Colorimetric, High-Throughput Assay for Screening Angiotensin I

Oct 19, 2009 - Angiotensin I-converting enzyme (ACE) plays a pivotal role in blood pressure regulation. A colorimetric assay to measure hippuric acid ...
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Anal. Chem. 2009, 81, 9388–9394

Colorimetric, High-Throughput Assay for Screening Angiotensin I-Converting Enzyme Inhibitors V. K. Jimsheena and Lalitha R. Gowda* Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Council of Scientific and Industrial Research, Mysore 570 020, India Angiotensin I-converting enzyme (ACE) plays a pivotal role in blood pressure regulation. A colorimetric assay to measure hippuric acid (HA) was transformed into a rapid ACE assay wherein the released HA from the substrate hippuryl-histidyl-leucine (HHL) is mixed with pyridine and benzene sulfonyl chloride. The resulting yellow color with a λmax at 410 nm is directly proportional to the released HA. The limit of detection and limit of quantitation were 1.46 × 10-7 and 4.43 × 10-7 M HA. ACE activities of different tissues using this method were comparable to the standard high-performance liquid chromatography (HPLC) method. Kinetic studies showed a Km of 30.8 ( 0.1 × 10-6 M for HHL and Vmax of 1.3 ( 0.01 × 10-6 mol/min for porcine lung ACE. This assay coupled with captopril and lisinopril showed IC50 values of 1.1 ( 0.05 × 10-9 and 2.5 ( 0.03 × 10-9 M, respectively. A 96-well microplate format of this method was used to screen the ACE inhibitory potential of peptides fractionated from an enzymatic hydrolysate of arachin. The precision, accuracy, reproducibility, and excellent correlation demonstrated between the colorimetric and the often-used HPLC method renders this extraction-free method a powerful tool for high-throughput screening of ACE inhibitors. Angiotensin I-converting enzyme (ACE; peptidyl dipeptide hydrolase, EC 3.4.15.1) is a membrane-bound glycoprotein localized in the epithelial cells of pulmonary capillaries.1 By virtue of its ability to convert the inactive decapeptide angiotensin I into the potent vasoconstricting octapeptide angiotensin II and abrogating the vasodilator function of bradykinin, ACE plays a pivotal role in the homeostatic mechanism of mammals being involved in the regulation of blood pressure and fluid balance. The synthetic ACE inhibitors captopril,2 enalapril, ramipril, and lisinopril, developed based on the Brazilian viper (Bothrops jararaca) venom

* To whom correspondence should be addressed. Phone: +91-821-2515331. Fax: +91-821-2517233. E-mail: [email protected], [email protected]. (1) Ryan, J. W.; Ryan, U. S.; Schultz, D. R.; Whitaker, C.; Chung, A. Biochem. J. 1975, 146, 497–499. (2) Cushman, D. W.; Cheung, H. S.; Sabo, E. F.; Ondetti, M. A. Biochemistry 1977, 16, 5484–5491.

