Fluorescence quenching of human orosomucoid. Accessibility to drugs and small quenching agents

Abstract

The fluorescence behaviour of human orosomucoid was investigated. The intrinsic fluorescence was more accessible to acrylamide than to the slightly larger succinimide, indicating limited accessibility to part of the tryptophan population. Although I- showed almost no quenching, that of Cs+ was enhanced, and suggested a region of negative charge proximal to an emitting tryptophan residue. Removal of more than 90% of sialic acid from the glycan chains led to no change in the Cs+, I-, succinimide or acrylamide quenching, indicating that the negatively charged region originates with the protein core. Quenching as a function of pH and temperature supported this view. The binding of chlorpromazine monitored by fluorescence quenching, in the presence and in the absence of the small quenching probes (above), led to a model of its binding domain on orosomucoid that includes two tryptophan residues relatively shielded from the bulk solvent, with the third tryptophan residue being on the periphery of the domain, or affected allotopically and near the negatively charged field.

Molecular Characterization of an rsmD -Like rRNA Methyltransferase from the Wolbachia Endosymbiont of Brugia malayi and Antifilarial Activity of Specific Inhibitors of the Enzyme

“…The IC 50 was determined by use of an Excel software-based line graphic template after plotting the concentration value of each sample against percent inhibition of motility of the parasite on the x and y axes, respectively (41). The motility data were considered for evaluating IC 50 s. An in vitro cytotoxicity assay with all the test samples was carried out for assessment of the 50% cytotoxicity concentration (CC 50 ), as described earlier (42). The selectivity index (SI) was calculated as CC 50 /IC 50 to determine the therapeutic window for safety for the use of the compounds in animals.…”

“…The motility data were considered for evaluating IC 50 s. An in vitro cytotoxicity assay with all the test samples was carried out for assessment of the 50% cytotoxicity concentration (CC 50 ), as described earlier (42). The selectivity index (SI) was calculated as CC 50 /IC 50 to determine the therapeutic window for safety for the use of the compounds in animals. The compounds with SIs of Ն10 were considered safe and further used for in vivo evaluation in animal models.…”

“…Slit widths for both excitation and emission were kept at 5 nm, and the photomultiplier voltage was 900 V. An enzyme concentration of 0.74 M in a 500-l volume in 20 mM Tris HCl buffer (pH 7.5) with 200 mM NaCl and 1 mM EDTA was used in the study. Quenching of the fluorescence intensity with increasing inhibitor concentrations (0 nm to 400 nm) was exploited in calculating the interaction constant, which is given by a Stern-Volmer plot (50). The Stern-Volmer relationship is represented by …”

Use of Photoaffinity Labeling and Site-directed Mutagenesis for Identification of the Key Residue Responsible for Extraordinarily High Affinity Binding of UCN-01 in Human α1-Acid Glycoprotein

“…2 of 3 Trp residues of hAGP are relatively shielded from the bulk solvent, whereas the third Trp residue is located on the periphery of the domain. It has been deduced that Trp-25 is located deep in the binding pocket, and that Trp-122 is located in the central hydrophobic pocket of the protein (65). This suggests that Trp-160 is the Trp residue that is exposed to the bulk solvent.…”

Mechanisms of retention of pyrrolidinyl norephedrine on immobilized ?1-acid glycoprotein

“…Researchers have proposed that the protein has the potential for stereoselective hydrogen bonding, 11,16,[19][20][21] as well as ion exchange. 10,12,22 Research has also shown that chiral retention of many classes of drugs are dominated by a hydrophobic interaction with the core of the AGP.…”