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.