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peptide scaffold3 are the drugs used to initiate a stepped care therapy to treat mild to moderate hypertension. Synthetic ACE inhibitors have established themselves in the therapy of hypertension and congestive heart failure.4 In addition, ACE inhibitors are used in therapeutics for the prophylactic control of diabetic neuropathy and treatment of heart failure.5 Synthetic inhibitors, although remarkably effective, render side effects which include allergic reactions, cough, taste disturbances, and skin rashes. As a consequence, the recent trend has been toward developing safer and natural inhibitors. Nutrition is a major factor that influences blood pressure.6 Therefore, many research groups have combed for novel ACE inhibitors from food components. Several peptides from a wide variety of foods are reported to inhibit ACE. In consequence, ACE inhibitory peptides derived from daily dietary food proteins as novel physiologically functional food additives afford a healthier and natural alternative to synthetic drugs for therapeutic purposes. A plethora of biologically active peptides from enzymatic hydrolysates of casein,7 fish,8 zein,9 soy sauce,10 fermented milk,11 mushroom,12 rapeseed,13 soy bean protein,14,15 and corn gluten16 have been demonstrated to inhibit ACE both in vivo and in vitro. Most of these antihypertensive peptides have been characterized by the rabbit lung ACE inhibitor assay based on the hydrolysis (3) Patchett, A. A.; Harris, E.; Tristram, E. W.; Wyvratt, M. J.; Wu, M. T.; Taub, D.; Peterson, E. R.; Ikeler, T. J.; ten Broeke, J.; Payne, L. G.; Ondeyka, D. L.; Thorsett, E. D.; Greenlee, W. J.; Lohr, N. S.; Hoffsommer, R. D.; Joshua, H.; Ruyle, W. V.; Rothrock, J. W.; Aster, S. D.; Maycock, A. L.; Robinson, F. M.; Hirschmann, R.; Sweet, C. S.; Ulm, E. H.; Gross, D. M.; Vassil, T. C.; Stone, C. A. Nature 1980, 288, 280–283. (4) Cheung, H. S.; Cushman, D. W. Biochim. Biophys. Acta 1973, 293, 451– 463. (5) Coppey, L. J.; Davidson, E. P.; Rinehart, T. W.; Gellett, J. S.; Oltman, C. L.; Lund, D. D.; Yorek, M. A. Diabetes 2006, 55, 341–348. (6) Houston, M. C. J. Am. Nutraceut. Assoc. 2002, (Suppl. No. I), 1–71. (7) Maruyama, S.; Suzuki, H. A. Agric. Biol. Chem. 1985, 49, 1405–1409. (8) Kohama, Y.; Matsumoto, S.; Oka, H.; Teramoto, T.; Okabe, M.; Mimura, T. Biochem. Biophys. Res. Commun. 1988, 155, 332–337. (9) Miyoshi, S.; Kaneko, T.; Ishizawa, Y.; Fukui, F.; Tanka, H.; Maruyama, S. Agric. Biol. Chem. 1991, 55, 1313–1318. (10) Okamoto, A.; Hanagata, H.; Matsumoto, E.; Kawamura, Y.; Koizumi, Y.; Yanagida, F. Biosci. Biotechnol. Biochem. 1995, 59, 1147–1149. (11) Gobbetti, M.; Ferranti, P.; Samacchi, E.; Goffredi, F. Appl. Environ. Microbiol. 2000, 66, 3898–3904. (12) Hyoung Lee, D.; Ho Kim, J.; Sik Park, J.; Jun Choi, Y.; Soo Lee, J. Peptides 2004, 25, 621–627. (13) Marczak, E. D.; Usui, H.; Fujita, H.; Yang, Y.; Yokoo, M.; Lipkowski, A. W.; Yoshikawa, M. Peptides 2003, 24, 791–798. (14) Mallikarjun Gouda, K. G.; Gowda, L. R.; Rao, A. G.; Prakash, V. J. Agric. Food Chem. 2006, 54, 4568–4573. (15) Wu, J.; Ding, X. Food Res. Int. 2002, 35, 367–375. (16) Yang, Y.; Tao, G.; Liu, P.; Liu, J. J. Agric. Food Chem. 2007, 55, 7891– 7895. 10.1021/ac901775h CCC: $40.75  2009 American Chemical Society Published on Web 10/19/2009

