SUPPLEMENTARY MATERIAL
Metallopeptide Promoted Inactivation of Angiotensin Converting Enzyme (ACE) and
Endothelin Converting Enzyme (ECE-1). Toward Dual Action Therapeutics.
Nikhil Gokhale and J. A. Cowan*
Correspondence to: Dr. J. A. Cowan, Evans Laboratory of Chemistry, Ohio State University, 100
West 18th Avenue, Columbus, Ohio 43210. Tel: 614 292 2703; Fax: 614 292 1685; e-mail:
cowan@chemistry.ohio-state.edu
(A) Histidine-Leucine calibration standard for evaluation of His-Leu product generated by
ACE activity (Figure SM1).
Florescence Intensity (emm 500nm)
250
Figure SM1. A 50 mM HEPES (pH 7.4)
solution containing 300 mM NaCl and
varying concentrations His-Leu in a final
volume of 0.2 mL. A standard curve was
established following reaction with ophthaldialdehyde.
200
150
100
50
0
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Histidine-Leucine (Moles)
Fluorescence Intensity (em= 500nm)
(B) Influence of ascorbate concentration on rACE activity (Figure SM2).
Figure SM2. A mixture of 1 g ACE,
and 1 mM HHL was incubated in the
presence of the indicated amount of
sodium ascorbate (freshly prepared) for
30 min in a solution containing 50 mM
HEPES (pH = 7.4) 300 mM NaCl, and 10
M ZnCl2.
Product formation was
evaluated
from
the
change
in
fluorescence intensity.
600
550
500
450
400
350
300
0.0
0.5
1.0
Ascorbate Concentration (mM)
1.5
1
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
1.2
4
6
8
10
12
14
16
18
20
Initial Velovity (v, RFU/min)
RFU
(C) Evaluation of the optimum substrate concentration for ECE-1 (Figures SM3 and
SM4).
1.0
0.8
0.6
0.4
0.2
0.0
0
10
20
30
40
50
60
0
Time (min)
Figure SM3. Initial velocities over a range
of substrate concentrations (4 to 20 M as
noted in the inset), and using 0.5nM hECE-1.
5
10
15
Flurogenic Peptide (M)
20
Figure SM4. Plot of the initial velocity
versus initial substrate concentration.
(D) Determination of Km for the fluorogenic peptide substrate Mca-Arg-Pro-Pro-Gly-PheSer-Ala-Phe-Lys(Dnp)-OH (Mca = 7-methoxycoumarin, Dnp = 2,4 dinitrophenyl) against
hECE-1 (Figure SM5).
-1
1/v (RFU . min)
2.0
Km = 7.3 M
-0.2
1.2
0.8
0.4
-1/Km = -0.136
-0.3
1.6
-0.1
0.0
0.0
0.1
0.2
0.3
-1
1/[s] (M )
Figure SM5. Line-Weaver Burk Plot showing evaluation of the Michaelis constant Km
for binding of the fluorogenic substrate to hECE-1. A substrate concentration range of 5
to 20 M was used.
2
% ECE-1 Activity
(E) Characterization of [Cu(KGHK)]+ as an inhibitor of hECE-1 under hydrolytic
conditions using Mca-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(Dnp)-OH as substrate
(Figure SM6).
Figure SM6. A solution containing 10 ng
enzyme (~1 nM) was pre-incubated over a
100
range of [Cu(KGHK)+] concentrations (0-100
80
M) for 1 h in MES buffer (pH = 6) in a 96
well plate assay (final vol. ~ 0.1 mL for each
60
well).
Reactions were initiated following
40
addition of 10 M substrate. Fluorescence
change was monitored up to 30 min and the
20
initial velocity (RFU/Min) determined both for
wells containing the inhibitor and without
0
inhibitor (control). The initial velocity was
0.1
1
10
100
converted to % ECE-1 activity and plotted as a
[Cu(KGHK)] (M)
function of inhibitor added (w.r.t. highest
activity obtained) and fitted to the dose
response curve to yield an IC50 = 4.9 M.
+
(F) Characterization of [Cu(KGHK)]+ as an inactivator of hECE-1 under oxidative
conditions. Aliquots were withdrawn over a range of time intervals and the activity of each
was evaluated by measuring the initial velocity (RFU/min) for ECE-1 mediated hydrolysis
of Mca-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(Dnp)-OH substrate (Figure SM7 to SM10).
