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Formation of long-lived hydroxyl free radical adducts of proline and hydroxyproline in a fenton reaction
94
Biochimica et Biophysica Acta, 790 (1984) 94-97
Elsevier
BBA Report BBA 30078 F O R M A T I O N O F L O N G - L I V E D H Y D R O X Y L FREE RADICAL A D D U C T S OF P R O L I N E AND H Y D R O X Y P R O L I N E IN A F E N T O N R E A C T I O N ROBERT A. FLOYD and IMRE ZS.-NAGY * Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104 (U.S.A.)
(Received March 2nd, 1984) (Revised manuscript received July 20th, 1984)
Key words: Free-radical scavenger," Fenton reaction," Nitroxyl radical; ESR; Proline; Hydroxyprofine
Proline and hydroxyproline when exposed to the hydroxyl free radical generating system of ADP-Fe(II)-H 202 yielded long-lived free radicals. An analysis of the electron paramagnetic resonance spectra of the long-lived hydroxyl free radical adducts of proline and hydroxyproline is consistent with a free electron on a nitroxyl group interacting with the nitrogen atom as well as with three separate protons. In the case of proline, nitroxide formation was observed under the influence of tert-butyl-hydroperoxide, giving a similar EPR spectrum (Lin, J.S., Tom, T.C. and Oicott, H.S. (1974) J. Alp'. Food Chem. 22, 526-528); however, the hydroxyl free radical adduct of hydroxyproline has not been described yet. In the case of the proline nitroxide radical, two of the three protons involved interact with the free electron equivalently. The coupling constants for the hydroxyl free radical adduct of proline are AN= 1.58 mT, A ~ I = A H 2 - 2 . 1 3 mT, A~t3 = 1.77 mT and for hydroxyproline are A N -~ 1.54 mT, A ~ I = 2.56 mT, A~ 2 = 2.03 and A ~ ~ 1.51. The data are consistent with the amine nitrogen of proline and hydroxyproline being oxidized to a nitroxyl group and the free electron of the nitroxyl interacting with the ~-protons of these amino acid hydroxyl free radical adducts.
It has been clear for some time that oxygen free radicals play a significant role in biological systems both as an etiological agent of certain pathological conditions as well as participate in many essential biochemical reactions [1]. Much of the work on oxidative damage up until recently has concentrated upon the lipid portion of membranes, with little attention directed toward the reaction of oxygen free radicals with proteins and amino acids, even though these are major constituents of the call. The recent demonstrations that seemingly minor oxidative changes in certain * Guest scientist from (present address): F. Verz~ International Laboratory for Experimental Gerontology (VILEG), Hungarian Section, University Medical School, H-4012 Debrecen, Hungary. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.
proteins such as glutamine synthetase and al-antiproteinase result in drastic biological consequences, for instance proteinase degradation of the former [2] and cessation of antiproteinase action in the case of the latter [3], if only one amino acid is oxidized, clearly point t o the importance of this area of research. Thus we have begun a study of the interaction of hydroxyl free radicals with proteins and amino acids [4]. In the process we have discovered that hydroxyl free radicals generated in a Fenton-type reaction, when allowed to interact with proline and hydroxyproline, as contrasted to other amino acids [4], produce long-lived free radicals. The formation of proline nitroxide has already been observed under the influence of tertbutylhydroperoxide [5-7], nevertheless, neither proline nor hydroxyproline nitroxide formation
95
