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 [Elb aum, D., Brzyska, M., Bacia, A. & Alkon, D. (2000) Biochem. Biophys. Res. C ommun. 267, 733-738]. In the present work we compare the ability of beta -a myloid to hydrolyse and oxidize model fluorescent derivatives of dichlorofl uorescein [dichlorodihydrofluorescein (H2DCF) or dichlorofluorescein diacet ate (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 modificat ion studies revealed that hydrolytic properties are related to a His, Ser a nd Asp/Glu triad, while residues of His, Tyr and Met are involved in the ox idative activity of amyloid. Studies with the rat homologue of human beta - amyloid (1-40), containing three amino-acid substitutions (Arg5 --> Gly, Ty r10 --> Phe and His13 --> Arg) confirmed a role of His in the studied proce sses. Reduction of the hydrolysis product caused by inhibitors of Ser ester ases (phenylmethylsulphonyl fluoride and eserine) suggests that beta -amylo id-mediated hydrolysis is Ser sensitive. Antioxidants and metal chelators t hat reduced H2DCF oxidation did not change or increase DCF-DA hydrolysis. S olvent isotope effects suggest the involvement of hydrogen bonds in the hyd rolysis reaction. Hydrolysis was inhibited by redox-active metal ions and w as practically oxygen independent while the oxidation process was redox-act ive-metal enhanced [Cu(II) and Fe(II) primarily], and oxygen dependent. Pro duct formation was significantly inhibited by catalase and superoxide dismu tase 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, sug gesting singlet oxygen intermediacy. Our data are consistent with superoxid e-mediated singlet oxygen intermediate in this Fenton mechanism-driven reac tion. These results indicate that hydrolytic and oxidative properties of be ta -amyloid are distinct features of this peptide and probably require diff erent mechanisms to occur, but both of them may contribute to beta -amyloid toxicity.