Determination of the Affinity of Drugs toward Serum Albumin by Measurement of the Quenching of the Intrinsic Tryptophan Fluorescence of the Protein

Abstract

Binding of new chemical entities to serum proteins is an issue confronting pharmaceutical companies during development of potential therapeutic agents. Most drugs bind to the most abundant plasma protein, human serum albumin (HSA), at two major binding sites. Excepting fluorescence spectroscopy, existing methods for assaying drug binding to serum albumin are insensitive to higher-affinity compounds and can be labour-intensive, time-consuming, and usually require compound-specific assays. This led us to examine alternative ways to measure drug-albumin interaction. One method described here uses fluorescence quenching of the single tryptophan (Trp) residue in HSA excited at 295 nm to measure drug-binding affinity. Unfortunately, many compounds absorb, fluoresce, or both, in this UV wavelength region of the spectrum. Several types of binding phenomenon and spectral interference were identified by use of six structurally unrelated compounds and the equations necessary to make corrections mathematically were derived and applied to calculate binding constants accurately. The general cases were: direct quenching of Trp fluorescence by optically transparent ligands with low or high affinities; binding of optically transparent, non-fluorescent ligands to two specific sites where both sites or only one site result in Trp fluorescence quenching; and chromophores whose absorption either overlaps the Trp emission and quenches by energy transfer or absorbs light at the Trp fluorescence excitation wavelength producing absorptive screening as well as fluorescence quenching. Unless identification of the site specificity of drug binding to serum albumin is desired, quenching of the Trp fluorescence of albumin by titration with ligand is a rapid and facile method for determining the binding affinities of drugs for serum albumin.

Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma

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Inhibitory activities of baicalin against renin and angiotensin-converting enzyme

“…Where k sv is the Stern−Volmer quenching constant with the unit of L mol −1 , [Q] is the concentration of the quencher, k q is the rate quenching constant of the biological macromolecule, and τ 0 is the average lifetime of the molecule without any quencher and the fluorescence lifetime of the biopolymer is 10 −8 s. K b is the binding constant, and n is the number of binding sites per macromolecule (Blatt et al, 1986;Epps et al, 1999).…”

Fluorescence Quenching of b-Casein by Silver Nanoparticles and Copper Nanoparticles in Presence of Sugars: A Quantitative Exploration

“…Metal nanoparticles based sensors are used for highly sensitive biomedical applications now-a-days. Fluorescence based sensing and binding can provide information about the micro environment of the protein, nanoparticles and sugar molecules [23][24][25][26][27][28][29][30][31][32]. Time resolved fluorescence measurement is a sophisticated technique to sense even a small change in protein, nanoparticles and sugar molecular interactions.…”