Research On The Interaction Between Heavy Metals And Keyhole Limpet Hemocyanin By Multispectroscopic Techniques

Protein is an important component of all living organisms, which plays a special role in the organisms. The metals especially the trace metal elements in living organisms also play a key role in the process of life. Hemocyanin plays an important role as dioxygen carriers in the hemolymph of many molluscs and arthropods, so far no concern is placed on the interaction mechanism of small molecules with hemocyanin in our knowledge, while the interactions between serum albumin and hemoglobin with many small molecules have been investigated successfully.In this study, the interactions between Keyhole Limpet Hemocyanin（KLH） and heavy metals（copper, chromium, zinc, cadmium, mercury） were investigated by mulispectropic techniques, such as fluorescence, UV-vis absorption and circular dichroism spectroscopy. The binding constants, the number of binding sites and conformational changes of KLH with metals were obtained. The main research content as follows:Chapter 1, spectroscopic investigation on the interaction between KLH and Cu²⁺. The results from UV-vis spectra showed that Cu²⁺ only interact with the polypeptide chain around the dinuclear copper of KLH, rather than replacing the copper ions in the KLH. The intrinsic fluorescence of KLH quenched by Cu²⁺ following static quenching process, and the number of binding sites was approximately equal to 1. Van der Waals force played a major role in the formation of Cu-KLH complex. Furthermore, the α-helix content of KLH decreased with the increase of Cu²⁺.Chapter 2, spectroscopic studies on the interaction between KLH and different species of Chromium. The experimental results showed that the different species of chromium interact with KLH by static quenching mechanism, formating 1:1 Cr-KLH complex. The conformation of KLH were altered by different species of chromium.The binding reaction of KLH with Cr（VI） was stronger than that of Cr（III）. Hydrophobic interaction force played a major role in stabilizing the complex in Cr（VI）-KLH system, while the binding of Cr（III） with KLH was strongly relied on the Van der Waals force.Chapter 3, spectroscopic studies on the interaction of KLH with Zn²⁺, Cd²⁺ and Hg²⁺. The intrinsic fluorescence of KLH quenched by Zn²⁺, Cd²⁺ and Hg²⁺ following static quenching process, and the number of binding sites was approximately equal to 1. The microenvironment and conformation of KLH were changed by Zn²⁺, Cd²⁺ and Hg²⁺. Hydrophobic interaction, electrostatic interaction and Van der Waals force played a major role in the formation of Zn-KLH, Cd-KLH and Hg-KLH complex, respectively.