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Solvent effects of dimethyl sulfoxide on the chemiluminescent reaction of luminol-H2O2 system catalyzed by horseradish peroxidase
Journal ofMolecular
Catalysis,
31 (1985)
39 - 48
39
SOLVENT EFFECTS OF DIMETHYL SULFOXIDE ON THE CHEMILUMINESCENT REACTION OF LUMINOL-H202 SYSTEM CATALYZED BY HORSERADISH PEROXIDASE YOSHIHITO IKARIYAMA, Institute
of Materials Science,
MASUO AIZAWA University
of Tsukuba,
Sakura-mura,
Zbaraki 305 (Japan)
and SHUICHI SUZUKI Research Laboratory of Resources Utilization, Midori-ku, Yokohama 227 (Japan)
Tokyo
Institute
of Technology,
Nagatsuta,
(Received June 15,1984)
Summary Dimethyl sulfoxide (DMSO) shows a remarkable promoting effect on the luminescent reaction of the luminol-H,O, system catalyzed by horseradish peroxidase. Two luminescent processes occur when a mixed solvent of water and DMSO is used. The total luminescence increases exponentially with increasing DMSO concentration. The initial-phase luminescence decreases when the DMSO concentration is increased, while the delayed luminescence increases. Participation of molecular oxygen and its reactive species (02’, O,*-) dissolved in the reaction medium was studied enzymatically. In the medium where oxygen had been removed, the rate of luminescent reaction decreased markedly. In the presence of superoxide dismutase the luminescence decreased by 15%. Addition of catalase to the medium during the course of the reaction resulted in complete disappearance of the luminescence. These facts suggest a new reaction scheme for the enzyme-induced luminescent reaction involving oxygen as well as its reactive species.
Introduction The reaction of the luminol-H202 system catalyzed by metal ions, metal complexes and metal enzymes emits light when luminol (1) is converted to aminophthalate (2) [ 1, 21. The stoichiometry can be summarized as follows :
0304-5102/85/$3.30
@ Elsevier Sequoia/Printed
in The Netherlands
40
::
P + 2H202
+
OH- +
>
WI
(2) +
+2H20 + N2 hv
Scheme 1.
Among many catalysts of the luminol reaction, horseradish peroxidase [HRP] is an interesting one since it proceeds according to a chain reaction. The mechanism of this reaction has been extensively studied to elaborate the reaction scheme involving several activated forms of luminol and oxygen [3]. In the reaction schemes proposed, five oxidation states of the enzyme are required to initiate the chain reaction between luminol (LH,) and hydrogen peroxide (H202). Among the five states, ferric enzyme (+3), Compound I (+5) and Compound II (+4) play important roles in the cycle of peroxidase catalysis as follows : Peroxidase(+3)
+ H202 +
Compound
1(+4) + LH2 --+
Compound
11(+5) + LH2 -
Compound Compound
1(+4) 11(+ 5) + LH’
Peroxidase(i3)
+ LH’
Scheme 2.
where the number in parentheses denotes the relative oxidizing equivalent retained in the enzyme on the basis of a valence of +3 for the natural (ferric) enzyme. The mechanism of complicated peroxidase-catalyzed reactions, as well as the versatile functions of peroxidase catalyst, seem to be explained [ 4 - 81, although its noncatalytic aspect is still open to much discussion. The kinetics of the peroxidase reaction are quite different, however, provided the enzyme-derived reactive in~rmediate(s) is (are) generated and stabilized in an aqueous-organic mixed solvent, In a similar way, the kinetics will vary markedly in the presence of enzymes which exploit the reactive%pecies. In our present study on the catalytic actions of peroxidase in the luminol-Hiz02 system, a new mechanism, where enzyme-energized species play impo~ant roles to initiate and promote the noncatalytic luminescent reaction, is proposed.