of the synthetic peptide hippuryl-histidyl-leucine (HHL) as described by Cushman and Cheung17 or with some modifications.18,19 HHL is hydrolyzed by ACE to hippuric acid (HA) and histidylleucine (HL). The extent of HA released is directly proportional to the ACE activity. The released HA requires extraction, evaporation, redissolution in water followed by an absorbance measurement at 228 nm, which does not lend itself to routine screening of the large number of potential ACE inhibitor peptides present in food protein hydrolysates. This method is time-consuming, and there exists the possibility of extracting unhydrolyzed HHL, thereby underestimating the inhibitory activity. Although this assay has been in use over decades by the pharmaceutical and food industry, the versatility is limited with respect to highthroughput screening. The products HA and HL have been separated from the substrate HHL by either reversed-phase high-performance liquid chromatography (RP-HPLC)20 or capillary electrophoresis21 requiring expensive instrumentation and a large volume of organic solvents. Direct, extraction-free, spectrophotometric methods involve derivatization of the product HL with 2,4,6-trinitrobenzene sulfonic acid22 or o-phthalaldehyde (OPA),23 which are nonspecific and react with all primary amino groups. Li et al.24 estimated the released HA by a chromogenic reaction that requires >2 h for the color to develop, with protection from light. In the present investigation a diagnostic colorimetric assay for urine HA25 was transformed in to an ACE and a high-throughput ACE inhibitor assay. The released HA when complexed with pyridine and benzene sulfonyl chloride (BSC) forms a yellow color, which is measured at 410 nm. This method is simple, sensitive, and rapid, requiring no solvent extraction and can therefore be used for high-throughput screening of ACE inhibitors. The limit of detection and limit of quantitation are at the nanomolar level. We further demonstrate application of this method for the detection of several ACE inhibitor peptides guaranteed by the enzymatic hydrolysis of arachin, the major storage protein of Arachis hypogaea. EXPERIMENTAL SECTION Materials. HHL, HA, HL, captopril, lisinopril, and bovine pepsin (EC 3.4.4.1) were obtained from Sigma-Aldrich Chemicals Co. St. Louis, MO. Peanut (A. hypogaea) variety TMV-2 was obtained from University of Agricultural Sciences, Gandhi Krishi Vignana Kendra (GKVK), Bangalore, India. Methanol (HPLC grade), acetonitrile (HPLC grade), trichloroacetic acid (TCA), pyridine, and BSC were from Spectrochem Pvt. Ltd., India. All other reagents and chemicals used were of analytical grade. (17) Cushman, D. W.; Cheung, H. S. Biochem. Pharmacol. 1971, 20, 1637– 1648. (18) Arihara, K.; Nakashima, Y.; Mukai, T.; Ishikawa, S.; Itoh, M. Meat Sci. 2001, 57, 319–324. (19) Hernandez-Ledesma, B.; Martin-Alvarez, P. J.; Pueyo, E. J. Agric. Food Chem. 2003, 51, 4175–4179. (20) Wu, J.; Aluko, R. E.; Muir, A. D. J. Chromatogr., A 2002, 950, 125–130. (21) Zhang, R.; Xu, X.; Chen, T.; Li, L.; Rao, P. Anal. Biochem. 2000, 280, 286– 290. (22) Matsui, T.; Matsufuji, H.; Osajima, Y. Biosci. Biotechnol. Biochem. 1992, 56, 517–518. (23) Chang, B. W.; Chen, R. L.; Huang, I. J.; Chang, H. C. Anal. Biochem. 2001, 291, 84–88. (24) Li, G. H.; Liu, H.; Shi, Y. H.; Le, G. W. J. Pharm. Biomed. Anal. 2005, 37, 219–224. (25) Umberger, C. J.; Fiorese, F. F. Clin. Chem. 1963, 9, 91–96.

Methods. Isolation of Arachin. Arachin was isolated as described earlier.26 A 10% (w/v) slurry of defatted peanut flour in 10% NaCl (w/v) was extracted by agitation for 4-6 h at 25 ± 2 °C. The extract was clarified by centrifugation at 8000 rpm for 45 min at 25 ± 2 °C. Arachin was precipitated by the addition of solid (NH4)2SO4 to a final concentration of 40% (w/v). The precipitate obtained after centrifugation (10 000 rpm, 30 min) was redissolved in 10% NaCl. The (NH4)2SO4 precipitation step was repeated three times to obtain pure arachin. The precipitated arachin dissolved in water was dialyzed extensively against water (1 L × 4), freeze-dried, and stored at 4 °C until further use. Preparation of Arachin-Derived ACE Inhibitor Peptides. One gram of arachin suspended in 10 mL of 0.01 M HCl was digested with bovine pepsin (13 000 U/mg of protein) using an enzyme substrate ratio of 4% (w/w) at 37 °C for 4 h. The pH of the reaction mixture was maintained by the addition of NaOH. The reaction was terminated by the addition of 1 N NaOH. Undigested protein was removed by 10% TCA (w/v) precipitation. The supernatant containing a suite of peptides was used as the source of ACE inhibitor peptides. This suite of peptides was fractionated by RPHPLC on a semipreparative C-18 Shimpak column (21.2 mm × 25 mm (i.d.), 10 µm) using a binary step gradient of 0.1% TFA and 70% acetonitrile in water containing 0.05% TFA at a flow rate of 15 mL/min traversing from 0-100% B with a hold of 4 min after every 10 min. The peptides were detected at 230 nm. Preparation of Porcine Kidney and Lung ACE. ACE was extracted from porcine lung and kidney acetone powder prepared in the laboratory. One gram of acetone powder was extracted with 10 mL of 0.05 M sodium borate buffer pH 8.2 containing 0.3 M NaCl and 0.5% Triton X-100 at 4 °C for 16-18 h. The extract was centrifuged at 15 000 rpm for 60 min at 4 °C. The supernatant was dialyzed against the same buffer without Triton X-100 (1 L × 3) for 24 h and stored at -20 °C until used. Protein Estimation. Protein concentration was determined by the method of Bradford27 or using bicinchoninic acid (BCA).28 Bovine serum albumin was used as the standard. Calibration Curve and Limit of Detection for Hippuric Acid. A stock standard of HA (1 mM) was prepared by dissolving HA in 0.05 M borate buffer pH 8.2 containing 0.3 M NaCl and stored at 4 °C. A series of working standards were obtained from the stock standard by serial dilution with the same buffer over the range of 3.125-100 µM. An amount of 200 µL of 1 M HCl was added to 0.2 mL of each working standard, followed by 0.4 mL of pyridine and then layered with 0.2 mL of BSC. The mixture was mixed by inversion, cooled on ice, and absorbance measured at 410 nm in a UV-vis spectrophotometer (Shimadzu UV 1601). Calibration curves were constructed over five different concentrations of HA, using five replicates of each concentration. The absorbance at 410 nm was plotted against the corresponding concentration. Linear regression analysis using Origin 4.1 was used to generate the standard curves. The limit of detection (LOD) and limit of quantitation (LOQ) were calculated based on the standard deviation of the A410 and slope of the linear calibration curves. Limit (26) Tombs, M. P. Biochem. J. 1965, 96, 119. (27) Bradford, M. M. Anal. Biochem. 1976, 72, 248–254. (28) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985, 150, 76–85.

