Separation, Purification And Identification Of The Protein Combined With Copper Complexes In Rat Hepatocytes

Metal complexes have been widely reported with therapeutic potential in the treatment of several types of diseases, such as diabetes, cancer, Alzheimer’s disease. Early studies about antitumor mechanism of metal complexes mainly focus on DNA as target molecules. With the development of proteomics, protein targets attract more and more attention.Recent studies have shown that copper complexes can decrease cancer cell growth by inhibiting the proteasomal chymotrypsin-like activity and protein tyrosine phosphatases. Some copper complexes are illustrated to inhibit GSK3β in the brains of APP/PS1transgenic AD model mice and decrease the abundance of0trimers and phosphorylated tau, having the potential to heal Alzheimer’s disease. These results demonstrate copper complexes can interact with various proteins. Then, how many proteins in vivo can combine with copper complexes? This problem attracts our interest. Here, we identified some proteins bond to copper or copper complexes in hepatocytes and studied the interaction between some copper complexes and the purified proteins.Firstly, hepatocytes were extracted and purified through situ digestion and percoll single density gradient centrifugation. The purity of hepatocytes was determined via glycogen staining and reached more than95%.Secondly,97proteins were separated through copper affinity chromatography and identified by mass spectrometry. Information about protein names, molecular weight, peptide number, and relative abundance were obtained. Then three proteins of catalase(protein A), undetermined protein (protein B) and ornithine carbamyl acyltransferase (protein C) were purified by ion exchange column.Thirdly, the interaction between purified protein A (catalase) and protein B (undetermined) and13copper complexes were investigated by protein fluorescence. The results showed that the model of fluorescence quenching of13copper complexes on the two proteins belonged to static quenching process. The combined ratio between the proteins and copper complexes was1:1. The binding constants indicated that the structure of copper complexes influenced the binding to proteins.