Abstract
The pH dependence of the dynamic quenching of the luminescence from Tb(III) and Eu(III) tris(pyridine-2,6-dicarboxylate≡DPA) chelates by the title proteins is studied. For Tb(DPA)3 3– also the quenching by the Lys 14→Glu and Lys99→Glu mutants of cytochrome c-550 (cytc-550) is investigated. The rate constants for quenching of the electronically excited Λ and Δ enantiomers of the luminophore by equine cytochrome c show a sharp decrease upon increasing the pH from 7 to 10, which can be described phenomenologically by deprotonation of a single acidic group with pK a of 9.2±0.1 for Eu and 9.4±0.1 for Tb. These values are similar to that found for the alkaline transition of the protein. The alkaline conformer(s) of the protein at pH>10 is found to be a very inefficient quencher of the lanthanide luminescence. For Tb, but not for Eu, a significant lowering of the degree of enantioselectivity (E q) in the quenching is found along with a reduction of the quenching rates. For cytc-550, the decrease of the quenching rate constants with increasing pH is described by pK a=9.8±0.1 and for the two mutants the same value is obtained. For the cytc-550 proteins the change of the quenching rates does not correlate with the alkaline transition, for which a pK a of 11.2 has been reported by other workers. For all proteins, the reduction of the quenching rates at high pH is ascribed to a reduction of the binding affinity of the excited lanthanide complex to the surface area of the protein near the exposed heme edge, caused by deprotonation of (presumably) several lysine residues.
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Received: 3 April 1998 / Accepted: 15 June 1998
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Meskers, S., Ubbink, M., Canters, G. et al. pH dependence of the enantioselective excited-state quenching of Λ,Δ-Tb(III) and Λ,Δ-Eu(III)tris(pyridine-2,6-dicarboxylate) chelates by ferricytochrome c from horse heart and ferricytochrome c-550 from Paracoccus versutus . JBIC 3, 463–469 (1998). https://doi.org/10.1007/s007750050256
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DOI: https://doi.org/10.1007/s007750050256