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Calculated background elimination in quantifying nitric-oxide synthase enzyme activity
J. Biochem. Biophys. Methods 50 Ž2001. 29–32 www.elsevier.comrlocaterjbbm
Calculated background elimination in quantifying nitric-oxide synthase enzyme activity Felix Stonek ) , Walter Tschugguel, Johannes C. Huber, Christian Schneeberger Department of Gynecology and Obstetrics, DiÕision of Gynecological Endocrinology and ReproductiÕe Medicine, UniÕersity Hospital Vienna, Waehringer Guertel 18-20 EBO5Q, A-1090 Vienna, Austria Received 17 November 2000; received in revised form 17 April 2001; accepted 1 May 2001
Abstract Measuring nitric-oxide synthase ŽNOS. activity by monitoring the conversion of L-arginine to is currently the standard assay for NOS activity. We describe a simple method of quantifying low values of NOS activity by removing the background mathematically. When performing NOS activity studies in samples with low protein amount Ž- 25 mgrml., we encountered the problem of sample values that can hardly be differentiated from blank values probably originating from radioactive-labeled arginine in the final eluate. Our method determines mathematically these background values and may be an improvement of the citrulline assay. q 2001 Elsevier Science B.V. All rights reserved.
L-citrulline
Keywords: Nitric oxide; Background elimination; NOS activity assay
1. Article Nitric oxide ŽNO., derived from L-arginine by the action of nitric-oxide synthase ŽNOS., is a cellular messenger with numerous biological functions w1–6x. NOS exists in a variety of isoforms. A constitutive, Ca2q-dependent form of the enzyme is found in endothelial cells ŽeNOS. and the brain ŽbNOS., whereas macrophages, neutrophiles, endothelial and epithelial cells express an inducible, Ca2q-independent form of the enzyme ŽiNOS. w7,8x. Numerous assays have been used to quantify the enzymatic reaction catalyzed by NOS. The Citrulline Assay, a frequently used activity assay for NOS developed by Bredt and Snyder w1,9x, monitors the conversion of radiolabeled )
Corresponding author. Tel.: q43-1-40400-7828; fax: q43-1-40400-7842. E-mail address: [email protected] ŽF. Stonek..
0165-022Xr01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X Ž 0 1 . 0 0 1 7 1 - 3
30
F. Stonek et al.r J. Biochem. Biophys. Methods 50 (2001) 29–32
arginine to citrulline, as this conversion is stoichiometric with the enzymatic formation of NO. In this assay, radioactive-labeled arginine is added to intact tissue or protein extracts. After incubation, the reaction is stopped by the addition of an EDTA-containing buffer, which chelates the calcium ions required by NOS and, consequently, inactivates the enzyme. Equilibrated resin, which binds to the arginine, is added to the sample reaction and transferred into spin cups. At a pH of 5.5, citrulline is ionically neutral and flows through the cups completely. The NOS enzyme activity can then simply be calculated by counting the radioactivity in the eluate. When performing NOS activity studies using a commercially available Citrulline Assay ŽNOSdetect assay Kit; Stratagene, La Jolla, CA., we encountered the problem of complete differentiation between sample and blank value Že.g., 700 vs. 900 cpm sample value., which made it impossible especially in samples with low protein amounts Ž- 25 mgrml. to calculate NOS activity. Although we switched from w3 Hx-labeled arginine, which is prone to radiolytic decay, to more stable w14 Cx-labeled arginine w10x and modified the manufacturer’s assay protocol by using more resin, different incubation times and different temperatures, lessened labeled arginine, varied pH levels of resin and reaction mixtures and used different types of spin cups, resins and reaction mixtures, we had no success in decreasing background levels. Even extensive purification of w14 Cxlabeled arginine on Dowex resin with 0.5 M ammonium chloride ŽNH 4 Cl. w11x failed to reduce background levels. From this data, we concluded that the observed background levels originated from a certain amount of radioactive-labeled arginine, which was not retained by the resin. Experiments for quantifying this amount of arginine were prepared as follows: 1 ml of w14 Cx-labeled arginine Ž296 mCirmmol, Amersham, Little Chalfont, UK. was mixed with 1 ml Microsint-20 Scintillation Cocktail ŽPackard, Meriden, CT. in a 24-well Scinticounterplate ŽPackard. and counted using the TopCount Microplate Scintillation counter ŽPackard.. Then, 200 ml of equilibrated resin ŽDowex 50WX8-400, Sigma; pH 5.5. was transferred into 500-ml microcentrifuge filter cups ŽSigma.. Different volumes Ž1.0, 0.9, 0.8, 0.7, 0.5, 0.3, 0.1 ml. of w14 Cx-labeled arginine were applied on each spin cup, tightly mixed and centrifuged ŽEppendorf