41
Experimental
Horseradish peroxidase (HRP, E.C.1,11.1.‘7) was purchased from Sigma (Type VI). HRP was prepared in 0.1 M phosphate buffer (pH 7.0), and its concentration was determined spectrophotometricaIly with an eM3 value of 90 mM_’ cm -‘. Luminol(5amino-2,3dihydro-1,4-phthalazinedione) and hemin were the products of Tokyo Kasei (Tokyo}, and were prepared in either M/l00 NaOH or DMSU solution, Hydrogen peroxide (30%) was a product of Mi~ubishi Gas Chemical Co. (Tokyo), and was diluted with 0.1 M phosphate buffer (pH 7.0). Organic solvents were laboratory grade reagents. Glucose oxidase (2400 units mg-i, E.C.1.1.3.4), superoxide dismutase (3500 units mg -I, E.C.1.15.1.1) and catalase (48 545 units mg-‘, 19 mg protein ml -I, E.C.1.11.1.6) were the products of Tokyo Kasei, Sigma (St.Louis, MO), and Miles (Elkhart, IN), respectively. Chemiluminescence measurements were performed with an ATP photometer from the American.Instrument Company (MD), and the signal (luminescence intensity) was immediately noted on a recorder (Riken Denshi, Tokyo) after the start of the reaction. Total luminescence was calculated by integrating the signal. Luminescent spectra were investigated with an opticdl multichannef analyzer (E. G. & G. Princeton Applied Research, Princeton, NJ). Absorbance measurements of the peroxidase-catalyzed luminol reaction were carried out with a Shimadzu double-beam absorption spectrometer. Absorbance at 410 nm was monitored for the product formation; fuminol has little absorbance at 410 nm, Oxygen and its reactive species were annihilated as follows. Dissolved oxygen was removed, if necessary, either by nitrogen gas bubbling or by nitrogen gas bubbling in the presence of an oxygen-consuming glucoseglucose oxidase system containing 1.64 mM glucose and 77 units ml-l gtucose oxidase. After pretreatment in a pyrogaaliof solution (1 M pyrogaIIo1 in 1 M NaOH), nitrogen gas was separately conducted both to a substrate solution (in which H,Oa and luminol were contained in a water-DMSO mixed solvent buffered with 0.1 M phosphate solution (pH 7.0)) and to an enzyme solution. Both solutions were then placed in a glass vial, which was then sealed with plastic film to prevent air contamination. In cases where the glucose-glucose oxidase system was used as an oxygen-scaveng~g system, an enzyme solution containing HRP and glucose oxidase and a substrate solution containing luminol, H202 and glucose were separately deoxygenated before initiation of the reaction. RemovaI of oxygen radicals (Oay) and peroxide (Oz2-) was performed with superoxide dismutase and cat&se as s~venging enzymes, respectivefy, Superoxide dismutase was added beforehand to the reaction medium containing the substrate and the enzyme fHRPf, whereas catalase was added
42
to the reaction medium when the luminescence intensity reached its maximum, because the presence of H202 is an absolute prerequisite as substrate to initiate the luminescent reaction.
Results
Effects of organic solvent concentration on the chemilumineseent of the luminol-H@, system
reaction
The reaction of the lumi~ol-H*O*-peroxid~e system was studied as a function of organic solvent concentration in the presence of 12 I.IM HzOz, 340 PM luminol and 0.5 nM (0.006 units ml-i) HRP, performed in 0.1 M phosphate buffer (pH 7.0) at 20 “C. The organic solvents investigated were acetone, ethanol, hexamethylphosphoric acid (HEMPA), DMSO and dimethyl formamide (DMF). The total luminescence in arbitrary units was plotted against organic solvent concen~ation (Fig. 1). The total luminescence, i.e. the integrated overall luminescence, of the system decreased rapidly with increasing concentration of HEMPA, while it decreased gradually with increasing concentration of acetone. In the case of HEMPA, the total luminescence decreased significantly, which suggested the inhibitory effect of HEMPA. Little luminescence was observed in the presence of only 10% HEMPA. Similar results were observed in mixed solvents of water and THF. Acetone showed no stimulatory effect on the luminescent reaction. In the cases of ethanol and DMF, however, the integrated luminescence gradually increased at low organic solvent concentrations. The total luminescence was significantly increased by increasing the concentration of DMSO (Fig. I).
