Using the LanthaScreen™ ToolBox of reagents to perform Protease Assays
To assay BACE1 catalyzed cleavage of a peptide corresponding to the cleavage site in
the Swedish mutation of APP, a peptide corresponding to the cleavage site (EVNLDAEF,
cleavage between residues L and D) was synthesized with a N-terminal biotin and a C-terminal
fluorescein. The fluorescein was attached via an –amino group of a lysine appended to the Cterminus of the peptide sequence.
To determine the amount of enzyme required for the assay, 100 nM of peptide substrate
was incubated with a dilution series of BACE1 enzyme in a 10 uL reaction volume of 50 mM
sodium acetate buffer, pH 4.5. Each reaction was performed in triplicate. After 1 hour, a 10 uL
solution containing 10 nM terbium-labeled streptavidin and 300 mM Tris, pH 8 in TR-FRET
dilution buffer was added. The high concentration of TRIS was used in order to raise the final pH
to slightly basic in order to stop the BACE1 reaction. After a short (~10 minute) incubation the
plate was read in TR-FRET mode on a Tecan Ultra plate reader and the data presented below in
figure 1.
 -Secretase Titration
into 100 nM TR-FRET Substrate
3.0
TR-FRET Ratio
2.5
2.0
1.5
1.0
0.5
0.0
0.01
0.1
1
10
[Enzyme] u/mL
Figure 1: Titration of BACE1 enzyme against 100 nM peptide substrate. Each datapoint represents the average of three
wells. Error bars are shown on the graph but are extremely small.
From the results of the enzyme titration experiment, a reaction concentration of 0.1
units/mL BACE1 was chosen for the assay. This concentration of enzyme is expected to give
approximately 50% cleavage of substrate in the absence of inhibitor. The inhibitor was a statine
containing peptide (Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Asn-(Statine)-Val-Ala-Glu-Phe-OH) was
titrated against enzyme in a two-fold dilution series beginning at 10 uM. After 60 minutes the
reaction was stopped as described previously by the addition of Tb-labeled streptavidin and Tris
buffer, pH 8, to a final concentration of 5 nM Tb-Streptavidin and 150 mM Tris. The plate was
read on a Tecan Ultra using standard instrument settings and the results are shown (Figure 2).
BACE Inhibition by
Statine Peptide
(Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Asn-(Statine)-Val-Ala-Glu-Phe-OH)
TR-FRET Ratio
1.4
1.2
1.0
0.8
0.6
0.1
1
10
100
[Inhibitor] nM
Figure 2: Inhibition of BACE1 by a statine containing peptide.
1000
10000