Oxidative and hydrolytic properties of beta-amyloid

beta-Amyloid protein is the major component of senile plaques found in the brains of Alzheimer's patients. Previously, a new biochemical property of amyloid, its ability to disrupt ester and peptide bonds, was described [Elbaum, D., Brzyska, M., Bacia, A. & Alkon, D. (2000) Biochem. Biophys. Res. Commun. 267, 733-738]. In the present work we compare the ability of beta-amyloid to hydrolyse and oxidize model fluorescent derivatives of dichlorofluorescein [dichlorodihydrofluorescein (H2DCF) or dichlorofluorescein diacetate (DCF-DA), respectively] to the same final product (dichlorofluorescein). Although there is accumulating evidence of oxidative properties of beta-amyloid, little is known about its hydrolytic abilities. Chemical modification studies revealed that hydrolytic properties are related to a His, Ser and Asp/Glu triad, while residues of His, Tyr and Met are involved in the oxidative activity of amyloid. Studies with the rat homologue of human beta-amyloid (1-40), containing three amino-acid substitutions (Arg5-->Gly, Tyr10-->Phe and His13-->Arg) confirmed a role of His in the studied processes. Reduction of the hydrolysis product caused by inhibitors of Ser esterases (phenylmethylsulphonyl fluoride and eserine) suggests that beta-amyloid-mediated hydrolysis is Ser sensitive. Antioxidants and metal chelators that reduced H2DCF oxidation did not change or increase DCF-DA hydrolysis. Solvent isotope effects suggest the involvement of hydrogen bonds in the hydrolysis reaction. Hydrolysis was inhibited by redox-active metal ions and was practically oxygen independent while the oxidation process was redox-active-metal enhanced [Cu(II) and Fe(II) primarily], and oxygen dependent. Product formation was significantly inhibited by catalase and superoxide dismutase as well as benzoquinone, a specific superoxide anion radical scavenger. Increase of fluorescence by oxidation was strongly inhibited by azide and His and enhanced in samples prepared with deuterated phosphate buffer, suggesting singlet oxygen intermediacy. Our data are consistent with superoxide-mediated singlet oxygen intermediate in this Fenton mechanism-driven reaction. These results indicate that hydrolytic and oxidative properties of beta-amyloid are distinct features of this peptide and probably require different mechanisms to occur, but both of them may contribute to beta-amyloid toxicity.