Solvent
content
(%,vlv)
Fig. 1. Luminol luminescence catalyzed by peroxidase in an aqueous-organic mixed solvent. Integrated overall luminescence (in arbitrary units) was plotted against organic solvent content. H202: 12 @I, luminol: 340 PM, peroxidase: 0.006 units ml-i. Organic solvent: DMSO (--Q-), DMF (- - - -* - - -), EtOH (- * + +-), acetone (- * -O- a-), HEMPA (+) and THF (*). Every mixed solvent was buffered at pH 7.0 with 0.1 M phosphate.
43
b, I\: ji :
a-._..,
i;’:
i
‘...,
j’:
‘.., “... \..,
‘\ ‘i, 1 i
i
i
:i. I
‘I...,,
'3 :
x.
I,
:
:
..c
10 Time
‘...., “...,
"...
30
20
(
mln
)
40
0
(b)
10
20
Dimethylsulfoxlde
30
40
content L%)
Fig. 2a. Luminol luminescence catalyzed by peroxidase in an aqueous-DMSO mixed solvent. HzOz: 15 PM, luminol: 180 /..&I, peroxidase: 0.32 units ml-‘, DMSO content: 0% (), 10% (-. -L 20% (- - - -), 30% (..o..*. ). The mixed solvent was buffered at pH 7.0 with 0.1 M phosphate. Fig. 2b. Correlation between total luminescence and DMSO content. Data from Fig. 2a are replotted in terms of integrated overall luminescence with respect to DMSO content.
Effects of dimethyl sulfoxide on the chemiluminescent reaction of the luminol-H,O,-peroxidase system The promoting effects of DMSO were studied for further details in the presence of 15 PM hydrogen peroxide, 180 ,uM luminol and 27 nM HRP. When DMSO content was increased up to 30 vol.%, the total luminescence increased markedly, as shown in Fig. 2a. On the other hand, the slope of the luminescence increase gradually became smaller with increasing DMSO concentration. The slope of the luminescence increase reached a maximum when 10 vol.% DMSO was placed in the reaction medium, and decreased when a concentration of DMSO greater than 10% was added. This may reflect a deactivation of peroxidase at the higher DMSO concentrations. Figure 2b shows a linear semilogarithmic relationship between total luminescence and DMSO concentration. A similar logarithmic increase in total luminescence was observed when 150 PM HzOz, 1.8 PM luminol and 0.32 units ml-’ peroxidase were incubated in the same mixed solvent. In this substrate ratio, i.e. [luminol]/[H,O,] = 0.012, the luminescent reaction lasted over several hours, whereas in the former case, where [luminol]/[HzOz] was approximately 6.0, the photon-generating reaction terminated within 1 h in the mixed solvent (30 vol.% DMSO). Hemin is also a catalyst for the luminol-HzOz system, and this reaction was likewise affected by DMSO. The total luminescence increased exponentially with increasing concentration of DMSO in a similar way. However, the initial luminescence was accelerated with increasing DMSO concentration, probably due to a nonproteinic organometallic complex whose catalytic action is not inhibited at high DMSO concentrations. Luminescence spectrum The luminescence spectrum of the photon-generating reaction catalyzed by peroxidase, both in the presence and absence of DMSO, was studied with
44 an
optical multichannel analyzer. Luminol and hydrogen peroxide concentrations were increased to strengthen luminescence, since the diode array shows lower sensitivity than the photon counter, Figure 3 shows the luminescence spectra of the peroxidase-luminol-HzOz system in the presence or absence of DMSO. This result is consistent with the luminescence spectrum of the luminol-H202 system in a water-DMSO mixed solvent [ 11. The luminescence spectrum in aqueous solution has peak luminescence at 430 nm; however, in the presence of DMSO the spectrum shows two peaks, at 430 nm and at 480 nm.
p, ; :,-A I
:
I
t’
:
:
:
‘\
i
?