For experiments in both Figures SM9 and SM10, 10 ng enzyme (~1 nM) was pre-incubated with
2 M [Cu(KGHK)+] for 1 h in MES buffer (pH = 6). The oxidative reaction was initiated by
addition of 500 M ascorbate. Hydrolytic controls lacked ascorbate. A 0.1 mL aliquot was
taken from the respective reaction tube at the indicated time intervals and the residual enzyme
activity was measured with 10 M final substrate concentration in a 96 well plate. The
fluorescence change (RFU) was monitored up to 40 min and fitted to linear fit in order to
calculate initial velocity. The initial velocity (RFU/Min) data was used to calculate % ECE-1
activity.
Hydrolytic Control
Hydrolytic Inhibition
50
0
3.55
7.10
10.65
14.20
17.75
21.30
24.85
28.40
31.95
RFU
60
40
Hydrolytic Control
0
3.55
7.10
10.65
14.20
17.75
21.30
24.85
28.40
31.95
40
30
RFU
80
20
20
Hydrolytic Inhibition
10
0
0
0
5
10
15
20
25
30
35
0
40
Time (min)
Figure SM7. The inset shows time when
aliquots were withdrawn
5
10
15
20
25
Time (min)
30
35
40
Figure SM8. The inset shows time when
aliquots were withdrawn
3
Oxidative Control
Oxidative Inhibition
70
0
3.55
7.10
10.65
14.20
17.75
21.30
24.85
28.40
31.95
100
RFU
80
60
40
Oxidative Control
Oxidative Inhibition
0
3.55
7.10
10.65
14.20
17.75
21.30
24.85
28.40
31.95
60
50
RFU
120
40
30
20
20
10
0
0
5
10
15
20
25
Time (min)
30
35
0
40
Figure SM9. The inset shows time when
aliquots were withdrawn
0
5
10
15
20
25
Time (min)
30
35
40
Figure SM10. The inset shows time when
aliquots were withdrawn
(G) Time dependent inhibition of hECE-1 by [Cu(KGHK)]+ in the presence of ascorbate
and substrate (Figure SM11 ).
200
Enzyme Control
Hydrolytic Inhibition
Oxidative inhibition
RFU
150
100
50
0
0
20
40
60
80
Time (min)
Figure SM11. Plot of RFU with time (min) under oxidative experimental conditions
(progress curve), where the enzyme control is a hydrolytic control, hydrolytic inhibition
represents enzyme inhibition by [Cu(KGHK)]+ under hydrolytic conditions, and
oxidative inhibition represents enzyme inhibition by [Cu(KGHK)]+ under oxidative
conditions. Final concentrations include 10 ng hECE-1, 2 M [Cu(KGHK)]+ and 10 M
substrate in 0.1 mL 0.1 M MES buffer containing 0.1 M NaCl (pH = 6). Oxidative
reactions have 500 M final concentration of ascorbate.
4
(H) Influence of ascorbate concentration on the activity of hECE-1 (Figure SM12)
240
% ECE-1 Activity
220
200
180
160
140
120
100
80
1
10
100
1000
[Ascorbate] M
Figure SM12. Enzyme activity was evaluated with 10 ng hECE-1 in the presence of the
indicated amount of L-ascorbic acid (freshly prepared). Fluorescence change (RFU/min)
was measured using 10 M of a fluorogenic substrate for 30 min in a solution containing
0.1 M MES (pH = 6), 0.1 M NaCl. The rate of change of fluorescence was converted to
% ECE-1 activity and plotted as a function of ascorbate concentration.
5
(I) Characterization of Cobalt(III) Complex of KGHK peptide by mass spectroscopy
Figure SM 13. The [KGHK-Co(NH3)2]Cl2 complex was prepared in 5 mM aqueous
ammonia solution (pH 9 adjusted by 1 M HCl). The solution was lyophilized and
reconstituted in de-ionized water and analyzed by mass spectroscopy.
6
(J) Characterization of [KGHK-Co(NH3)2]2+ by UV-Vis spectroscopy
0.14
[Co(NH3)6]
0.12
3+
[KGHK-Co(NH3)2]
2+
abs
0.10
0.08
0.06
0.04
0.02
0.00
300
400
500
600
 (nM)
Figure SM 14. UV-VIS spectral features of (—)1 mM [KGHK-Co(NH3)2]2+ and (------) 1 mM
[Co(NH3)6]3+ in aqueous ammonia (pH = 9)
(K) SDS PAGE gels showing the protein bands for hACE under various experimental
conditions
Figure SM 15. The protein was pre-incubated with ascorbate in the presence and absence of
complex for 6 h at 37 C prior to electrophoresis. Lane 1: 0.78 M hACE + 25 M [KGHK-Cu]+
+ 500 M ascorbic acid. Lane 2: 0.78 M hACE + 500 M ascorbic acid. Lane M: Molecular
weight marker.
7