have been reported in a Fenton reaction previously. Hydroxyl free radicals were generated in an ADP-Fe(II)-H202 system as describeed by Floyd and Lewis [8]. A typical experiment was carried out as follows: to 10 #1 20 mM ADP, 10 #1 FeC12 (1 mM in 0.0012 M HC1) was added with rapid mixing followed by incubation at 37 °C for 30 s. This was followed by the addition of 20 #1 250 mM proline or hydroxyproline and then 50 #1 buffer (10 mM NaC1/25 mM N a H C O 3 (pH 7.1)). Finally 10 #1 0.3% H 2 0 2 w a s added with rapid mixing and then incubated for 30 s at 37 ° C. This mixture yields a concentration of OH radicals of greater than 20 # M [8]. About 30 s after the final incubation, an EPR spectrum was begun on this mixture. The spectra were obtained on a Varian E-109 X-band spectrometer with the following instrumental parameters: field set 323.7 mT, scan range 10 or 20 m T in an 8 or 16 min scan with a time constant of 1 or 3 s, modulation frequency 100 kHz, modulation amplitude 0.2 mT, receiver gain 1.25.104 , incident microwave frequency 9.14 G H z with a incident power level of 25 mW. The samples were at room temperature (about 25 o C) during the EPR recording. Fig. 1 presents the EPR spectra obtained on proline and hydroxyproline solutions exposed to the ADP-Fe(II)-H202 hydroxyl free radical generating system. The free radicals are present as soon as the EPR spectra can be obtained and persist for several hours. The amount of these radicals pre-
Fig. 1. Electron paramagnetic resonance spectra of hydroxyl free radical adducts of proline (top) and hydroxyproline (bottom).
sent was estimated to be about 9.0 and 8.7 # M for the hydroxyproline and proline adducts, respectively. No detectable free radicals are present when hydroxyproline and proline are absent in the hydroxyl free radical generating system. The spectra have been analyzed and the stick diagrams of our interpretation are shown above the two spectra in Figs. 2 and 3. In each case it is possible to explain the EPR spectra by the interaction of the electron of a nitroxyl free radical with one nitrogen atom and three different protons. The coupling constants for the hydroxyl free radical adduct of proline are A~ = 1.58 roT, A~ 1 = Aft2 = 2.13 mT a n d A~I3 = 1.77 mT, where H1, H2 and H3 designate three different protons, but H1 and H2 are equivalent with respect to the manner the free electron interacts with them. We have designated these protons as fl strictly based upon the results and conventions utilized in spin-trapping studies [9,10] where the protons are linked to an a-carbon atom adjacent to the nitroxyl nitrogen. The coupling constants for the hydroxyl free radical adduct of hydroxyproline are Ar~ -- 1.54 mT, A~ 1 = 2.56 mT, A~ 2 = 2.03 m T a n d A~ 3 = 1.51 mT. Fig. 4 presents our interpretation of the hydroxyl free radical adducts of proline and hydroxyproline. We have assigned protons 1, 2 and 3 as designated in the figure for the following reasons. They are in the 13 position near the nitroxyl and the angle subtended between the Pz orbital of the nitroxyl bond and the proton-carbon bond governs the magnitude of the coupling constant [9,10]. Thus in the case of the OH adduct of proline there are two equivalent protons and we
Fig. 2. Electron paramagnetic resonance spectrum and stick diagram of the hydroxyl free radical adduct of proline.
96
1 mT
Fig. 3. Electron paramagnetic resonance spectrum and stick diagram of the hydroxyl free radical adduct of hydroxyprofine.
have designated these H1 and H2 and assigned them as shown. The other fl-proton, i.e., that associated with the a-carbon to which the carboxyl group is attached, has been assigned and designated as shown. Thus based on the logic of the assignments made in the case of proline we have assigned the protons designated in the free radical of hydroxyproline. The H1 and H2 protons are unequivalent in the latter case, but the magnitude of the values as compared to proline dictates our assignment. The free radicals of proline and hydroxyproline are quite stable as is usually the case for nitroxyl free radicals and also it is clear from the spectrum that there is a nitrogen atom interaction with the free electron. For all of the above reasons we propose the chemical structures shown in Fig. 4 to account for our results.