\
\
:
‘\
,’ #’
,,I’
400
WAVELENGTH
500
\
‘.
‘...
600
(Nfi)
Fig. 3. Luminescence spectra of the luminol-HOOD system in aqueous solution (-) and a mixed solvent containing 1:3 H20:DMS0 (- - - -). Every component was increased, to 50 mM (HzOz), 5 mM (luminol) and 40 units ml‘-” (peroxidase), to produce luminescence spectra measurable with the optical multichannel analyzer. The number of scans was 5000.
Effects of LIMSO on the initiuL rate of the ~u~~neseent reaction The initial rate of the luminol reaction catalyzed by HRP was spectrophotometrically studied in medium containing 2.90 mM HzOz, 240 PM luminol and 0.28 units ml-’ peroxidase. The effect of DMSO on the initial increase in absorbance at 410 nm was studied by increasing DMSO concentration from 0 to 30 vol.%. Figure 4 presents the relation of absorbance change at 410 nm to the solvent concentration. The rate of aminophthalate formation decreased markedly when DMSO concentration increased. A similar result was obtained when the slope of luminescence increase was plotted against the DMSO concentration, as illustrated in Fig. 5. The initial change in luminescence intensity corresponds to the rate of aminophth~a~ formation, as shown in Fig. 4. Active role of dissolved oxygen and its reactive species in the luminol reaction The role of dissolved oxygen in the luminol-H,Oz system was studied with a medium which contained 1.46 mM HzO, and 180 ,uM luminol. The reactions were carried out at 20 “C with 0.026 units ml-“ peroxidase in 0.1 M phosphate buffer (pH 7.0) in the presence of 10 vol.% DMSO. The
45
\
0
\ %,
Ob 0
5 10 15 DMSO CONTENT(Z)
M
0
10
20
JJ
4J
DNSO CONTENT (X,v/v)
Fig. 4. Effect of DMSO content on the initial rate of reaction in the luminol-HZOZperoxidase system. Absorbance change at 410 nm is plotted us. DMSO content. The reaction medium was buffered at pH 7.0 with 0.1 M phosphate. HzOz: 2.90 mM, luminol: 240 /_&I,peroxidase: 0.28 units ml-‘. Fig. 5. Effect of DMSO concentration on the initial slope of the luminescent reaction. Initial change in luminescence intensity was plotted against DMSO content. Hz02: 6 PM, luminol: 370 /.&I, peroxidase: 0.012 units ml-‘.
concentrations of substrates and enzyme were adjusted in order to perform the experiments under the most suitable conditions, i.e. so that the slope of the luminescence increase could be clearly observed. Molecular oxygen dissolved in the medium was removed by nitrogen gas bubbling. Further removal of dissolved oxygen was performed by nitrogen gas bubbling in the presence of an oxygen-consuming glucose-glucose oxidase system. In the presence of dissolved oxygen, the luminescence intensity increased gradually after the onset of the reaction, as shown in Fig. 6, whereas in the absence of oxygen, i.e. in the medium in which dissolved oxygen was purged with nitrogen gas, the intensity was smaller. This finding led us inevitably to perform experiments where thorough removal of the oxygen formed from the decomposition of HzOz during the redox reaction between luminol and H,02 was required. Addition of the oxygen-consuming glucoseglucose oxidase system to the nitrogen gas-treated medium resulted in an intensity much smaller than that of the former two systems. The change in intensity is also shown in Fig. 6. These findings suggest that oxygen plays an essential part in the luminescent reaction of the luminol-H202 system. Disappearance of the luminescence was also observed in the hemincatalyzed luminescent reaction. In this reaction, however, the removal of oxygen resulted in an extremely small luminescence. The role of oxygen dissolved in the luminol-H,O, system was confirmed by the demonstration that oxygen is a key substance which accelerates the luminescent reaction. Oxygen may be reduced to either the superoxide radical or hydrogen peroxide in the course of the luminescent reaction. The existence of the superoxide radical potentially generated during the luminol reaction can be
(A)
/._-------
.NH .’
