Vol. 188, No. 2, 1992 October 30, 1992
Oxidation
BIOCHEMICAL
of Nitric
Oxide
AND BIOPHYSICAL
by Oxygen
by Porphyrinic Ziad Taha, Frederick
Kiechlel
in Biological
RESEARCH COMMUNICATIONS Pages 734-739
Systems
Monitored
Sensor and Tadeusz Malinski*
Department. of Chemistry, Oakland University, Rochester, MI 48309-4401 IDepartment Received
of Clinical Pathology, Wm. Beaumont Hospital, Royal Oak, MI 48072
September
2,
1992
Summary. A porphyrinic sensor was used to monitor the reaction of nitric oxide (NO) with oxygen. In the absence of biological material, the reaction rate is independent of the initial concentration of NO (zero order) and depends only on 02 concentration (first order). At physiologic concentration of NO and 02, the half-life of nitric oxide is in order of minutes and decreased to seconds only in the presence of biological material (intact cells). 0 1992 Academic Press, Inc.
Nitric oxide (NO) plays a role in vasodilatory responses which maintain normal blood pressure and may also mediates the antitumor effects of cytotoxic activated macrophages (1, 2). Recently, NO release has been detected, in the central nervous system and may function as a retrograde messenger (3). The formation of NO also has been demonstrated in neutrophils (4), hepatocytes (5), and adrenal glands (6). The reactivity of NO with different components of biological materials is still unclear, and different conclusions concerning the stability of NO have appeared in literature (7). The reported half-life for NO varies from 3-30s. It has been assumed in most of the studies describing NO release that the short half-life of NO is due to its high reactivity with oxygen. The NO-02 reactivity in biological systems has been based on extrapolation of data obtained for the reaction of nitric oxide with oxygen at liquid air temperature (8). The problems associated with the kinetics of NO release and reactivity are attributed to the lack of a direct real-time analytical procedure for its detection in cells and tissues. Chemiluminiscence, which is widely used, requires purging the sample solution with inert gas to remove NO from its environment. This
*To whom correspondence should be addressed. 0006
?51X/92
Copyright All rights
$4.00
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
134
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
procedure is tedious and time-consuming and most importantly, does not permit the quantitation of nitric oxide in its physiologic environment in the presence of other constituents. The oxygen probe adopted for NO detection (9) has a very limited linear range (l-3 @I), and does not discriminate against NO;. To our knowledge, no report has appeared in the literature concerning the reaction of nitric oxide with oxygen in aqueous media in the absence and also in the presence of biological materials. We have developed a porphyrinic based microsensor for NO detection in a single cell (10). This sensor combines the electrocatalytic properties of conductive polymeric porphyrins and the selectivity of Nafion film which excludes nitrite, a major interferent. This sensor is sensitive enough to monitor changes in NO concentration in a biological system with a response time of less than 10 ms. In this communication we report in situ monitoring of the reaction of NO with 02 in the presence and absence of biological materials. EXPERIMENTAL Reagents and Materials Nickel(H) tetrakis (3-methoxy-4-hydroxy-phenyl) porphyrin (Ni(II)TMHPP) was synthesized according to a procedure described previously (11). Supporting electrolytes for electrochemical measurements was provided by 0.05 M phosphate buffer (pH = 7.4). Nitric oxide standard solutions were prepared by saturating a 25 ml degassed-siliconrubber sealed phosphate buffer with nitric oxide gas (Matheson). Sodium hydroxide (0.1 M) was used on-line to trap other oxides. Standard solutions of oxygen were prepared by adding an equivalent amount of hydrogen peroxide with a micro-syringe to a degassed solution of potassium permanganate in 0.1 M hydrochloric acid. Nitric
