Fig. 2.

(A) The Gd³⁺-binding affinity measurement of native ProCA1 was determined by a competing titration using Fluo-5N as a Gd³⁺ indicator. The inset shows the fluorescence spectra (λ_ex = 488 nm) of Fluo-5N with a gradual increase of ProCA1 from 0 µM (top) to 160 µM (bottom). The fluorescence intensity at 520 nm was normalized and the apparent constant (K_app = 18 ± 3 µM) was estimated by a curve-fitting. The dissociation constant (K_d(Gd-ProCA1) = 3.5 × 10⁻¹² M) was calculated based on the K_d measured in previous report [17]. The Gd³⁺-binding dissociation constants K_ds of PEGylated ProCA1 variants were measured with the same method and summarized in Table 1. (B) Hydrodynamic radii of ProCA1 (◆) and various PEGylated ProCA1 with PEG chains PEG0.6k (●), PEG2.4k (■) and PEG12k (▲), were determined by pulse-field-gradient diffusion on 600 MHz NMR. PEGylation increased the radii gradually based on PEG size increased. Data were fitted by a one component equation, exp (−m1*m0^ 2), and the final results are shown in Table 1.