0
0
^l'-- AI' : 2.1a
A I,. 2 5 e
Ama = 1 . 7 7
Amt - 2 . 0 3
A m = 1.55
A~I= 1.51 A m = 1.54
Fig. 4. Chemical structures and coupling constants (mT) of the hydroxyl free radical adduct of proline (left) and hydroxyproline (fight).
Proline nitroxides have been studied by others [5-7,10], both in water and in organic solvents. There is a remarkable resemblance between the spectra shown by others [5,6] for proline nitroxide and that observed during the present experiments. Although under various experimental conditions the coupling constants proved to be somewhat varying, there is no doubt that our interpretation is basically correct. This statement is supported by computer simulation of the EPR spectra of both the proline and hydroxyproline free radicals (personal communication from one of the reporters of this paper). Another set of reasons that strengthens our interpretation of the results is that in the presence of H20 z without added ADP or Fe(II), there is a slow (over a period of hours) accumulation of the same free radicals as observed when the complete hydroxyl free radical generating system is present. Hydrogen peroxide is used routinely to synthesize nitroxyl free radicals from amines [11]. Regarding chemical mechanisms, it is possible to explain the formation of the nitroxyl from the amine by two sequential additions of hydroxyl free radicals to the amine nitrogen followed by the subsequent loss of water yielding the N-hydroxy compound which is then rapidly oxidized to the nitroxyl [11]. As a matter of fact, proline nitroxide was also synthesized by starting from N-hydroxyL-proline [7]. The biological significance of the observations reported here is not known. It is probable that the amine nitrogen of proline is not available for oxidation to the nitroxyl if it is in a peptide linkage, unless perhaps proline were to be the terminal amino acid in the protein chain. However, it appears necessary to study the action of OH on proline or hydroxyproline when it is in a peptide linkage. Our preliminary experiments with some randomly chosen commonly available proteins has shown that hydroxyl free radicals do not yield free radicals of nitroxyl type even with collagen being rich in proline and hydroxyproline. Nevertheless, it seems to be of importance that proline and hydroxyproline may be hydroxyl free radical scavengers under certain biological conditions and in specific microenvironments where these amino acids as well as hydrogen peroxide and available iron are present and can react with each other.
97 W e would like to t h a n k K a y Wallace, Lisa Gropf, A n i t a Hill, Pat Carnes a n d S a n d r a N a n k for help in the p r e p a r a t i o n of this m a n u s c r i p t a n d for technical assistance. T h e research was supported b y N I H G r a n t AG02599. We also would like to express thanks to one of the reporters of this paper for c o m p u t e r s i m u l a t i o n of the proline a n d h y d r o x y p r o l i n e nitroxyl free radicals.
References 1 Fridovich, I. (1978) Science 201,875-880 2 Fucci, L., Ohver, C.N., Coon, M.J. and Stadtman, E.R. (1983) Proc. Natl. Acad. Sci. USA 80, 1521-1525 3 Johnson, D. and Travis, J. (1979) J. Biol. chem. 254, 4022-4026
4 Zs.-Nagy, I. and Floyd, R.A. (1984) Biochim. Biophys.Acta 790, in the press 5 Van der Veen, J., Weil, J.T., Kennedy, T.E. and Olcott, H.S. (1970) Lipids 5, 509-512 6 Janzen, E.G. (1971) in Topics in Stereochemistry(Alhnger, N.L. and Eliel, E.L., eds.), Vol. 6, pp. 177-217, Wiley Interscience, New York 7 Lin, J.S., Tom, T.C. and Olcott, H.S. (1974) J. Agr. Food Chem. 22, 526-528 8 Floyd, R.A. and Lewis, C.A. (1983) Biochemistry 22, 2645-2649 9 Janzen, E.G. (1971) Acc. Chem. Res. 4, 31-40 10 Janzen, E.G., Evans, C.A. and Liu, J.I-P. (1973) J. Magn. Reson. 9, 513-516 11 Rozantsev, E.G. (1970) in Free Nitroxyl Radicals (translated from Russian by B.J. Hazzard) (Ulrich, H. ed.), Plenum Press, New York
Biochimica et Biophysica Acta, 790 (1984) 94-97
Elsevier
BBA Report BBA 30078 F O R M A T I O N O F L O N G - L I V E D H Y D R O X Y L FREE RADICAL A D D U C T S OF P R O L I N E AND H Y D R O X Y P R O L I N E IN A F E N T O N R E A C T I O N ROBERT A. FLOYD and IMRE ZS.-NAGY * Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104 (U.S.A.)