,’
_--
(B).- /-*---
/.I ,,..=0
2
4 Time
6
8
IO
( min1
Fig. 6. Effect of dissolved oxygen on the peroxidase-catalyzed luminescent reaction. HzOz: 1.46 mM, luminol: 180 PM, peroxidase: 0.026 units ml-‘, DMSO content: 10%. (A) No treatment to remove dissolved oxygen was performed. (B) The reaction medium was bubbled with nitrogen gas before reaction. (C) The reaction medium was bubbled with nitrogen gas before reaction in the presence of oxygen-consuming glucose (1.64 mM)-glucose oxidase (7 7 units ml-’ ) system.
enzymatically prevented by annihilating the radical with superoxide dismutase. The luminescent reaction was then studied in a medium where 15 PM H202, 180 PM luminol and 0.32 units ml-’ of peroxidase were mixed with various concentrations of superoxide dismutase. Table 1 shows that the total luminescence decreased upon increasing the concentration of superoxide dismutase from 0 to 360 units ml-‘. Enzymatic annihilation of the superoxide radical did not result in complete disappearance of luminescence. However, superoxide is easily dismutated to O2 and H,Oz in the presence of the dismutase, both of which in turn are involved in the HRP-catalyzed luminescent reaction. This is why complete disappearance of luminescence was not observed upon addition of superoxide dismu tase .
TABLE 1 Effect of superoxide dismutase on the luminescent reaction. The reaction was performed in the presence of HzOz (15 /.&I), luminol (180 PM), peroxidase (0.32 units ml-‘) and superoxide dismutase Superoxide dismutase (u ml-’ ) Luminescence (%)
0 100
90 87
180 81
270 78
360 77
These results indicate that the luminescent reaction proceeds via two pathways, i.e., catalyst-initiated reaction and delayed reaction. The enzyme-catalyzed luminescent reaction is not initiated until HzOz is added to the reaction medium. However, whether HzOz is involved in delayed
47
luminescence has not been fully clarified; this may be studied by the use of an annihilating enzyme of H202, catalase. Therefore, after the luminescence intensity reached its maximum, catalase (4.7 units) was added to the reaction medium (0.5 ml) containing 12 FM H,O,, 350 PM luminol and 6.0 nM (0.073 units ml-‘) HRP. The catalase addition caused ~s~n~eous disappearance of the luminescence from the luminol-H,O,-peroxidase system, which clearly proves the indispensable role of H20, in the delayed luminescence. The degree of luminescence disappearance could be correlated to a change in the amount of catalase.
Discussion Several schemes have been proposed concerning luminol luminescence [9 - 151; however, they lack clear evidence due to unsuccessful efforts to identify the reactive intermediates. In our previous report [16], the fact that nonenzymatic luminescence was created in the HRP-initiated luminescent reaction of the luminolHzO, system was clearly shown by applying immobilized HRP to the luminescent reaction. Since in this work the oxygen molecule and its radical as well as HzOz were shown to be essential reactive species, causing a noncatalytic luminescent reaction, thus the scheme illustrated in Fig. 7 was postulated, wherein molecular oxygen is required to oxygenate the luminol radical anion. The enzyme directly generates energized species such as the luminol radical (LH’ ), which has the potential to emit light and to generate 02; from oxygen. The 0,: radical can oxidize a luminol radical nonenzymatic~ly, with the resultant formation of 0 22- (H202). The luminol radical is oxygenated, which might be followed by the generation of an oxygenated luminol anion as illustrated in Fig. 7. The luminol radical anion is not oxygenated provided that oxygen is removed from the reaction medium. Therefore, the complete removal of oxygen molecules by the oxygen-consum~g glucoseglucose oxidase system led to a considerable disappearance of luminescence. DMSO displayed disparate effects on the rate of the luminescence reaction, mainly due to the effect of solvent on stabilization of such intermediates as the luminol radical anion and the oxygen radical anion generated by the catalysts described. Both radicals are species essential to the luminescence reaction, and their accessability to substrates is assumed to be increased by increasing the concentration of DMSO. Although we have not yet fully succeeded in identifying the reactive intermediates of the luminol reaction, our new findings that oxygen, oxygen radicals and hydrogen peroxide play quite important roles in the luminol luminescence, especially in a water-DMSO mixed solvent system, very strongly indicate the postulated nonca~ytic process of the luminescence reaction.
