Angiotensin-converting enzyme (ACE) plays a key role in regulating blood pressure in the body by converting the angiotensin I (AI) into angiotensin II (AII). Angiotensin II is a potent vaso-active peptide that causes arterioles to constrict, resulting in increased blood pressure. A rapid and sensitive method for the identification of inhibitors of ACE was developed, and optimized employing HPLC-ESI-QqQ-MS. In this assay, angiotensin I substrate was converted into the product angiotensin II with the catalytic action of ACE. A calibration curve for depleting concentration of angiotensin I was developed and linearity of R 2 = 0.999 with a remarkably low concentration of substrate range 20–200 nM. The limit of detection and quantification of angiotensin I was found to be 1.93 and 5.84 nM, respectively. The enzymatic reaction was optimized for incubation time, concentration, and volume of enzyme and substrate. All reactions were performed at 37 °C at pH 7.5 with standard incubation time of 20 min. Two standard inhibitors, Captopril and Lisinopril, were checked through the newly developed method for their inhibitory potential, and their IC 50 values were found to be 3.969 and 0.852 μM, respectively. Reproducibility and precision analysis of different experiments showed < 9.9% RSD. The developed method can be used for the identification of new ACE inhibitors.
Angiotensin-converting enzyme (ACE) is a di-peptidyl carboxypeptidase (EC 3.4.15.1) with a zinc atom with a mass in range of 150–180 kDa. It catalyzes the conversion of angiotensin I (AI) to the potent vasoconstrictor angiotensin II (AII) (Balasuriya and Rupasinghe, 2011, Elased et al., 2006, Lapointe and Rouleau, 2002). It plays an important role in the renin angiotensin aldosterone system which regulates the blood pressure in the body (Wu et al., 2002). Inhibition of ACE leads to a decrease in the concentration of angiotensin II which causes the reduction in blood pressure (Elased et al., 2006, Lu et al., 2011, Mason et al., 2012). Initially ACE peptide inhibitors were isolated from snake venom, and their role in the treatment of hypertension was recognized immediately. Other potent synthetic inhibitors such as Captopril and Lisinopril were developed and now used widely as drugs for the treatment of hypertension (Chen et al., 2013, Hsieh et al., 1998, Lapointe and Rouleau, 2002, Wu et al., 2002).
Several methods involving the measurement of ACE activity and inhibition activity of drugs have been described, including spectrophotometric (Holmquist et al., 1979, Li et al., 2005), biochemical (van Elswijk et al., 2003), fluorimetric (Cheung et al., 1980, Kang et al., 2002), high-performance liquid chromatography (HPLC) (Chen et al., 2013, Lahogue et al., 2010, Wu et al., 2002), internally quenched fluorogenic (Araujo et al., 1999) and mass spectrometric methods (Greis et al., 2006, Hsieh et al., 1998, Xiao et al., 2006). Most of the previously mass spectrometric method used the LC-MS or MALDI-TOF but this developed method used the LC-MS/MS approach to monitor the enzymatic reaction. A comparison of this method with previously developed methods is given in Table 5.
High performance liquid chromatography-mass spectrometry (HPLC-MS) has high selectivity and high sensitivity. It is a useful tool for biological analysis and widely used for the identification and quantification of proteins and peptides. A combination of LC-MS with MS/MS allows sensitive and unambiguous analysis of peptides in complex sample matrices (de Boer et al., 2007, de Rond et al., 2015, Greis, 2007, Liesener and Karst, 2005, Rathore et al., 2008).
During this study a HPLC-ESI-MS/MS based method was developed for simultaneous analysis and quantification of angiotensin I in an enzymatic mixture by using multiple reaction monitoring (MRM) mode. The conditions for HPLC separation and MS detections were optimized using a standard mixture of angiotensin I, angiotensin II and Bradykinin (internal standard). The method was evaluated for reproducibility, precision, limit of quantification and detection. Two commercially available antihypertensive drugs, Captopril and Lisinopril, were checked to validate the method. The IC 50 values of both standard drugs (Captopril and Lisinopril) were determined and used as reference for the screening of other inhibitors (drugs and compounds). The newly developed assay offers the possibility to use only 20 pico-mole amount of substrate (angiotensin I) per well, as this low quantity was found to be sufficient to perform a complete time dependent study and to determination the IC 50 value. This in turn also decreased the amount of inhibitors (drugs and compounds) used in inhibition assay, up-to pico-mole level quantity.
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Angiotensin-converting enzyme, its substrate angiotensin I, product angiotensin II, and internal standard bradykinin were purchased from Sigma Aldrich (USA). Standard inhibitors Captopril and Lisinopril were purchased from Tokyo Chemical Industries Co, Ltd. (Japan), both > 98% pure. Tris buffer (research grade) was purchased from Serva (Germany). The concentration of this buffer was prepared 20 mM with 3 mM dithiothreitol (DTT) for the pH 7.5 at 37 °C. Formic acid and acetonitrile were purchased
The data for the three peptides including angiotensin I (substrate), angiotensin II (product) and bradykinin (internal standard) was acquired using optimized LC-MS/MS conditions. The ratio of analyte-to-internal standard only for angiotensin I was used for further calculation, calibration curve, and enzymatic analysis.
LC-ESI-QqQ-MS offers a valid readout for enzyme inhibition screening assays. The speed, sensitivity, reduced cost, and reproducibility of this approach makes it an ideal assay for obtaining dose-response curves for comparative IC 50 measurements. Direct measurement ensures the minimization of the false positive and false negative results, and the compounds can be screened without the need for labels, which can limit the other screening approaches. Newly developed enzyme inhibition assay provides
The authors wish to acknowledge Dr. Arslan Ali (Research Officer) and Mr. Junaid-ul-Haq (mass spectroscopist), H.E.J. Research Institute of Chemistry, for their help throughout this project. One of the authors, Mr. M. Salman Bhatti, acknowledges the financial support of the Higher Education Commission (HEC), Pakistan.
