 Method

Peptides. The peptide ACAAKAAAKHAAAHKWA was synthesized with the N-terminus

blocked by acetylation and the C-terminus by amidation on a PS3 synthesizer
(Protein Technologies, UK). Diluted to a concentration of 20 mM in MOPS buffer (260
mM NaCl and 60 mM MOPS, pH 7.2), the peptide was labeled with 100 mM
fluorescein 5-maleimide (F5M) for 2 hr at room temperature. The labeled peptide
was purified by HPLC (Agilent 1100 series, San Clara, CA) by using a reverse phase
C18 column (Discovery® BIO wide pore 18, Sigma, USA), and characterized by
MALDI-TOF mass spectrometry (Bruker, Germany).
Fluorometry. The fluorescently labeled peptide was diluted to a concentration of 80
nM in MOPS buffer. To obtain the Cu(II)-binding curves, the HPLC-purified peptide
was mixed with different concentrations of copper chloride solution in aqueous
buffer at pH 7.4. Steady-state fluorescence emission spectra were acquired on a
Hitachi F4500 fluorescence spectrophotometer (Tokyo, Japan). The F5M fluorophore
was excited at 494 nm and the fluorescence was detected at 520 nm. The change of
the emission of the F5M-labeled peptide with increasing concentrations of Cu 2+
(Cu2+/peptide ratios from 1/80 to 31/1) was recorded in separate experiments.
Finally, the change of the emission of the F5M-labeled peptide (80nM) with fixed
concentration of Cu2+ (Cu2+/peptide ratios of 1/80, 1/40, and 1/16) and increasing
concentration of ascorbic acid (from 0.5 nM to 5 nM) was also recorded.
Fluorescence data analysis of peptides. Fluorescence values were averaged over a 5
nm window surrounding the peak of the spectrum. All spectra were corrected with
based line. Data for the Cu(II) titrated with F5M-modified peptide was fitted to a
single-site binding model with following equation:
2+
ΔF [ P⋅Cu] [Cu ]
= =
ΔF max CP 1
+[Cu2+ ]
Ka (1)

From this fitted curve, ΔFmax can be obtained, where ΔF is the fluorescence
variation in the presence of Cu(II), C p is the total concentration of peptide, and the
concentration of bound Cu(II)([ P⋅Cu ]), and its stability constant (1/K a) can be
determined. The molar extinction coefficient of fluorescein F5M : ≥ 80,000 M -1cm-1.

 Results
Metal induced quenching of fluorescein. In order to obtain the quantity of Cu(II) on
peptide, the fluorescence study of peptide was carried out. Peptide with F5M exhibit
fluorescence emission around 520 nm. When Cu(II) was added to the solution of the
peptide, fluorescence intensity at 520 nm will be quenched due to intersystem
crossing mechanism F5M on peptide and peptide bound Cu(II). Therefore, the
fluorescence quenching for the peptide was described as the extent of Cu(II) bound
with peptide by eq 1. Figure 1 shows the fitted result forΔFmax is 6650. In Table 1,
ΔF is the value of 82.5, when final condition were in 1 nM Cu(II) and 80 nM peptide
solution. According to eq 1, ΔF/ΔFmax is the value of 0.012, and [ P⋅Cu ] is 0.99 nM.

Figure 1. The plot of ΔF against [Cu2+]. The determination of ΔFmax by fitting the
fluorescence quenching curve with eq 1 ( figure insert ).

Table 1. The experimental and calculated results of ΔF, ΔF/ΔF max, [P·Cu], and [Cu2+]
for 80 nM peptide in the presence of various concentration of Cu(II).
a. Concentration value of this table is in nM scale.
Fluorescence restore by reduce of metal ion. To further study the concentration of
Cu(I) released from the peptide, the recovery of fluorescence intensity was titrated
with ascorbic acid. Peptide in the presence of Cu(II) were quenched. After ascorbic
acid was added, the fluorescence emission at 520 nm appeared again. This is
because Cu(II) that quenches the fluorescence of peptide was reduced to Cu(I). In
addition, Cu(I) is d10 configuration, which cannot bind with peptide, so as not to
quench the fluorescence of F5M. As the result, the enhancement of fluorescence
intensity for peptide are described as the extent of Cu(I) released from peptide.
Again, the variation of fluorescence can be fitted to eq 1. Table 2 shows fitted results
for ΔFmax are 549.71, 2108, and 2338.89 for 1, 2, and 5 nM Cu(II) peptide solution,
respectively. In the case of 1 nM Cu(II), when 5 nM ascorbic acid was added, ΔF is
the value of 475. According to eq 1, ΔF/ΔFmax is the value of 0.86, and and [Cu+] is
0.86 nM.

Figure 2. The plot of ΔF against [Cu2+]. The determination of ΔFmax by fitting the
fluorescence quenching curve with eq 1. Overlay are three variation of Cu(II)
concentration. (a) Ascorbic acid titrated curve with 1, (b) 2, and (C) 5nM Cu(II) peptide
solution.
Table 2. The environment existed ascorbic acid, the amount of unbound Cu(I) and bound
Cu(II) were calculated.
a. Are the bound Cu(II) concentration from Table 1. of 1nM, 2nM and 5nM Cu(II) added peptide solution, respectively.

b. Concentration value of this table is in nM scale.

In the future, we would like to observe the reducing amount of Cu(II) to Cu(I) in E-
cluster of pmob. If E-cluster undergo electron transfer to C-cluster, Cu(II) will be
generated in E-cluster. As the Cu(II) ion can quench the fluorescence intensity,
[Cu2+] can be calculated from the fluorescence experiment. By knowing [Cu2+], we
can know the extent of electron transfer from E-cluster to C-cluster.