(Received March 2nd, 1984) (Revised manuscript received July 20th, 1984)
Key words: Free-radical scavenger," Fenton reaction," Nitroxyl radical; ESR; Proline; Hydroxyprofine
Proline and hydroxyproline when exposed to the hydroxyl free radical generating system of ADP-Fe(II)-H 202 yielded long-lived free radicals. An analysis of the electron paramagnetic resonance spectra of the long-lived hydroxyl free radical adducts of proline and hydroxyproline is consistent with a free electron on a nitroxyl group interacting with the nitrogen atom as well as with three separate protons. In the case of proline, nitroxide formation was observed under the influence of tert-butyl-hydroperoxide, giving a similar EPR spectrum (Lin, J.S., Tom, T.C. and Oicott, H.S. (1974) J. Alp'. Food Chem. 22, 526-528); however, the hydroxyl free radical adduct of hydroxyproline has not been described yet. In the case of the proline nitroxide radical, two of the three protons involved interact with the free electron equivalently. The coupling constants for the hydroxyl free radical adduct of proline are AN= 1.58 mT, A ~ I = A H 2 - 2 . 1 3 mT, A~t3 = 1.77 mT and for hydroxyproline are A N -~ 1.54 mT, A ~ I = 2.56 mT, A~ 2 = 2.03 and A ~ ~ 1.51. The data are consistent with the amine nitrogen of proline and hydroxyproline being oxidized to a nitroxyl group and the free electron of the nitroxyl interacting with the ~-protons of these amino acid hydroxyl free radical adducts.
It has been clear for some time that oxygen free radicals play a significant role in biological systems both as an etiological agent of certain pathological conditions as well as participate in many essential biochemical reactions [1]. Much of the work on oxidative damage up until recently has concentrated upon the lipid portion of membranes, with little attention directed toward the reaction of oxygen free radicals with proteins and amino acids, even though these are major constituents of the call. The recent demonstrations that seemingly minor oxidative changes in certain * Guest scientist from (present address): F. Verz~ International Laboratory for Experimental Gerontology (VILEG), Hungarian Section, University Medical School, H-4012 Debrecen, Hungary. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.
proteins such as glutamine synthetase and al-antiproteinase result in drastic biological consequences, for instance proteinase degradation of the former [2] and cessation of antiproteinase action in the case of the latter [3], if only one amino acid is oxidized, clearly point t o the importance of this area of research. Thus we have begun a study of the interaction of hydroxyl free radicals with proteins and amino acids [4]. In the process we have discovered that hydroxyl free radicals generated in a Fenton-type reaction, when allowed to interact with proline and hydroxyproline, as contrasted to other amino acids [4], produce long-lived free radicals. The formation of proline nitroxide has already been observed under the influence of tertbutylhydroperoxide [5-7], nevertheless, neither proline nor hydroxyproline nitroxide formation
95
have been reported in a Fenton reaction previously. Hydroxyl free radicals were generated in an ADP-Fe(II)-H202 system as describeed by Floyd and Lewis [8]. A typical experiment was carried out as follows: to 10 #1 20 mM ADP, 10 #1 FeC12 (1 mM in 0.0012 M HC1) was added with rapid mixing followed by incubation at 37 °C for 30 s. This was followed by the addition of 20 #1 250 mM proline or hydroxyproline and then 50 #1 buffer (10 mM NaC1/25 mM N a H C O 3 (pH 7.1)). Finally 10 #1 0.3% H 2 0 2 w a s added with rapid mixing and then incubated for 30 s at 37 ° C. This mixture yields a concentration of OH radicals of greater than 20 # M [8]. About 30 s after the final incubation, an EPR spectrum was begun on this mixture. The spectra were obtained on a Varian E-109 X-band spectrometer with the following instrumental parameters: field set 323.7 mT, scan range 10 or 20 m T in an 8 or 16 min scan with a time constant of 1 or 3 s, modulation frequency 100 kHz, modulation amplitude 0.2 mT, receiver gain 1.25.104 , incident microwave frequency 9.14 G H z with a incident power level of 25 mW. The samples were at room temperature (about 25 o C) during the EPR recording. Fig. 1 presents the EPR spectra obtained on proline and hydroxyproline solutions exposed to the ADP-Fe(II)-H202 hydroxyl free radical generating system. The free radicals are present as soon as the EPR spectra can be obtained and persist for several hours. The amount of these radicals pre-
Fig. 1. Electron paramagnetic resonance spectra of hydroxyl free radical adducts of proline (top) and hydroxyproline (bottom).