Microcentrifuge, 10,000 rpm, 30 s.. The eluate was mixed with 1 ml of Scintillation Cocktail and again counted. The results of this experiment can be seen in Fig. 1. Every data point from Fig. 1 was measured in triplicate and is presented as mean value. The measurement of NOS activity in an unknown tissue sample was performed using the NOSdetect Assay Kit ŽStratagene. according to the manufacturer’s instructions. In brief, after transferring into homogenization buffer, the tissue was disrupted by repeated pipetting. After centrifugation ŽEppendorf Microcentrifuge, 14,000 rpm, 2 min. the supernatant was removed and the protein concentration was measured using BCA Protein Assay Reagent Kit ŽPierce; Rockford, IL.. Ten microliters of protein extract, containing less than 25 mg proteinrml, was mixed with 40 ml of reaction buffer and 1 ml of w14 Cx-labeled arginine. After incubation for 1 h at 37 8C, the reaction was stopped by adding 400 ml of stop buffer. A microcentrifuge filter cup was prepared with resin as described above. The stopped reaction solution was applied on the filter cup, gently mixed and centrifuged within 30 s at full speed. The eluate was collected, transferred to a 24-well plate, combined with 1 ml of Scintillation Cocktail and counted. To mathemat-
F. Stonek et al.r J. Biochem. Biophys. Methods 50 (2001) 29–32
31
Fig. 1. The figure shows an example of the calculation of eluted w14 Cx citrulline using our method for background elimination.
ically eliminate the radioactive background, which originates from nonretained radioactive-labeled arginine in the eluate, one needs to calculate the value of arginine in the eluate by using the diagram shown in Fig. 1 Žalso see the example in Fig. 1.. Due to the low sensitivity of commercially available assays, quantitative detection of low NOS enzyme activities, e.g., in samples with low protein concentrations Žless than 25 mgrml., is extremely difficult to perform. One way to gain scientifically correct results in such samples would be to lower the radioactive background caused by nonretained arginine. In our hands, neither lesser amounts of radioactive arginine, different incubation times and temperatures, nor purification w11x of w14 Cx-labeled arginine Žrecommended by the assay manufacturer. could further reduce the background level significantly. In contrast, our method of background elimination makes it possible to quantify low values of NOS activity by removing the background mathematically. This paper shows a way to measure low NOS enzyme activity by calculated background elimination. The improvement of the citrulline assay was developed and used in our laboratory for studies
32
F. Stonek et al.r J. Biochem. Biophys. Methods 50 (2001) 29–32
of NOS activity in cervical tissue w12,13x and in cultured human endothelial cells, but it can also be used in any other question of interest.
Acknowledgements This work was supported in part by the Jubilaeumsfonds der Oesterreichischen Nationalbank ŽProject 6832..
References w1x Bredt DS, Snyder SH. Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem 1994;63:175–95. w2x Ignarro LJ. Signal transduction mechanism involving nitric oxide. Biochem Pharmacol 1991;41:485–90. w3x Marletta MA. Nitric oxide synthase structure and mechanism. J Biol Chem 1993;268:12231–4. w4x Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993;329:2002–12. w5x Nathan C, Xie QQ. Regulation of biosynthesis of nitric oxide. J Biol Chem 1994;269:13725–8. w6x Schmidt HHHW, Lohmann SM, Walter U. The nitric oxide and cGMP signal transduction system: regulation and mechanism of action. Biochim Biophys Acta 1993;1178:153–75. w7x Knowles RG, Moncada S. Nitric oxide synthase in mammals. Biochem J 1994;298:249–58. w8x Robbins RA, Barns PJ, Springall DR, Warren JB, Kwon OJ, Buttery LD, et al. Expression of inducible nitric oxide synthase in human lung epithelial cells. Biochem Biophys Res Commun 1994;204:209–18. w9x Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci U S A 1990;87:682–5. w10x Bredt DS, Schmidt HHHW. The citrullin assay. In: Fellisch M, Stamler JS, editors. Methods in nitric oxide research. Chichester: Wiley 1996:249–55. w11x Salter M, Knowles RG, Moncada S. Widespread tissue distribution, species distribution and changes in activity of Ca-dependent and Ca-independent nitric oxide synthases. FEBS Lett 1991;291:145–9. w12x Tschugguel W, Schneeberger C, Lass H, Stonek F, Zaghlula MB, Czerwenka K, et al. Human cervical ripening is associated with an increase in cervical inducible nitric oxide synthase expression. Biol Reprod 1998;60:1367–72. w13x Tschugguel W, Schneeberger C, Unfried G, Brautigam G, Stonek F, Wieser F, et al. Elevation of ¨ inducible nitric oxide synthase activity in human endometrium during menstruation. Biol Reprod 1999;60:297–304.
