The strict regulation of the enzyme activity is essential for the proper functioning of all living organisms, due to the known connection of the extreme, either minimum or maximum, activity of the enzymes to the numerous pathological conditions. A group of papain-like proteases, namely cysteine cathepsins, has awoken a special interest amongst researchers. Cathepsin B is a cysteine peptidase with a catalytic dyad consisting of a cysteine and a histidine residue. It is expressed in all types of cells and it commonly degrades proteins and peptides non-specifically. The activity of the cathepsin B is closely associated with a long list of conditions such as neurological, autoimmune, skeletal, cardiovascular diseases and the formation and metastasis of various malignant tumors. For this reason, cathepsins are a very popular subject of many studies of complete or partial inhibition of enzyme activity. Different protein molecules, organic molecules, metal ions and metal complexes are dynamically studied as inhibitors of the activity of the cathepsin B.
During the research for the Master thesis, we have studied the inhibitory effect of the caffeic acid in combination with iron(III) ions on the activity of cathepsin B. In previous studies, the inhibitory effects of both these elements separately have been proven. However, our research focuses on understanding of the effect of the complex of caffeic acid and the selected metal ion on the activity of enzyme, and determing whether the complex, rather then it's separated components, presents a more powerful inhibitory effect on cathepsin B's activity. In the initial experiments, I have checked the effect of caffeic acid in the presence of different metal ions, in order to determine which ion most strongly inhibits the activity of cathepsin B when combined with caffeic acid. Iron(III) ions were proven to be the best choice. I have measured the effect of the caffeic acid and iron(III) ions on the activity of the enzyme in three different pH values by flourometric tracking of the hydrolysis of the synthetic substrate Z-FR-AMC. Further on I analized the data with GraphPad Prism program and determined the values of the inhibition constants and the EC50 and the mechanism of action of the in situ prepared complex. We found that the inhibitory effects of caffeic acid are improved when an appropriate amount of iron(III) ions is added to the reaction mixtures. Determined values of the EC50 for the inhibition of cathepsine B with the in situ prepared complex in different pH are 6,9 ± 5,3 µM (pH 4,5), 7,1 ± 2,3 µM (pH 5,5), 24,9 ± 5,4 µM (pH 7,4), and are lower when compared to the EC50 values of the inhibition by individual components of the complex. The complex was proven to be an enzyme activity inhibitor acting by a uncompetitivne inhibition mechanism, with the constant of uncompetitive inhibition Kiu= 4,27± 0,29 µM.
I have also attempted to synthesize and characterize the complex of caffeic acid and iron(III) ion in the ratio 3:1, however the synthesis and, consequently, the characterization of the complex showed only partial success. The problems appeared either in the purity or in the solubility of the formed products. Certainly, continuing research in the direction of the synthesis of the complex of caffeic acid and metal ions and the preparation of the crystal structure of the complex makes sense, because this may provide a better insight and a more concise explanation of the mode of action of the complex of caffeic acid and iron(III) ions in the inhibition of cathepsin B.
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