Oxide Sensor Fabrication The nitric oxide sensor was prepared according to the procedure described in detail previously (10). Polymeric film of NiTMHPP was deposited on a glassy carbon electrode (GCE) from a solution of 0.1 M sodium hydroxide containing 5 x 10-S M Ni(II)TMHPP by cyclic scanning between -0.2 to 1.2 V vs saturated calomel electrode (SCE) with a scan rate of 100 mV/s. A response with El/2 = 0.5 V appeared due to Ni(II)/Ni(III) couple. The number of deposited monolayers depends on the initial monomer concentration and the number of cyclic voltammetric scans. After the film formation, the electrode was rinsed and immersed in 0.1 M NaOH solution. The sensor surface was coated with a Nafion film by placing 8 ~1 of Nafion (5% alcohol, Aldrich) using a micropipette and left to dry for 5 min. Differential pulse voltammetry (potential range 0.4 - 0.9 V) or amperometry (constant potential 0.7 V) were used to monitor an analytical signal (voltammetric analyzer IBM EC270). 735
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Procedure All measurements were performed using an air tight conventional threeelectrode system in a glass cell fitted with a Teflon cap. The working electrode was a Nafion coated poly-NiTMHPP-GCE, a platinum wire was used as auxiliary electrode and a saturated calomel electrode (SCE) as a reference electrode. Five ml phosphate buffer solution was introduced and bubbled with purified nitrogen for 15 min and nitrogen was kept over the solution throughout the measurements. This purging period was found to be enough to reduce the oxygen level to lower than the sub-micromolar level. After recording the background a standard aliquots of NO and 02 were added. The decrease of NO concentration was followed by a series of voltammograms recorded periodically. The same procedure was repeated for different NO:02 ratios. This procedure was repeated using Hank’s balanced salt solution (HBSS) in the presence and absence of endothelial cells. Peak current yg time curves were analyzed using a Cricket program. An initial reaction rate was obtained by differentiating the current-time equation. RESULTS
AND
DISCUSSION
Figure 1 shows a series of differential pulse voltammograms of 20 @I nitric oxide before and after the addition of 50 @I oxygen. The peak current decreased
160 a
10
120
POTENTIAL.
Figure
1.
Figure
2.
V
’
02
TIME,
a
Voltammetric response of 20 PM nitric oxide before (a) and after the addition of 50 m oxygen at; b) 40 s, c) 100 s, d) 165 s, e) 225 s, f) 285 s, g) 410 s, h) 660 s and i) 725 s. Phosphate buffer (pH = 7.41, scan rate; 10 mV/s and 40 mV pulse (0.5 s). The decay of 10 $.!I NO (a) and 20 PM NO (b) in the presence of 50 w 02. Other conditions as in Figure 1. 736
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
significantly during the first four minutes, after which a slight decrease was observed. The resulted peak-current, i 2 time, t, plot was constructed (Figure 2). This curve, which was analyzed by the least square method fit polynomial of i = a-bt + c@ - dt3 where a =273.6, b = 0.379, c = 2.931 x 104 and d = 4.905 x 10-8, for the mixture of 20 PM NO and 50 @I 02; a = 140.9, b = 0.369, c = 6.870x 104, and d =4.763 x 10-7, for a mixture of 10 @I NO and 50 @I 02. The initial rate of the NO + 02 reaction is di z = b+2ct-3dt2
(2)
Since ct and dt2 are small for short reaction time (t c 100s) they can be neglected and di/dt = b. Calculated initial rates for the NO - 02 reactions are collected in Table 1. As shown in Table 1, there is no effect on the initial reaction rate upon increasing the concentration of nitric oxide at constant oxygen concentration. However, changing the oxygen concentration at a fixed concentration of nitric oxide leads to a dramatic change in the reaction rate. Based on these data, the reaction order can be calculated:
dCN01
di
-dt- - - dt = KLNO]X [Oz]Y (3) From Table 1, it can be also shown that x = 0 and y = 1; i.e. the reaction rate is independent of the initial concentration of NO (reaction is zero order) and it is first order for oxygen. Such independence of NO concentration was observed for different ratios of reactants which excludes the possibility of a pseudo- second order reaction. This suggests that the oxidation of NO by 02 proceeds through a multistep reaction mechanism in which NO is not involved in the rate limiting step. The analytical procedure presented here, unlike other procedures, can be used to quantitate nitric oxide in its natural environment and monitor continuously the change in its concentration with the small consumption of the substrate. The results reported here do not agree with that observed in the gas
Table
1. The observed rate constants for the reaction of nitric with oxygen in phosphate buffer pH 7.4
NO (PM)
02
ww
dlNOl/dt(@/s)
la
xl
0.369
lo
loo
0.730
20
50
0.379
al
100
0.720
737
oxide
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1
1
c !L .I
i TIME
Figure
3.