Calculated background elimination in quantifying nitric-oxide synthase enzyme activity Felix Stonek ) , Walter Tschugguel, Johannes C. Huber, Christian Schneeberger Department of Gynecology and Obstetrics, DiÕision of Gynecological Endocrinology and ReproductiÕe Medicine, UniÕersity Hospital Vienna, Waehringer Guertel 18-20 EBO5Q, A-1090 Vienna, Austria Received 17 November 2000; received in revised form 17 April 2001; accepted 1 May 2001
Abstract Measuring nitric-oxide synthase ŽNOS. activity by monitoring the conversion of L-arginine to is currently the standard assay for NOS activity. We describe a simple method of quantifying low values of NOS activity by removing the background mathematically. When performing NOS activity studies in samples with low protein amount Ž- 25 mgrml., we encountered the problem of sample values that can hardly be differentiated from blank values probably originating from radioactive-labeled arginine in the final eluate. Our method determines mathematically these background values and may be an improvement of the citrulline assay. q 2001 Elsevier Science B.V. All rights reserved.
L-citrulline
Keywords: Nitric oxide; Background elimination; NOS activity assay
1. Article Nitric oxide ŽNO., derived from L-arginine by the action of nitric-oxide synthase ŽNOS., is a cellular messenger with numerous biological functions w1–6x. NOS exists in a variety of isoforms. A constitutive, Ca2q-dependent form of the enzyme is found in endothelial cells ŽeNOS. and the brain ŽbNOS., whereas macrophages, neutrophiles, endothelial and epithelial cells express an inducible, Ca2q-independent form of the enzyme ŽiNOS. w7,8x. Numerous assays have been used to quantify the enzymatic reaction catalyzed by NOS. The Citrulline Assay, a frequently used activity assay for NOS developed by Bredt and Snyder w1,9x, monitors the conversion of radiolabeled )
Corresponding author. Tel.: q43-1-40400-7828; fax: q43-1-40400-7842. E-mail address: [email protected] ŽF. Stonek..
0165-022Xr01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X Ž 0 1 . 0 0 1 7 1 - 3
30
F. Stonek et al.r J. Biochem. Biophys. Methods 50 (2001) 29–32
arginine to citrulline, as this conversion is stoichiometric with the enzymatic formation of NO. In this assay, radioactive-labeled arginine is added to intact tissue or protein extracts. After incubation, the reaction is stopped by the addition of an EDTA-containing buffer, which chelates the calcium ions required by NOS and, consequently, inactivates the enzyme. Equilibrated resin, which binds to the arginine, is added to the sample reaction and transferred into spin cups. At a pH of 5.5, citrulline is ionically neutral and flows through the cups completely. The NOS enzyme activity can then simply be calculated by counting the radioactivity in the eluate. When performing NOS activity studies using a commercially available Citrulline Assay ŽNOSdetect assay Kit; Stratagene, La Jolla, CA., we encountered the problem of complete differentiation between sample and blank value Že.g., 700 vs. 900 cpm sample value., which made it impossible especially in samples with low protein amounts Ž- 25 mgrml. to calculate NOS activity. Although we switched from w3 Hx-labeled arginine, which is prone to radiolytic decay, to more stable w14 Cx-labeled arginine w10x and modified the manufacturer’s assay protocol by using more resin, different incubation times and different temperatures, lessened labeled arginine, varied pH levels of resin and reaction mixtures and used different types of spin cups, resins and reaction mixtures, we had no success in decreasing background levels. Even extensive purification of w14 Cxlabeled arginine on Dowex resin with 0.5 M ammonium chloride ŽNH 4 Cl. w11x failed to reduce background levels. From this data, we concluded that the observed background levels originated from a certain amount of radioactive-labeled arginine, which was not retained by the resin. Experiments for quantifying this amount of arginine were prepared as follows: 1 ml of w14 Cx-labeled arginine Ž296 mCirmmol, Amersham, Little Chalfont, UK. was mixed with 1 ml Microsint-20 Scintillation Cocktail ŽPackard, Meriden, CT. in a 24-well Scinticounterplate ŽPackard. and counted using the TopCount Microplate Scintillation counter ŽPackard.. Then, 200 ml of equilibrated resin ŽDowex 50WX8-400, Sigma; pH 5.5. was transferred into 500-ml microcentrifuge filter cups ŽSigma.. Different volumes Ž1.0, 0.9, 0.8, 0.7, 0.5, 0.3, 0.1 ml. of w14 Cx-labeled arginine were applied on each spin cup, tightly mixed and centrifuged ŽEppendorf Microcentrifuge, 10,000 rpm, 30 s.. The eluate was mixed with 1 ml of Scintillation Cocktail and again