iH2
6
NH2 0
NonenzymaticProcess
Chemiluminescent Process Fig. 7. Postulated scheme for a nonenzymatic luminescent pathway where the essential involvements of oxygen molecule, superoxide radical and hydrogen peroxide are shown with respect to luminol and its radical species. The luminol radical is also produced in enzyme-catalyzed process.
References 1 2 3 4 5 6 7 8 9 10 11 12’ 13 14 15 16
E. H. White and M. M. Bursey, J. Am. Chem. Sot., 86 (1964) 941. E. H. White,Acc. Chem. Res., 3 (1970) 54. M. J. Cormier and P. Prichard, J. Biol. Chem., 243 (1968) 4706. K. Yokota and L Yamazaki, Biochim. Biophys. Ado, 105 (1965) 301. P. Douzou and F. Leterrier, Biochim. Biophys. Acta, 220 (1970) 338. I. Yamazaki and K. Yokota, Mol. Cell. Biochem., 2 (1973) 39. B. W. Griffin and P. L. Ting, Biochem., 17 (1978) 2206. Y. Hayashi and I. Yamazaki, J. Biol. Chem., 254 (1979) 9101. M. M. Rauhut, A. M. Semsel and B. G. Roberts,J. Org. Chem., 31 (1966) 2431. R. B. Shevlin and H. A. Neufeld, J. Org. Chem., 35 (1970) 2178. E. H. White, E. G. Nash, D. R. Roberts and 0. C. Zafiriou, J. Am. Chem. Sot., (1968) 5932. Y. Omote, T. Miyake and N. Sugiyama, Bull. Chem. Sot. Jpn., 40 (1967) 2466. J. R. Totter and G. E. Philbrook, Photochem. Photobiol., 5 (1966) 177. F. McCapra and P. D. Leeson, J. Chem. Sot., Chem. Commun., (1979) 114. N. Suzuki, S. Wakatsuki and Y. Izawa, Tetrahedron Left., (1980) 2313. Y. Ikariyama, S. Suzuki and M. Aizawa, Appl. Biochem. Biotech., 6 (1981) 223.
90
Catalysis,
31 (1985)
39 - 48
39
SOLVENT EFFECTS OF DIMETHYL SULFOXIDE ON THE CHEMILUMINESCENT REACTION OF LUMINOL-H202 SYSTEM CATALYZED BY HORSERADISH PEROXIDASE YOSHIHITO IKARIYAMA, Institute
of Materials Science,
MASUO AIZAWA University
of Tsukuba,
Sakura-mura,
Zbaraki 305 (Japan)
and SHUICHI SUZUKI Research Laboratory of Resources Utilization, Midori-ku, Yokohama 227 (Japan)
Tokyo
Institute
of Technology,
Nagatsuta,
(Received June 15,1984)
Summary Dimethyl sulfoxide (DMSO) shows a remarkable promoting effect on the luminescent reaction of the luminol-H,O, system catalyzed by horseradish peroxidase. Two luminescent processes occur when a mixed solvent of water and DMSO is used. The total luminescence increases exponentially with increasing DMSO concentration. The initial-phase luminescence decreases when the DMSO concentration is increased, while the delayed luminescence increases. Participation of molecular oxygen and its reactive species (02’, O,*-) dissolved in the reaction medium was studied enzymatically. In the medium where oxygen had been removed, the rate of luminescent reaction decreased markedly. In the presence of superoxide dismutase the luminescence decreased by 15%. Addition of catalase to the medium during the course of the reaction resulted in complete disappearance of the luminescence. These facts suggest a new reaction scheme for the enzyme-induced luminescent reaction involving oxygen as well as its reactive species.
