화학공학소재연구정보센터
Journal of Electroanalytical Chemistry, Vol.438, No.1-2, 55-60, 1997
Quenching as a four-dimensional experiment : application to the multi-domain binding of cytochrome c by cytochrome c peroxidase
We have presented an expanded view of quenching titration experiments as generating four-dimensional data sets, yielding not curves but 2-D surfaces. To determine the stoichiometry and reactivity for the [CcP, Cc] complex, quenching titration experiments were performed using both the conventional substitution mode (C-mode) where Zn-substituted cytochrome c peroxidase (ZnCcP) is the photoprobe and the inverse substitution mode (I-mode) where Zn-substituted cytochrome c (ZnCc) is the photoprobe. For both modes, titrations were performed in either the normal (N) way where aliquots of the quencher are added into a solution containing a fixed concentration of the probe, or the reverse (R) way where aliquots of the probe are titrated into a solution where the concentration of the quencher is fixed. This leads to the expansion of quenching protocols to include four types of titrations : C-N, C-R, I-N, and I-R. Together, the four titrations demonstrate that : (i) Cc reacts at two distinct and non-exclusive surface domains of CcP; (ii) two molecules of Cc can bind simultaneously to CcP; and (iii) the ternary complex is more reactive than the binary complex for the heme-heme ET reaction.