counted. The results of this experiment can be seen in Fig. 1. Every data point from Fig. 1 was measured in triplicate and is presented as mean value. The measurement of NOS activity in an unknown tissue sample was performed using the NOSdetect Assay Kit ŽStratagene. according to the manufacturer’s instructions. In brief, after transferring into homogenization buffer, the tissue was disrupted by repeated pipetting. After centrifugation ŽEppendorf Microcentrifuge, 14,000 rpm, 2 min. the supernatant was removed and the protein concentration was measured using BCA Protein Assay Reagent Kit ŽPierce; Rockford, IL.. Ten microliters of protein extract, containing less than 25 mg proteinrml, was mixed with 40 ml of reaction buffer and 1 ml of w14 Cx-labeled arginine. After incubation for 1 h at 37 8C, the reaction was stopped by adding 400 ml of stop buffer. A microcentrifuge filter cup was prepared with resin as described above. The stopped reaction solution was applied on the filter cup, gently mixed and centrifuged within 30 s at full speed. The eluate was collected, transferred to a 24-well plate, combined with 1 ml of Scintillation Cocktail and counted. To mathemat-
F. Stonek et al.r J. Biochem. Biophys. Methods 50 (2001) 29–32
31
Fig. 1. The figure shows an example of the calculation of eluted w14 Cx citrulline using our method for background elimination.
ically eliminate the radioactive background, which originates from nonretained radioactive-labeled arginine in the eluate, one needs to calculate the value of arginine in the eluate by using the diagram shown in Fig. 1 Žalso see the example in Fig. 1.. Due to the low sensitivity of commercially available assays, quantitative detection of low NOS enzyme activities, e.g., in samples with low protein concentrations Žless than 25 mgrml., is extremely difficult to perform. One way to gain scientifically correct results in such samples would be to lower the radioactive background caused by nonretained arginine. In our hands, neither lesser amounts of radioactive arginine, different incubation times and temperatures, nor purification w11x of w14 Cx-labeled arginine Žrecommended by the assay manufacturer. could further reduce the background level significantly. In contrast, our method of background elimination makes it possible to quantify low values of NOS activity by removing the background mathematically. This paper shows a way to measure low NOS enzyme activity by calculated background elimination. The improvement of the citrulline assay was developed and used in our laboratory for studies
32
F. Stonek et al.r J. Biochem. Biophys. Methods 50 (2001) 29–32
of NOS activity in cervical tissue w12,13x and in cultured human endothelial cells, but it can also be used in any other question of interest.
Acknowledgements This work was supported in part by the Jubilaeumsfonds der Oesterreichischen Nationalbank ŽProject 6832..
References w1x Bredt DS, Snyder SH. Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem 1994;63:175–95. w2x Ignarro LJ. Signal transduction mechanism involving nitric oxide. Biochem Pharmacol 1991;41:485–90. w3x Marletta MA. Nitric oxide synthase structure and mechanism. J Biol Chem 1993;268:12231–4. w4x Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993;329:2002–12. w5x Nathan C, Xie QQ. Regulation of biosynthesis of nitric oxide. J Biol Chem 1994;269:13725–8. w6x Schmidt HHHW, Lohmann SM, Walter U. The nitric oxide and cGMP signal transduction system: regulation and mechanism of action. Biochim Biophys Acta 1993;1178:153–75. w7x Knowles RG, Moncada S. Nitric oxide synthase in mammals. Biochem J 1994;298:249–58. w8x Robbins RA, Barns PJ, Springall DR, Warren JB, Kwon OJ, Buttery LD, et al. Expression of inducible nitric oxide synthase in human lung epithelial cells. Biochem Biophys Res Commun 1994;204:209–18. w9x Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci U S A 1990;87:682–5. w10x Bredt DS, Schmidt HHHW. The citrullin assay. In: Fellisch M, Stamler JS, editors. Methods in nitric oxide research. Chichester: Wiley 1996:249–55. w11x Salter M, Knowles RG, Moncada S. Widespread tissue distribution, species distribution and changes in activity of Ca-dependent and Ca-independent nitric oxide synthases. FEBS Lett 1991;291:145–9. w12x Tschugguel W, Schneeberger C, Lass H, Stonek F, Zaghlula MB, Czerwenka K, et al. Human cervical ripening is associated with an increase in cervical inducible nitric oxide synthase expression. Biol Reprod 1998;60:1367–72. w13x Tschugguel W, Schneeberger C, Unfried G, Brautigam G, Stonek F, Wieser F, et al. Elevation of ¨ inducible nitric oxide synthase activity in human endometrium during menstruation. Biol Reprod 1999;60:297–304.