Injection
of 10 pM of NO to HBSS
solution
a) in the absence of oxygen b) in the presence of oxygen (50 pM) c) in the presence of oxygen (50 u.M) and endothelial cells (5 x lo7 cells, HBSS solution).
phase reaction (12, 13) and at liquid-air temperature (14) which suggests a second order of the NO-02 reaction. The half-life for NO (10 @VU in the presence of 50 @I of 02 is 4 min. This is significantly longer than that reported in biological systems. Figure 3 shows a change of NO concentration in HBSS medium as measured by porphyrinic sensor. An injection of 50 nmole of NO to HBSS medium in close proximity to the sensor caused a decrease of local concentration due to diffusion and reached a plateau of 10 @l after 180 s (Figure 2 a). In the presence of 20 PM of 02, a concentration of NO 6 @l was observed at 180 s (Figure 2 b). However, in the presence of the endothelial cells (5 x 107 cells in HBSS solution) (other condition the same as 2 b) after 70 s the concentration of NO is not detectable by the sensor (below 10 -8 Ml. This data indicate that oxygen plays a minor role in the direct oxidation process of NO in biological systems. The consumption of nitric oxide by endothelial cells can be attributed to different reaction pathways such as reactions with amines and thiols as suggested by Moncada (8) and reaction with 0:. 2
ACKNOWLEDGMRNT This work was supported Research Institute.
in part by a grant from William 738
Beaumont
Hospital
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Buchmuller-Rouiller, Y., Cowadin, S. and Mauel, J. (1992). Biochem. J., 284: 387-392. Xie, Q., Cho, H., Calaycay, J., Mumford, R., Swiderek, K., Lee, T., Ding, A., Troso, T. and Nathan C. (1992). Science, 256: 225228. Schuman, E. and Madison, D. (1991). Science, 254: X03-1507. Schmidt, H., Seifert, R. and Bohme, E. (1989). FEBS, Lettr. 244: 357-260. Billiar, T., Curran, R., Harbrecht, B., Stadler, J., Williams, D., Ochoa, J., Silvio, M., Simmons, R., and Murray S. (1992). Am. J. Physiol. (Cell Physiol. 31) 261: C1077C1082. Palacios, M., Knowles, R., Palmer, R. and Moncada S. (1989). Biochem. Biophys. Res. Comnnm 165: 802-809. Furchgott, R. and Vanhoutte, P. (1989). FASEB J. 3: 2007-2013. Knowles, R., Palacios, M., Palmer, R. and Moncada, S. (1990). Biochem. J. 269: 207-212. Shibuki, K., (1990). Neurosci. Res. 9: 69-76. Mahnski, T. and Taha 2. (1992). Nature 358: 676-678. Malinski, T., Ciszewski, A., Bennett, J. and Fish, J. (1991). J.Electrochem. Sot. 138: 2008-2015. Guillory, W. and Johnson, H. (1965). J. Chem. Phys. 47: 2457-2461. Shaaban, A. (1990). J. Chem. Educ. 67: 869-871. Temkin M. and Puizhov, V. (1935). Acta Physicochim, U.R.S.S. 2: 473-486.