sent was estimated to be about 9.0 and 8.7 # M for the hydroxyproline and proline adducts, respectively. No detectable free radicals are present when hydroxyproline and proline are absent in the hydroxyl free radical generating system. The spectra have been analyzed and the stick diagrams of our interpretation are shown above the two spectra in Figs. 2 and 3. In each case it is possible to explain the EPR spectra by the interaction of the electron of a nitroxyl free radical with one nitrogen atom and three different protons. The coupling constants for the hydroxyl free radical adduct of proline are A~ = 1.58 roT, A~ 1 = Aft2 = 2.13 mT a n d A~I3 = 1.77 mT, where H1, H2 and H3 designate three different protons, but H1 and H2 are equivalent with respect to the manner the free electron interacts with them. We have designated these protons as fl strictly based upon the results and conventions utilized in spin-trapping studies [9,10] where the protons are linked to an a-carbon atom adjacent to the nitroxyl nitrogen. The coupling constants for the hydroxyl free radical adduct of hydroxyproline are Ar~ -- 1.54 mT, A~ 1 = 2.56 mT, A~ 2 = 2.03 m T a n d A~ 3 = 1.51 mT. Fig. 4 presents our interpretation of the hydroxyl free radical adducts of proline and hydroxyproline. We have assigned protons 1, 2 and 3 as designated in the figure for the following reasons. They are in the 13 position near the nitroxyl and the angle subtended between the Pz orbital of the nitroxyl bond and the proton-carbon bond governs the magnitude of the coupling constant [9,10]. Thus in the case of the OH adduct of proline there are two equivalent protons and we
Fig. 2. Electron paramagnetic resonance spectrum and stick diagram of the hydroxyl free radical adduct of proline.
96
1 mT
Fig. 3. Electron paramagnetic resonance spectrum and stick diagram of the hydroxyl free radical adduct of hydroxyprofine.
have designated these H1 and H2 and assigned them as shown. The other fl-proton, i.e., that associated with the a-carbon to which the carboxyl group is attached, has been assigned and designated as shown. Thus based on the logic of the assignments made in the case of proline we have assigned the protons designated in the free radical of hydroxyproline. The H1 and H2 protons are unequivalent in the latter case, but the magnitude of the values as compared to proline dictates our assignment. The free radicals of proline and hydroxyproline are quite stable as is usually the case for nitroxyl free radicals and also it is clear from the spectrum that there is a nitrogen atom interaction with the free electron. For all of the above reasons we propose the chemical structures shown in Fig. 4 to account for our results.
0
0
^l'-- AI' : 2.1a
A I,. 2 5 e
Ama = 1 . 7 7
Amt - 2 . 0 3
A m = 1.55
A~I= 1.51 A m = 1.54
Fig. 4. Chemical structures and coupling constants (mT) of the hydroxyl free radical adduct of proline (left) and hydroxyproline (fight).