Introduction The reaction of the luminol-H202 system catalyzed by metal ions, metal complexes and metal enzymes emits light when luminol (1) is converted to aminophthalate (2) [ 1, 21. The stoichiometry can be summarized as follows :
0304-5102/85/$3.30
@ Elsevier Sequoia/Printed
in The Netherlands
40
::
P + 2H202
+
OH- +
>
WI
(2) +
+2H20 + N2 hv
Scheme 1.
Among many catalysts of the luminol reaction, horseradish peroxidase [HRP] is an interesting one since it proceeds according to a chain reaction. The mechanism of this reaction has been extensively studied to elaborate the reaction scheme involving several activated forms of luminol and oxygen [3]. In the reaction schemes proposed, five oxidation states of the enzyme are required to initiate the chain reaction between luminol (LH,) and hydrogen peroxide (H202). Among the five states, ferric enzyme (+3), Compound I (+5) and Compound II (+4) play important roles in the cycle of peroxidase catalysis as follows : Peroxidase(+3)
+ H202 +
Compound
1(+4) + LH2 --+
Compound
11(+5) + LH2 -
Compound Compound
1(+4) 11(+ 5) + LH’
Peroxidase(i3)
+ LH’
Scheme 2.
where the number in parentheses denotes the relative oxidizing equivalent retained in the enzyme on the basis of a valence of +3 for the natural (ferric) enzyme. The mechanism of complicated peroxidase-catalyzed reactions, as well as the versatile functions of peroxidase catalyst, seem to be explained [ 4 - 81, although its noncatalytic aspect is still open to much discussion. The kinetics of the peroxidase reaction are quite different, however, provided the enzyme-derived reactive in~rmediate(s) is (are) generated and stabilized in an aqueous-organic mixed solvent, In a similar way, the kinetics will vary markedly in the presence of enzymes which exploit the reactive%pecies. In our present study on the catalytic actions of peroxidase in the luminol-Hiz02 system, a new mechanism, where enzyme-energized species play impo~ant roles to initiate and promote the noncatalytic luminescent reaction, is proposed.
41
Experimental
Horseradish peroxidase (HRP, E.C.1,11.1.‘7) was purchased from Sigma (Type VI). HRP was prepared in 0.1 M phosphate buffer (pH 7.0), and its concentration was determined spectrophotometricaIly with an eM3 value of 90 mM_’ cm -‘. Luminol(5amino-2,3dihydro-1,4-phthalazinedione) and hemin were the products of Tokyo Kasei (Tokyo}, and were prepared in either M/l00 NaOH or DMSU solution, Hydrogen peroxide (30%) was a product of Mi~ubishi Gas Chemical Co. (Tokyo), and was diluted with 0.1 M phosphate buffer (pH 7.0). Organic solvents were laboratory grade reagents. Glucose oxidase (2400 units mg-i, E.C.1.1.3.4), superoxide dismutase (3500 units mg -I, E.C.1.15.1.1) and catalase (48 545 units mg-‘, 19 mg protein ml -I, E.C.1.11.1.6) were the products of Tokyo Kasei, Sigma (St.Louis, MO), and Miles (Elkhart, IN), respectively. Chemiluminescence measurements were performed with an ATP photometer from the American.Instrument Company (MD), and the signal (luminescence intensity) was immediately noted on a recorder (Riken Denshi, Tokyo) after the start of the reaction. Total luminescence was calculated by integrating the signal. Luminescent spectra were investigated with an opticdl multichannef analyzer (E. G. & G. Princeton Applied Research, Princeton, NJ). Absorbance measurements of the peroxidase-catalyzed luminol reaction were carried out with a Shimadzu double-beam absorption spectrometer. Absorbance at 410 nm was monitored for the