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Bovine α‐Lactalbumin: Identification of two metal‐ion‐binding sites using the europium (III) luminescent probe
Author(s) -
Bünzli JeanClaude G.,
Pfefferlé JeanMarc
Publication year - 1994
Publication title -
helvetica chimica acta
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.74
H-Index - 82
eISSN - 1522-2675
pISSN - 0018-019X
DOI - 10.1002/hlca.19940770132
Subject(s) - chemistry , luminescence , lanthanide , europium , population , binding site , crystallography , metal ions in aqueous solution , titration , fluorescence , binding constant , metal , ion , inorganic chemistry , analytical chemistry (journal) , organic chemistry , biochemistry , physics , demography , optoelectronics , sociology , quantum mechanics
Abstract The luminescent Eu III ion has been used to probe the metal‐binding sites of bovine α‐lactalbumin (BLA) in D 2 O. Upon addition of apo‐BLA to an Eu III ‐containing solution, the intrinsic luminescence of the protein is quenched, and the Eu III luminescence is enhanced. Luminescent titrations point to there being at least two different metal‐binding sites in the apo‐protein. Curve analysis of the high resolution 5 D 0 ← 7 F 0 excitation spectra reveals the existence of three different environments for the bonded Eu III ions. Two environments, labelled I a and I b , give 5 D 0 ← 7 F 0 bands very close in energy; they contain four negatively charged groups and are assigned to one site we identify as the calcium‐binding site. Site I is protected from solvent influences and is somewhat rigid, since it displays selectivity towards lanthanide ions. The origin of the two similar environments I a and I b could not be determined unambiguously. The third environment is ascribed to a nonspecific metal‐binding site in which the Eu III ion is more exposed to the solvent (site II). It is sequentially populated after saturation of site I, and its population is pH‐dependent. The affinity constant of Eu III for this site was estimated from the excitation spectra: log K 2 app 3.5(1). Assignment of the metal binding sites has been facilitated by comparison with model compounds, [Eu(dota)] − (dota 1,4,7,10‐tetraazacyclododecane N,N′,N″, N‴ ‐tetraacetate), [Eu(dtpa)] 2− (dtpa diethylenetriamine tetraacetate), and [Eu(bsa)] (bsa bovine serum albumin). The usefulness and limits of the use of curve‐analysis procedures to unravel the various components of 5 D 0 ← 7 F 0 excitation spectra in biological materials are also discussed.