Anti-TYrosinase polyclonal antibody

Cysteine dioxygenase (CDO) structurally resembles cupin enzymes that use a 3-His/1-Glu coordination scheme. However, the glutamate ligand is substituted with a cysteine (Cys93) residue, which forms a thioether bond with tyrosine (Tyr157) under physiological conditions. The reversion variant, C93E CDO, was generated in order to reestablish the more common 3-His/1-Glu metal ligands of the cupin superfamily. This variant provides a framework for testing the structural and functional significance of Cys93 and the cross-link in CDO. Although dioxygen consumption was observed with C93E CDO, it was not coupled with L-cysteine oxidation. Substrate analogues (D-cysteine, cysteamine, and 3-mercaptopropionate) were not viable substrates for the C93E CDO variant, although they showed variable coordinations to the iron center. The structures of C93E and cross-linked and non-cross-linked wild-type CDO were solved by X-ray crystallography to 1.91, 2.49, and 2.30 angstrom, respectively. The C93E CDO variant had similar overall structural properties compared to cross-linked CDO; however, the iron was coordinated by a 3-His/1-Glu geometry, leaving only two coordination sites available for dioxygen and bidentate L-cysteine binding. The hydroxyl group of Tyr157 shifted in both non-cross-linked and C93E CDO, and this displacement prevented the residue from participating in substrate stabilization. Based on these results, the divergence of the metal center of cysteine dioxygenase from the 3-His/1-Glu geometry seen with many cupin enzymes was essential for effective substrate binding. The substitution of Glu with Cys in CDO allows for a third coordination site on the iron for bidentate cysteine and monodentate oxygen binding.

This study systematically investigated the binding mechanism between capsaicin (CAP) and beta-lactoglobulin (beta-lg) through the method of multi-spectroscopic, thermodynamics, molecular docking, and molecular dynamics simulation at pH 7.4. The results showed that CAP could interact with beta-lg to enhance the fluorescence intensity of beta-lg. Simultaneously, the complex formed by CAP and beta-lg enhanced the hydrophobicity of the microenvironment of Trp and Tyr in beta-lg. The change in particle size without changing the secondary structure of beta-lg indicates a transition between the large aggregate particle to small aggregate particle of beta-lg. Isothermal titration calorimetric (ITC) results show that hydrophobic interactions play an important role in the formation of the complex. The MD simulation results showed that the RMSD of the systems reached equilibrium and wiggle around the mean value after 30 ns of simulation time. Analysis of Rg indicated that beta-lg and beta-lg/CAP complex was stabilized around 30 ns. Secondary structure analysis results showed that CAP has no distinct effect on the beta-lg structure. Furthermore, the van der Waals interactions are also involved in binding between beta-lg and CAP according to the result of MD simulation. The calculational results indicated that CAP preferred to bind to the hydrophobic pocket of beta-lg. The obtained results could provide some new clues to the interaction mechanism of beta-lg and CAP, which proved that beta-lg possesses the ability to apply in functional food as the vehicle of capsaicin.