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:

(A) Histidine-Leucine calibration standard for evaluation of His-Leu product generated by ACE activity (Figure SM1).

/ 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 o-phthaldialdehyde.

(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.

(C) Evaluation of the optimum substrate concentration for ECE-1 (Figures SM3 and SM4).

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. / 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-Phe-Ser-Ala-Phe-Lys(Dnp)-OH (Mca = 7-methoxycoumarin, Dnp = 2,4 dinitrophenyl) against hECE-1 (Figure SM5).


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.

(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 range of [Cu(KGHK)+] concentrations (0-100 M) for 1 h in MES buffer (pH = 6) in a 96 well plate assay (final vol. ~ 0.1 mL for each well). Reactions were initiated following addition of 10 M substrate. Fluorescence change was monitored up to 30 min and the initial velocity (RFU/Min) determined both for wells containing the inhibitor and without inhibitor (control). The initial velocity was converted to % ECE-1 activity and plotted as a 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

Figure SM7. The inset shows time when aliquots were withdrawn
Oxidative Control

Figure SM9. The inset shows time when aliquots were withdrawn /
Figure SM8. The inset shows time when aliquots were withdrawn
Oxidative Inhibition

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 ).


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.

(H) Influence of ascorbate concentrationon the activity of hECE-1 (Figure SM12)

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.

(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.

(J) Characterization of [KGHK-Co(NH3)2]2+ by UV-Vis spectroscopy

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.

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