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of detection was expressed as 3.3d/s and LOQ as 10d/s where d ) the standard deviation of the y-intercept of the regression line and s ) the average slope of the regression line. In Vitro Colorimetric Assay of ACE. ACE activity was assayed by monitoring the release of HA from the hydrolysis of the substrate HHL. The assay mixture contained 0.125 mL of 0.05 M sodium borate buffer pH 8.2 containing 0.3 M NaCl, 0.05 mL of 5 mM HHL, and 0.025 mL of ACE enzyme extract. The reaction was arrested after incubation at 37 °C for 30 min by the addition of 0.2 mL of 1 M HCl. After stopping the reaction, 0.4 mL of pyridine was added followed by 0.2 mL of BSC, and the solution was mixed by inversion for 1 min and cooled on ice. The yellow color developed was measured at 410 nm. One unit of ACE activity is defined as the amount of enzyme that releases 1 µmol of HA per min at 37 °C and pH 8.2. Conventional HPLC Assay for ACE. The ACE activity was conventionally assayed by RP-HPLC as reported by Wu and Ding.15 The product HA was separated from HHL by RP-HPLC on a Waters Symmetryshield octadecyl column (4.6 mm × 150 mm (i.d.), 5 µm) by isocratic elution with 50% methanol containing 0.1% TFA at a flow rate of 0.8 mL/min and detected at 228 nm. Method Validation. The method validation procedures for the present colorimetric assay included evaluation of linearity, intraand inter-run precision, accuracy, and precision and correlation between methods. Linearity was assessed by plotting a curve of the rate of HA released versus enzyme concentration. Three different concentrations of porcine lung ACE were used to assess the intra- and inter-run precision by coefficient of variation obtained from 10 replicates in 10 consecutive runs. The precision between the colorimetric and established HPLC method15 was carried out by Fisher’s test of ratio between the variances of the two methods estimated from the semilogarithmic residual ACE activity curve used to determine the IC50 for captopril and lisinopril. Recovery of HA (25 nmol) spiked in to the ACE assay solutions was used to assess accuracy. Correlation between the colorimetric and HPLC method was estimated by Pearson coefficient, employing two sets of data of ACE assay of different tissues and the percent inhibition obtained with 26 peptide fractions of an arachin hydrolysate. Determination of Vmax and Km. Various concentrations of the substrate HHL were incubated with either porcine lung or kidney ACE. The released HA was determined colorimetrically and by RP-HPLC as described above. The initial reaction velocities were calculated from the released HA. The Eadie-Hofstee plot (v vs v/[S]) was used to calculate the Km and Vmax. In Vitro Colorimetric Assay of ACE Inhibitor Activity. Porcine lung or kidney ACE was preincubated at 37 ± 2 °C for 10 min with either the drugs captopril or lisinopril or peptide fractions, and the residual ACE activity was determined. The IC50 value is defined as the concentration of the inhibitor required to decrease the ACE activity by 50%. The percent inhibition curves were plotted using a minimum of five determinations for each inhibitor concentration, and IC50 values computed from the semilogarithmic plots. A linear regression analysis was performed using Origin 4.1. High-Throughput Microassay for the Screening of ACE Inhibitor Peptides. A high-throughput assay was optimized in a 96-well quartz microtiter plate in a total volume of 0.25 mL. The first two 9390

