Characterization of the Calcium Binding Domain of Dual Oxidase and Purification of Tri-N-acetylglucosamine from Colloidal Chitin

Hydrogen Peroxide produced by Dual Oxidase 2 is essential for cellular processes including hormone synthesis and bacterial defense. The H2O2 generating activity is regulated by its calcium binding domain (CaBD) which contains two EF-hands that bind Ca2+. Previously, we have studied the Ca2+ binding in the N-terminal of NADPH Oxidase 5's CaBD (N-CaBD) which also contains only two EF-hands. In order to elucidate the mechanism of activation in the CaBD two different protein constructs were generated, trx-Duox (809-902) and Duox (623-1041). These constructs' Ca2+ binding was characterized with intrinsic and extrinsic fluorescence, isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and size exclusion chromatography (SEC). There was no observed increase in Try fluorescence upon binding with Ca2+ for either Duox construct. In contrast of N-CaBD, both Duox constructs showed an increase in ANS fluorescence when bound to Ca2+. ITC studies of trx-Duox (809-902) showed Ca2+ binding occurs first with an exothermic reaction, then an endothermic reaction, indicating that the second Ca2+ ion binding to Duox is an entropicly driven binding. DSC studies show trx-Duox (809-902) to be more stable upon binding with Ca2+ with a DeltaTm of 38°. SEC experiments of trx-Duox (809-902) do not show any influence of Ca2+ binding on dimerization.;Lysozyme has caught much attention due to its aggregation that causes amyloidosis and is a perfect model for studies in protein binding and refolding. To initiate projects answering those critical questions, here we isolated its inhibitor tri-N-acetylglucosamine (NAG3) from colloidal chitin. The chitin polymer was hydrolyzed with 11 M HCl followed by size exclusion chromatography. The fraction rich in NAG3 was further separated by a reverse phase high performance liquid chromatography (RP HPLC). The NAG3 produced was characterized with thin layer chromatography (TLC) and nuclear magnetic resonance (NMR).