Abstract: Detailed circular dichroism(CD) and Fourier transform infrared(FTIR) studies have been carried out to monitor thermal unfolding of horseradish peroxidase isoenzyme C(HRP) inhibited by CN^-(HRP-CN). The results suggest that HRP CNis quite different from native HRP with different_spin states of Fe of heme and different coordinated states. Cyanide becomes the sixth ligand of Fe(Ⅲ) of heme and the hydrogen binding network is destroyed partly at the same time, which cause the drastic decrease of thermal stability of HRP. The FTIRand Soret-CD spectra analysis demonstrate that during the heating process there is an intermediate state(I') which has both partly destroyed secondary and tertiary structures of native HRP, then it is the appearance of protein aggregation state(A) after fully unfolding. The unfolding pathway thus can be shown as follows: I——I'——U——A.

Key words: Horseradish peroxidase(HRP), Cyanide adduct, Circular dichroism(CD), FTIR, Thermal denaturation