Fig. 3.

Determination of Tb³⁺, Gd³⁺, Zn²⁺ and Ca²⁺ affinity to ProCA32. PSMA. (a) Determined Tb³⁺ affinity to ProCA32.PSMA using the Tb-DTPA chelator buffer system. Free [Tb³⁺] was maintained in a range between 10⁻²³ and 10⁻¹⁷ M by a tightly controlled concentration ratio between Tb-DTPA and free DTPA. The interaction between Tb³⁺ and ProCA32. PSMA was quantified by fluorescence intensity increase mediated by the luminescence resonance energy transfer between Trp in ProCA32.PSMA and bounded Tb³⁺. (b) Determined Gd³⁺ affinity by using Gd³⁺ competition assay. Different concentrations of Gd³⁺ were incubated with Tb³⁺-loaded ProCA32.PSMA. The fluorescence intensity caused by the luminescence resonance energy transfer between Trp in ProCA32.PSMA and bounded Tb³⁺ decrease when Gd³⁺ competed Tb³⁺ out of the metal binding pocket. (c) Determined Zn²⁺ affinity using Fluozin-1 competition assay. (d) Determined Ca²⁺ affinity to ProCA32.PSMA using the Ca²⁺-EGTA buffer system. Free [Ca²⁺] was maintained in a range between 10⁻¹⁰ and 10⁻⁴ M by the tightly controlled concentration of Ca²⁺ and EGTA. The interaction between Ca²⁺ and ProCA32.PSMA was monitored by the Trp fluorescence intensity increase by increasing Ca²⁺ concentration.