A dye-binding assay for measurement of the binding of Cu(II) to proteins.

Autor: Wilkinson-White LE; School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW 2006, Australia., Easterbrook-Smith SB
Jazyk: angličtina
Zdroj: Journal of inorganic biochemistry [J Inorg Biochem] 2008 Oct; Vol. 102 (10), pp. 1831-8. Date of Electronic Publication: 2008 Jun 20.
DOI: 10.1016/j.jinorgbio.2008.06.008
Abstrakt: We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values. An apparent affinity constant for Cu(II) binding to alpha-synuclein of approximately 1 x 10(9)M(-1) was obtained from measurements of tyrosine fluorescence quenching by Cu(II). This value was in good agreement with that obtained using 5-Br-PSAA. Our analysis and data therefore show that measurement of changes in the equilibria between Cu(II) and 5-Br-PSAA by Cu(II)-binding proteins provides a general procedure for estimating the affinities of proteins for Cu(II).
Databáze: MEDLINE
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