Binding of Cibacron blue F3GA to proteins containing the dinucleotide fold.

Abstract

A simple, convenient, and sensitive spectrophotometric procedure is described for quantitative measurement of nucleoside phosphate binding sites constructed by the dinucleotide fold. The procedure involves difference spectral titration of such enzymes with the dye Cibacron blue F3GA in a spectral region remote from the intrinsic absorbance of proteins or natural ligands. The dehydrogenase, and glyceraldehyde-phosphate dehydrogenase, respectively. The concentrations of phosphoglycerate kinase and subtilisin were determined using E ' values of 4.9 (11) and 11.7 (12) and molecular weights of 47,000 (11) and 27,500 (12) for the two proteins, respectively. The dye concentration was also measured spectrophotometrically at 610 nm using an extinction coefficient of 13.6 mM-1 cm1 determined by dry weight measurements.Difference spectra and difference spectral titrations were performed at room temperature in cuvettes having 10 mm light paths using a Cary Model 14 and a Gilford spectrophotometer, respectively. Exactly 1 ml of a protein solution and 1 ml of the protein solvent were placed in the sample and reference cuvettes, respectively, and the baseline difference spectrum or difference absorbance was measured. Identical volume increments of a concentrated solution of Cibacron blue F3GA dye were added to both cuvettes and the difference spectrum or abs6rbance was obtained after each incremental addition. [PL]

Orientation of Electron Transport Activities in the Membrane of Intact Glyoxysomes Isolated from Castor Bean Endosperm

“…The sensitivity of NADH:CCR to Cibacron blue 3GA indicated that the enzyme contains a pyridine-nucleotide binding site (23). NADH:CCR also contains (a) sulfhydryl group(s) sensitive to attack by the mercurial reagent mersalyl.…”

Adenylate Cyclase Toxin (ACT) fromBordetella hinzii: Characterization and Differences from ACT ofBordetella pertussis

“…Calmodulin is the eukaryotic protein that binds within a subdomain of the N terminus of B. pertussis ACT and stimulates its adenylate cyclase activity (51,92). To address this affinity issue, we evaluated binding of ACT to Blue-Sepharose, a reactive dye matrix that interacts with proteins through their nucleotide binding sites (54,80). This method has been previously utilized to determine ACT binding affinities based on the differential binding ability of ACT for Blue-Sepharose in the presence and absence of CaM (50).…”

Purification of Phytochrome by Affinity Chromatography on Agarose-Immobilized Cibacron Blue 3GA

“…This is one of several triazine dyes which, when covalently linked to an insoluble porous support matrix such as agarose, is useful in the purification of a variety of enzymes (2,3,(8)(9)(10)12). The mode of purification by blue agarose affmity chromatography is comparable to purification by affmity chromatography in which the covalently linked ligand is a biological ligand, including, among others, substrates, cofactors, activators, antigens, antibodies, and competitive inhibitors.…”