstract A method is described for purification of Clq, Cl[rbar] and Cl[sbar] from Cohn Fraction I paste. Initial adsorption of the Cl complex is done by affinity chromatography on an IgG-Sepharose resin in which the IgG is attached to the agarose polymer by an azo-benzyloxyethylsulfono-ethoxy [-N=N-C6H4-CH2O-(CH2)2 -SO2-(CH2)2-O-] “arm”. The Cl[rbar] and Cl[sbar] are co-eluted from this resin by 100 mM EDTA, pH 7.4. The Cl[qbar] is eluted by a buffer containing 200 mM diaminopropane - 1 M NaCl - 200 H3BO3, pH 7.4. The Cl[rbar] and Cl[sbar] are then resolved by DEAE ion exchange chromatography. The Cl[sbar] is purified by rechromatography on DEAE, but the Cl[rbar] requires adsorption with an anti-Cl[sbar] antibody resin to remove the remaining Cl[sbar]. The Clq is purified in two further steps, affinity chromatography on IgG-Sepharose and gel filtration chromatography. Between 100 and 200 mg each of Clq, Cl[rbar] and Cl[sbar] are obtained in a high degree of purity from 8 to 10 kg of Cohn I paste. Biochem...

[9] Design of exo affinity labeling reagents

Publisher Summary Exo affinity labeling reagents are those that covalently bind to an amino acid outside the active center and consist of three regions of activity, S-B-X. The group S is designed to be complementary with the enzyme active site. The chemically reactive group X covalently binds the label to the enzyme. The bridging group, B, links S to X and ensures that X reacts with an amino acid outside the enzyme active site. Endo affinity labeling reagents interfere directly with, and thus identify, amino acids involved in the catalytic process or in the binding of the specific ligand. This chapter details the experiments done to determine and capture the design of exo affinity labeling reagents. It details the synthesis of Benzamidine Sulfonyl Fluorides. It lists the primary features of exo reagents and synthesis of bridging groups. It also considers the secondary considerations, and concludes that essential components of an exo affinity labeling reagent are a substrate analog with a high affinity for the enzyme binding site, a bridging group that has the potential for interaction with secondary sites on the enzyme, and a reactive group for covalent binding to the protein.

Purification of the human complement protein C1s̄ by affinity chromatography

A subunit of the first component of human complement C1q, was purified by the technique of affinity chromatography. The chromatographic resin was cyanogen bromide-activated Sepharose covalently linked to human IgG. For the removal of traces of IgM it was necessary to subject further the C1q obtained from the chromatographic step to ultracentrifugation in sucrose gradients. The highly purified C1q was characterized immunochemically and according to its electrophoretic mobility in polyacrylamide gel. The purified material was capable of combining with C1r and C1s to reconstitute fully active macromolecular C1.

Preparation and characterization of 2,4-diaminovaleric acid: An intermediate in the anaerobic oxidation of ornithine☆

Abstract 2,4-Diaminovaleric acid was purified in gram quantities, three derivatives were crystallized and characterized, and the structure was verified by infrared and nuclear magnetic resonance spectroscopy.

A simplified method for the centrifugation of liposomes

Abstract A method is described by which liposomes may be removed from solution by low speed centrifugation and washed with an isotonic salt solution. Little loss of the trapped galactose marker occurs indicating little destruction of the liposome vesicles.