1. Steady-state and time-resolved fluorescence measurements for studying molecular interactions: interaction of a calcium-binding probe with proteins
- Author
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Beverly Z. Packard, Ludwig Brand, Dmitri Toptygin, Gopal Grandhige, Kim Marie Hirshfield, and Hans Kim
- Subjects
Glucose-6-phosphate isomerase ,Biophysics ,Serum albumin ,chemistry.chemical_element ,Calcium ,Ligands ,Biochemistry ,Fluorescence ,Gel permeation chromatography ,medicine ,Humans ,Bovine serum albumin ,Serum Albumin ,Chelating Agents ,Fluorescent Dyes ,Chromatography ,biology ,Chemistry ,Organic Chemistry ,Calcium-Binding Proteins ,Human serum albumin ,Spectrometry, Fluorescence ,Energy Transfer ,biology.protein ,Aminoquinolines ,Chromatography, Gel ,Anisotropy ,Fluorescence anisotropy ,Software ,medicine.drug ,Protein Binding - Abstract
The binding of 2-[(2-bis[carboxymethyl]amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis[carboxymethyl] aminoquinoline, the fluorescent calcium probe Quin2, to serum albumin and several other proteins has been investigated. Changes in fluorescence emission spectra and fluorescence anisotropy revealed interactions between Quin2 and several proteins including human serum albumin, bovine serum albumin, aldolase, phosphoglucose isomerase, glyceraldehyde-3-phosphate dehydrogenase, and alkaline phosphatase. Protein-probe interactions were inhibited by the presence of calcium. Binding was also measured by resonance energy transfer and gel permeation chromatography. Equilibrium binding constants for Quin2 were quantitated by the application of the recently-developed ‘SPECTRABIND’ program to spectroscopic data (D. Toptygin and L. Brand. Anal. Biochem., 224 (1995) 330–338). Binding of Quin2 to human serum albumin is discussed in terms of the published X-ray crystal structure of human serum albumin (X.M. He and D.C. Carter, Nature, 358 (1992) 209–215).
- Published
- 1996