739
Oxidation
BIOCHEMICAL
of Nitric
Oxide
AND BIOPHYSICAL
by Oxygen
by Porphyrinic Ziad Taha, Frederick
Kiechlel
in Biological
RESEARCH COMMUNICATIONS Pages 734-739
Systems
Monitored
Sensor and Tadeusz Malinski*
Department. of Chemistry, Oakland University, Rochester, MI 48309-4401 IDepartment Received
of Clinical Pathology, Wm. Beaumont Hospital, Royal Oak, MI 48072
September
2,
1992
Summary. A porphyrinic sensor was used to monitor the reaction of nitric oxide (NO) with oxygen. In the absence of biological material, the reaction rate is independent of the initial concentration of NO (zero order) and depends only on 02 concentration (first order). At physiologic concentration of NO and 02, the half-life of nitric oxide is in order of minutes and decreased to seconds only in the presence of biological material (intact cells). 0 1992 Academic Press, Inc.
Nitric oxide (NO) plays a role in vasodilatory responses which maintain normal blood pressure and may also mediates the antitumor effects of cytotoxic activated macrophages (1, 2). Recently, NO release has been detected, in the central nervous system and may function as a retrograde messenger (3). The formation of NO also has been demonstrated in neutrophils (4), hepatocytes (5), and adrenal glands (6). The reactivity of NO with different components of biological materials is still unclear, and different conclusions concerning the stability of NO have appeared in literature (7). The reported half-life for NO varies from 3-30s. It has been assumed in most of the studies describing NO release that the short half-life of NO is due to its high reactivity with oxygen. The NO-02 reactivity in biological systems has been based on extrapolation of data obtained for the reaction of nitric oxide with oxygen at liquid air temperature (8). The problems associated with the kinetics of NO release and reactivity are attributed to the lack of a direct real-time analytical procedure for its detection in cells and tissues. Chemiluminiscence, which is widely used, requires purging the sample solution with inert gas to remove NO from its environment. This
*To whom correspondence should be addressed. 0006
?51X/92
Copyright All rights
$4.00
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
134
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
procedure is tedious and time-consuming and most importantly, does not permit the quantitation of nitric oxide in its physiologic environment in the presence of other constituents. The oxygen probe adopted for NO detection (9) has a very limited linear range (l-3 @I), and does not discriminate against NO;. To our knowledge, no report has appeared in the literature concerning the reaction of nitric oxide with oxygen in aqueous media in the absence and also in the presence of biological materials. We have developed a porphyrinic based microsensor for NO detection in a single cell (10). This sensor combines the electrocatalytic properties of conductive polymeric porphyrins and the selectivity of Nafion film which excludes nitrite, a major interferent. This sensor is sensitive enough to monitor changes in NO concentration in a biological system with a response time of less than 10 ms. In this communication we report in situ monitoring of the reaction of NO with 02 in the presence and absence of biological materials. EXPERIMENTAL Reagents and Materials Nickel(H) tetrakis (3-methoxy-4-hydroxy-phenyl) porphyrin (Ni(II)TMHPP) was synthesized according to a procedure described previously (11). Supporting electrolytes for electrochemical measurements was provided by 0.05 M phosphate buffer (pH = 7.4). Nitric oxide standard solutions were prepared by saturating a 25 ml degassed-siliconrubber sealed phosphate buffer with nitric oxide gas (Matheson). Sodium hydroxide (0.1 M) was used on-line to trap other oxides. Standard solutions of oxygen were prepared by adding an equivalent amount of hydrogen peroxide with a micro-syringe to a degassed solution of potassium permanganate in 0.1 M hydrochloric acid. Nitric
