A dye-binding assay for measurement of the binding of Cu(II) to proteins.
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).
Wilkinson-White
Lorna E
LE
School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW 2006, Australia.
Easterbrook-Smith
Simon B
SB
D000818:Animals; D001665:Binding Sites; D004396:Coloring Agents / Q000737:chemistry / Q000378:metabolism; D003300:Copper / Q000378:metabolism*; D006801:Humans; D011228:Prealbumin / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D051844:alpha-Synuclein / Q000378:metabolism*
D004396:Coloring Agents; D011228:Prealbumin; D011506:Proteins; C497604:SNCA protein, human; D051844:alpha-Synuclein; D012710:Serum Albumin, Bovine; D003300:Copper