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rows of the microtiter plate contained HA standards (0.78-25 µM) in a total volume of 0.050 mL in 0.05 M sodium borate buffer, pH 8.2. The consecutive two rows were used to assay ACE at different dilutions in a total reaction volume of 0.0375 mL. The penultimate and last rows were used to assay the inhibitory activity of the RP-HPLC peptide fractions. A preincubation step at 37 °C for 10 min was used to establish contact between inhibitor and ACE. The ACE reaction was initiated by adding 0.0125 mL of 0.005 M HHL and incubating the mixture at 37 °C for 30 min. The reaction was stopped by adding 0.05 mL of 1 M HCl to all the wells including the standard HA. The color was developed by adding 0.1 mL of pyridine followed by 0.05 mL of BSC and mixing thoroughly. The yellow color formed was measured at 410 nm in an ELISA plate reader Spectramax Plus-384 (Molecular Devices, U.S.A.). Data Analysis. For all measurements, a minimum of 3-5 replicates were taken for data analysis. Using the software Origin 4.1, all of the values were averaged and mean values reported. The standard deviation of five different replicates data is tabulated. RESULTS Absorption Spectra of HA under ACE Assay Conditions. ACE hydrolyses the synthetic substrate HHL to HA and the dipeptide HL. The released HA is taken as a measure of the enzyme activity. HA is a metabolic product normally occurring in human urine, which is clinically measured by the yellow color (λmax ) 410 nm) produced when mixed with equal volumes of pyridine and BSC, added in that order.25 This color reaction for HA was adapted to measure the HA released by the action of ACE on HHL. The methodology presented in this work is based on this colored complex, which can be routinely detected using a UV-vis spectrophotometer. ACE assay is carried out in 0.05 M borate buffer, pH 8.2 containing 0.3 M NaCl, and the reaction is terminated by acidification with 1 M HCl. The absorption spectrum of the yellow-colored HA-pyridine-BSC complex under these conditions is shown in Figure 1A. The spectrum showed a single absorption maximum at 410 nm similar to that of urine HA. Both the substrate HHL and the second product, dipeptide HL, did not produce any color when mixed with pyridine and BSC (Figure 1A, curves 2 and 3). These results indicate that neither HHL nor HL interfere with A410 used to measure intensity of the yellow color formed. Further, these results indicate that the whole assay mixture can be used, unlike the conventional Cushman’s method wherein HA requires extraction into ethyl acetate.17 The stability of the yellow color at 410 nm over a period of 2 h showed that the color formed was stable up to 2 h (Figure 1B) and did not require protection from light. Linearity of the Colorimetric Assay for HA. The calibration curve for HA was linear in the range of 3.125-100 µM. The dilutions of HA standard to obtain the required concentration were made in conditions that simulate the pH of the final ACE assay mixture. Regression analysis revealed a coefficient of linearity, R2 ) 0.998, and the intercept was not significantly different from zero. The calibration curve for HA under standard ACE assay conditions follows Beer-Lambert’s law to concentrations as high as 3 × 10-4 M. The LOD is defined as the concentration that gives an increase in absorbance that is still significantly different from the background. The LOD for this method was

Figure 1. (A) Absorbance spectra of the pyridine-BSC complex with (1) HA, (2) HL, and (3) HHL under standard ACE assay conditions (0.05 M sodium borate buffer, pH 8.2 acidified with 1 M HCl). (B) Effect of time on the stability of the HA-pyridine-BSC complex under assay conditions at 410 nm.

Figure 2. (A) Porcine lung ACE activity as a function of time, (B) the effect of varying porcine lung ACE concentration on the rate of HA released, (C) the effect of varying substrate concentrations on the initial velocity of porcine lung ACE, and (D) Eadie-Hofstee plot to evaluate Km and Vmax.

0.146 µM, and LOQ is 0.44 µM of HA. The LOD value of this method is far superior to 29 µM determined by a method using BSC and quinoline.24 The relative standard deviations (RSD) of