A novel insulin-disubstituted Co（Ⅲ） protoporphyrin Ⅸ , CoPI, where Ⅰ is insulin and CoP is Co（Ⅲ） protoporphyrin Ⅸ, was prepared by covalently coupling the propionate groups on the porphyrin ring to the e-amino groups of B29-Lys on insulin via amide linkages. The FTIR spectra in the amide Ⅰ region （1600---1700 cm^-1） and circular dichroism study show that CoPI has a conformation similar to the insulin at pH 6.9, whereas it exhibits significant conformational changes in structure as compared with the insulin self at pH 8.2. The pH absorption titration indicates that the alkaline conditions （pH≥8.0） are required for the formation of complexes between the free CoP and the insulin. The thermodynamic and kinetic data reveal that free CoP is bound to either the zinc-insulin or free insulin with a dissociation constant of （2.0±0.3） × 10^-5 or （2.2±0.3） × 10^-5 mol/L.
