Figure 3. Site of ferric ion binding to CCK₈SO₄.

At pH 4.0 and 298 K addition to 50 μM CCK₈PO₄ (A) or CCK₈SO₄ (C) of 1 (spectrum II) or 3 mol/mol (spectrum III) of FeCl₃ resulted in a substantial loss of signal intensity compared to the spectrum without ferric ions (spectrum I). These results indicate the formation of insoluble Fe³⁺-peptide complexes over the duration of the NMR experiment. For CCK₈SO₄ remaining in solution, shifts were observed in several resonances on addition of ferric ions. The largest change was 0.04 ppm for the Asp7 CHβ and CHβ′ resonances, while the change in the corresponding Asp1 peaks was only half as great. The Asp1 and Asp7 CHβ and CHβ′ resonances each appear as a doublet of doublets because of geminal coupling of each proton to its twin and vicinal coupling to the CHα proton. No such shifts were observed for CCK₈PO₄ remaining in solution. At pH 6.5 and 298 K addition to 140 μM CCK₈PO₄ (B) of 0, 1, 2 or 4 mol/mol of ferric ions (spectra I-IV, respectively) resulted in a slight broadening of all peaks in the spectrum. As the same amounts of ferric citrate were added to CCK₈SO₄ (D) there was a downfield shift in the Asp1 CHβ and CHβ′ resonances to a maximum of 0.02 ppm. Parallel but smaller changes in chemical shifts to a maximum of 0.01 ppm were observed for the Asp7 CHβ and CHβ′ resonances. The chemical shifts of other resonances were unaffected by addition of ferric citrate.