Oxide Sensor Fabrication The nitric oxide sensor was prepared according to the procedure described in detail previously (10). Polymeric film of NiTMHPP was deposited on a glassy carbon electrode (GCE) from a solution of 0.1 M sodium hydroxide containing 5 x 10-S M Ni(II)TMHPP by cyclic scanning between -0.2 to 1.2 V vs saturated calomel electrode (SCE) with a scan rate of 100 mV/s. A response with El/2 = 0.5 V appeared due to Ni(II)/Ni(III) couple. The number of deposited monolayers depends on the initial monomer concentration and the number of cyclic voltammetric scans. After the film formation, the electrode was rinsed and immersed in 0.1 M NaOH solution. The sensor surface was coated with a Nafion film by placing 8 ~1 of Nafion (5% alcohol, Aldrich) using a micropipette and left to dry for 5 min. Differential pulse voltammetry (potential range 0.4 - 0.9 V) or amperometry (constant potential 0.7 V) were used to monitor an analytical signal (voltammetric analyzer IBM EC270). 735
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Procedure All measurements were performed using an air tight conventional threeelectrode system in a glass cell fitted with a Teflon cap. The working electrode was a Nafion coated poly-NiTMHPP-GCE, a platinum wire was used as auxiliary electrode and a saturated calomel electrode (SCE) as a reference electrode. Five ml phosphate buffer solution was introduced and bubbled with purified nitrogen for 15 min and nitrogen was kept over the solution throughout the measurements. This purging period was found to be enough to reduce the oxygen level to lower than the sub-micromolar level. After recording the background a standard aliquots of NO and 02 were added. The decrease of NO concentration was followed by a series of voltammograms recorded periodically. The same procedure was repeated for different NO:02 ratios. This procedure was repeated using Hank’s balanced salt solution (HBSS) in the presence and absence of endothelial cells. Peak current yg time curves were analyzed using a Cricket program. An initial reaction rate was obtained by differentiating the current-time equation. RESULTS
AND
DISCUSSION
Figure 1 shows a series of differential pulse voltammograms of 20 @I nitric oxide before and after the addition of 50 @I oxygen. The peak current decreased
160 a
10
120
POTENTIAL.
Figure
1.
Figure
2.
V
’
02
TIME,
a
Voltammetric response of 20 PM nitric oxide before (a) and after the addition of 50 m oxygen at; b) 40 s, c) 100 s, d) 165 s, e) 225 s, f) 285 s, g) 410 s, h) 660 s and i) 725 s. Phosphate buffer (pH = 7.41, scan rate; 10 mV/s and 40 mV pulse (0.5 s). The decay of 10 $.!I NO (a) and 20 PM NO (b) in the presence of 50 w 02. Other conditions as in Figure 1. 736
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
significantly during the first four minutes, after which a slight decrease was observed. The resulted peak-current, i 2 time, t, plot was constructed (Figure 2). This curve, which was analyzed by the least square method fit polynomial of i = a-bt + c@ - dt3 where a =273.6, b = 0.379, c = 2.931 x 104 and d = 4.905 x 10-8, for the mixture of 20 PM NO and 50 @I 02; a = 140.9, b = 0.369, c = 6.870x 104, and d =4.763 x 10-7, for a mixture of 10 @I NO and 50 @I 02. The initial rate of the NO + 02 reaction is di z = b+2ct-3dt2
(2)
Since ct and dt2 are small for short reaction time (t c 100s) they can be neglected and di/dt = b. Calculated initial rates for the NO - 02 reactions are collected in Table 1. As shown in Table 1, there is no effect on the initial reaction rate upon increasing the concentration of nitric oxide at constant oxygen concentration. However, changing the oxygen concentration at a fixed concentration of nitric oxide leads to a dramatic change in the reaction rate. Based on these data, the reaction order can be calculated:
dCN01
di
-dt- - - dt = KLNO]X [Oz]Y (3) From Table 1, it can be also shown that x = 0 and y = 1; i.e. the reaction rate is independent of the initial concentration of NO (reaction is zero order) and it is first order for oxygen. Such independence of NO concentration was observed for different ratios of reactants which excludes the possibility of a pseudo- second order reaction. This suggests that the oxidation of NO by 02 proceeds through a multistep reaction mechanism in which NO is not involved in the rate limiting step. The analytical procedure presented here, unlike other procedures, can be used to quantitate nitric oxide in its natural environment and monitor continuously the change in its concentration with the small consumption of the substrate. The results reported here do not agree with that observed in the gas
Table
1. The observed rate constants for the reaction of nitric with oxygen in phosphate buffer pH 7.4
NO (PM)
02
ww
dlNOl/dt(@/s)
la
xl
0.369
lo
loo
0.730
20
50
0.379
al
100
0.720
737
oxide
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1
1
c !L .I
i TIME
Figure
3.
