Kinetics Investigations Of The Interactions Between Several Biomolecules Using Surface Plasmon Resonance

Cytochrome C (Cyt c) and cytochrome C oxidase (COX) are a couple of terminal enzymes on the mitochondrial respiratory chain. Cyt c, as an electron transportor on the mitochondrial respiratory chain, plays an important role in the rapid enzymatic redox reactions in tissues. COX, locating at the end of the cytochrome system, is capable of accepting four electrons from four Cyt c molecules, and transfering these electrons to oxygen atoms, causing the reduction of oxygen to H2O. Meanwhile, four protons are transported across the membrane, forming a transmembrane potential difference which can be used to synthesis ATP (a basic energy molecule in organisms). The relationship between mitochondrial metabolism and neurological diseases and tumors has caused widespread concern. Understanding the kinetics of Cyt c/COX interactions is very important for the related pathology research. Surface plasmon resonance (SPR) is a powerful tool on the interaction studies of biomolecules, due to its highly sensitive, label-free, and real-time detection. The combination of SPR and electrochemistry, EC-SPR, is capable of stimultaneous detection of both strutural and electrochemical signals from the surface reactions. Moreover, a better control on the redox form of immobillized proteins can be achieved. Here, we immobillized COX onto the surface of the SPR gold film using the amine coupling method. Then, the redox form of injected Cyt c was controlled by the surface potential. After nonlinear curve fitting of SPR signals, we obtain the value of association rate constant ka is7.68×105mol-1·L·s-1for reduced Cyt c/COX and4.06×104mol-1·L·s-1for oxidized Cyt c/COX.The dissociation rate constant kd value is0.0266s-1for reduced Cyt c/COX and0.00593s-1for oxidized Cyt c/COX. Additionally, equilibrium adsorption Ka/desorption constant Kd could calculated to be6.84×105mol-1·L and1.46×10-6mol·L-1for reduced Cyt c/COX,3.01×107mol-1·L and3.32×10-8mol·L-1for oxidized Cyt c/COX, respectively.A(3is the major constitute of senile plaque, a hallmark in the brain of AD victims. It is well-known that Aβ is crucial for the development of AD. The interactions of A(3with neurotransmitters (DA and NE) may affect the neurons, the neurobiological processes and pathogenesis of AD. Here, we preliminarily investigated the mechanisms of Aβ interacting with DA and NE. The results show that in the interaction of DA/Aβ12-28, the binding site in Aβ12-28is Lys28, and the dissociation constant K&of DA/Aβ12-28is2.92pmol·L-1. In the interaction of NE/Aβ1-16, the binding site in Aβ1-16is Tyr10, and the dissociation constant Kd of NE/Aβ1-16is0.59μmol·L-1.