Interaction of europium and curium with alpha-amylase

The complexation of Eu(iii) and Cm(iii) with the protein α-amylase (Amy), a major enzyme in saliva and pancreatic juice, was investigated over wide ranges of pH and concentration at both ambient and physiological temperatures. Macroscopic sorption experiments demonstrated a strong and fast binding of Eu(iii) to Amy between pH 5 and 8. The protein provides three independent, non-cooperative binding sites for Eu(iii). The overall association constant of these three binding sites on the protein was calculated to be log K = 6.4 ± 0.1 at ambient temperature. With potentiometric titration, the averaged deprotonation constant of the carboxyl groups (the aspartic and glutamic acid residues) of Amy was determined to be pKa = 5.23 ± 0.14 at 25 °C and 5.11 ± 0.24 at 37 °C. Time-resolved laser-induced fluorescence spectroscopy (TRLFS) revealed two different species for both Eu(iii) and Cm(iii) with Amy. In the case of the Eu(iii) species, the stability constants were determined to be log β11 = 4.7 ± 0.2 and log β13 = 12.0 ± 0.4 for Eu : Amy = 1 : 1 and 1 : 3 complexes, respectively, whereas the values for the respective Cm(iii) species were log β11 = 4.8 ± 0.1 and log β13 = 12.1 ± 0.1. Furthermore, the obtained stability constants were extrapolated to infinite dilution to make our data compatible with the existing thermodynamic database.


Figures
Figure S1.Left: Excitation spectra of Eu-Amy at pH 5.2 and 7.2.Right: Emission spectra of Eu-Amy at pH 7.2 after direct excitation at 579.30 nm.

Experimental
Suspensions of 5 g/L Amy in 0.1 M NaCl with 1 × 10 −4 M Eu(III) were prepared and the pH was adjusted to 5.2 and 7.2.The mixtures were shaken for 24 hours at ambient temperature and afterwards centrifuged for 20 min at 4000 rpm.The Eu-Amy solids were separated from the aqueous solutions and dried at ambient temperature.Measurements were performed at < 20 K using a pulsed flash lamp pumped Nd:YAG-OPO laser system from Spectra Physics (Mountain View, USA), combined with a spectrograph M270 and an ICCD camera system Spectrum One (Horiba-Jobin Yvon).The excitation wavelength was varied between 577 and 582 nm, that cover the range of the 7 F 0 → 5 D 0 transition, with a precision of 0.01 nm, and the luminescence emission was recorded between 585 and 780 nm.

Results
Since the 7 F 0 transition of Eu(III) is a non-degenerate transition, every non-equivalent Eu(III) species yields a single emission line.Therefore the direct excitation of the 7 F 0 → 5 D 0 transition enables us to determine the number of existing Eu(III) species.The excitation spectra of Eu(III)-Amy samples at pH 5.2 and 7.2 exhibit two peak maxima at 578.70 nm (pH 5.2) / 578.90 nm (pH 7.2) and 579.30nm, indicating the presence of two different Eu(III)-Amy complexes at both pH values (Figure S1, left).The intensities of these peaks vary depending on the pH.At pH 5.2 both excitation maxima are pronounced similarly, while at pH 7.2 the peak at 579.30 nm becomes dominant.The species distribution in Figure 4 shows that at pH ~ 5 both 1:1 and 1:3 complexes are formed with similar fractions.On the other hand at pH ~ 7 the 1:3 complex is the dominating species with ~90% fraction, while the 1:1 complex represents only about 10% fraction.Therefore, the excitation maximum at 579.30 nm can be assigned to the 1:3 complex and that at 578.70 nm to the 1:1 complex.However, the luminescence spectra of the single species could not be extracted from the data collected at the relevant excitation wavelengths.The luminescence decay remains mostly biexponential, except for the excitation of the pH ~ 7 sample at 579.70 nm, which shows monoexponential luminescence decay.The static luminescence spectrum in Figure S1 (right) is similar to the spectra with the highest pH and Amy concentration at ambient and physiological temperature (see Figures 3, S2 and S3).Also the luminescence lifetime of 580 ± 10 µs is comparable to that measured at ambient temperature for the 1:3 species.All other static spectra seem to be sum spectra of both species.Nevertheless, the average values of the shorter and longer lifetimes (280 ± 20 µs and 700 ± 50 µs) are comparable to those measured at ambient temperature for the 1:1 and 1:3 species.

Figure S2 .
Figure S2.Deconvoluted luminescence spectra of individual Eu(III) species in the Eu(III)-Amy complexation system.

Figure S4 .
Figure S4.Deconvoluted luminescence spectra of individual Cm(III) species in the Cm(III)-Amy complexation system.

Figure S1 .
Figure S1.Left: Excitation spectra of Eu-Amy at pH 5.2 and 7.2.Right: Emission spectra of Eu-Amy at pH 7.2 after direct excitation at 579.30 nm.

Figure S2 .
Figure S2.Deconvoluted luminescence spectra of individual Eu(III) species in the Eu(III)-Amy complexation system as obtained from SPECFIT.