Injection
of 10 pM of NO to HBSS
solution
a) in the absence of oxygen b) in the presence of oxygen (50 pM) c) in the presence of oxygen (50 u.M) and endothelial cells (5 x lo7 cells, HBSS solution).
phase reaction (12, 13) and at liquid-air temperature (14) which suggests a second order of the NO-02 reaction. The half-life for NO (10 @VU in the presence of 50 @I of 02 is 4 min. This is significantly longer than that reported in biological systems. Figure 3 shows a change of NO concentration in HBSS medium as measured by porphyrinic sensor. An injection of 50 nmole of NO to HBSS medium in close proximity to the sensor caused a decrease of local concentration due to diffusion and reached a plateau of 10 @l after 180 s (Figure 2 a). In the presence of 20 PM of 02, a concentration of NO 6 @l was observed at 180 s (Figure 2 b). However, in the presence of the endothelial cells (5 x 107 cells in HBSS solution) (other condition the same as 2 b) after 70 s the concentration of NO is not detectable by the sensor (below 10 -8 Ml. This data indicate that oxygen plays a minor role in the direct oxidation process of NO in biological systems. The consumption of nitric oxide by endothelial cells can be attributed to different reaction pathways such as reactions with amines and thiols as suggested by Moncada (8) and reaction with 0:. 2
ACKNOWLEDGMRNT This work was supported Research Institute.
in part by a grant from William 738
Beaumont
Hospital
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Buchmuller-Rouiller, Y., Cowadin, S. and Mauel, J. (1992). Biochem. J., 284: 387-392. Xie, Q., Cho, H., Calaycay, J., Mumford, R., Swiderek, K., Lee, T., Ding, A., Troso, T. and Nathan C. (1992). Science, 256: 225228. Schuman, E. and Madison, D. (1991). Science, 254: X03-1507. Schmidt, H., Seifert, R. and Bohme, E. (1989). FEBS, Lettr. 244: 357-260. Billiar, T., Curran, R., Harbrecht, B., Stadler, J., Williams, D., Ochoa, J., Silvio, M., Simmons, R., and Murray S. (1992). Am. J. Physiol. (Cell Physiol. 31) 261: C1077C1082. Palacios, M., Knowles, R., Palmer, R. and Moncada S. (1989). Biochem. Biophys. Res. Comnnm 165: 802-809. Furchgott, R. and Vanhoutte, P. (1989). FASEB J. 3: 2007-2013. Knowles, R., Palacios, M., Palmer, R. and Moncada, S. (1990). Biochem. J. 269: 207-212. Shibuki, K., (1990). Neurosci. Res. 9: 69-76. Mahnski, T. and Taha 2. (1992). Nature 358: 676-678. Malinski, T., Ciszewski, A., Bennett, J. and Fish, J. (1991). J.Electrochem. Sot. 138: 2008-2015. Guillory, W. and Johnson, H. (1965). J. Chem. Phys. 47: 2457-2461. Shaaban, A. (1990). J. Chem. Educ. 67: 869-871. Temkin M. and Puizhov, V. (1935). Acta Physicochim, U.R.S.S. 2: 473-486.
739