Proline nitroxides have been studied by others [5-7,10], both in water and in organic solvents. There is a remarkable resemblance between the spectra shown by others [5,6] for proline nitroxide and that observed during the present experiments. Although under various experimental conditions the coupling constants proved to be somewhat varying, there is no doubt that our interpretation is basically correct. This statement is supported by computer simulation of the EPR spectra of both the proline and hydroxyproline free radicals (personal communication from one of the reporters of this paper). Another set of reasons that strengthens our interpretation of the results is that in the presence of H20 z without added ADP or Fe(II), there is a slow (over a period of hours) accumulation of the same free radicals as observed when the complete hydroxyl free radical generating system is present. Hydrogen peroxide is used routinely to synthesize nitroxyl free radicals from amines [11]. Regarding chemical mechanisms, it is possible to explain the formation of the nitroxyl from the amine by two sequential additions of hydroxyl free radicals to the amine nitrogen followed by the subsequent loss of water yielding the N-hydroxy compound which is then rapidly oxidized to the nitroxyl [11]. As a matter of fact, proline nitroxide was also synthesized by starting from N-hydroxyL-proline [7]. The biological significance of the observations reported here is not known. It is probable that the amine nitrogen of proline is not available for oxidation to the nitroxyl if it is in a peptide linkage, unless perhaps proline were to be the terminal amino acid in the protein chain. However, it appears necessary to study the action of OH on proline or hydroxyproline when it is in a peptide linkage. Our preliminary experiments with some randomly chosen commonly available proteins has shown that hydroxyl free radicals do not yield free radicals of nitroxyl type even with collagen being rich in proline and hydroxyproline. Nevertheless, it seems to be of importance that proline and hydroxyproline may be hydroxyl free radical scavengers under certain biological conditions and in specific microenvironments where these amino acids as well as hydrogen peroxide and available iron are present and can react with each other.
97 W e would like to t h a n k K a y Wallace, Lisa Gropf, A n i t a Hill, Pat Carnes a n d S a n d r a N a n k for help in the p r e p a r a t i o n of this m a n u s c r i p t a n d for technical assistance. T h e research was supported b y N I H G r a n t AG02599. We also would like to express thanks to one of the reporters of this paper for c o m p u t e r s i m u l a t i o n of the proline a n d h y d r o x y p r o l i n e nitroxyl free radicals.
References 1 Fridovich, I. (1978) Science 201,875-880 2 Fucci, L., Ohver, C.N., Coon, M.J. and Stadtman, E.R. (1983) Proc. Natl. Acad. Sci. USA 80, 1521-1525 3 Johnson, D. and Travis, J. (1979) J. Biol. chem. 254, 4022-4026
4 Zs.-Nagy, I. and Floyd, R.A. (1984) Biochim. Biophys.Acta 790, in the press 5 Van der Veen, J., Weil, J.T., Kennedy, T.E. and Olcott, H.S. (1970) Lipids 5, 509-512 6 Janzen, E.G. (1971) in Topics in Stereochemistry(Alhnger, N.L. and Eliel, E.L., eds.), Vol. 6, pp. 177-217, Wiley Interscience, New York 7 Lin, J.S., Tom, T.C. and Olcott, H.S. (1974) J. Agr. Food Chem. 22, 526-528 8 Floyd, R.A. and Lewis, C.A. (1983) Biochemistry 22, 2645-2649 9 Janzen, E.G. (1971) Acc. Chem. Res. 4, 31-40 10 Janzen, E.G., Evans, C.A. and Liu, J.I-P. (1973) J. Magn. Reson. 9, 513-516 11 Rozantsev, E.G. (1970) in Free Nitroxyl Radicals (translated from Russian by B.J. Hazzard) (Ulrich, H. ed.), Plenum Press, New York