product formation; fuminol has little absorbance at 410 nm, Oxygen and its reactive species were annihilated as follows. Dissolved oxygen was removed, if necessary, either by nitrogen gas bubbling or by nitrogen gas bubbling in the presence of an oxygen-consuming glucoseglucose oxidase system containing 1.64 mM glucose and 77 units ml-l gtucose oxidase. After pretreatment in a pyrogaaliof solution (1 M pyrogaIIo1 in 1 M NaOH), nitrogen gas was separately conducted both to a substrate solution (in which H,Oa and luminol were contained in a water-DMSO mixed solvent buffered with 0.1 M phosphate solution (pH 7.0)) and to an enzyme solution. Both solutions were then placed in a glass vial, which was then sealed with plastic film to prevent air contamination. In cases where the glucose-glucose oxidase system was used as an oxygen-scaveng~g system, an enzyme solution containing HRP and glucose oxidase and a substrate solution containing luminol, H202 and glucose were separately deoxygenated before initiation of the reaction. RemovaI of oxygen radicals (Oay) and peroxide (Oz2-) was performed with superoxide dismutase and cat&se as s~venging enzymes, respectivefy, Superoxide dismutase was added beforehand to the reaction medium containing the substrate and the enzyme fHRPf, whereas catalase was added
42
to the reaction medium when the luminescence intensity reached its maximum, because the presence of H202 is an absolute prerequisite as substrate to initiate the luminescent reaction.
Results
Effects of organic solvent concentration on the chemilumineseent of the luminol-H@, system
reaction
The reaction of the lumi~ol-H*O*-peroxid~e system was studied as a function of organic solvent concentration in the presence of 12 I.IM HzOz, 340 PM luminol and 0.5 nM (0.006 units ml-i) HRP, performed in 0.1 M phosphate buffer (pH 7.0) at 20 “C. The organic solvents investigated were acetone, ethanol, hexamethylphosphoric acid (HEMPA), DMSO and dimethyl formamide (DMF). The total luminescence in arbitrary units was plotted against organic solvent concen~ation (Fig. 1). The total luminescence, i.e. the integrated overall luminescence, of the system decreased rapidly with increasing concentration of HEMPA, while it decreased gradually with increasing concentration of acetone. In the case of HEMPA, the total luminescence decreased significantly, which suggested the inhibitory effect of HEMPA. Little luminescence was observed in the presence of only 10% HEMPA. Similar results were observed in mixed solvents of water and THF. Acetone showed no stimulatory effect on the luminescent reaction. In the cases of ethanol and DMF, however, the integrated luminescence gradually increased at low organic solvent concentrations. The total luminescence was significantly increased by increasing the concentration of DMSO (Fig. I).
Solvent
content
(%,vlv)
Fig. 1. Luminol luminescence catalyzed by peroxidase in an aqueous-organic mixed solvent. Integrated overall luminescence (in arbitrary units) was plotted against organic solvent content. H202: 12 @I, luminol: 340 PM, peroxidase: 0.006 units ml-i. Organic solvent: DMSO (--Q-), DMF (- - - -* - - -), EtOH (- * + +-), acetone (- * -O- a-), HEMPA (+) and THF (*). Every mixed solvent was buffered at pH 7.0 with 0.1 M phosphate.
43
b, I\: ji :
a-._..,
i;’:
i
‘...,
j’:
‘.., “... \..,
‘\ ‘i, 1 i
i
i
:i. I
‘I...,,
'3 :
x.
I,
:
:
..c
10 Time
‘...., “...,
"...
30
20
(
mln
)
40
0
(b)
10
20
Dimethylsulfoxlde
30
40
content L%)
Fig. 2a. Luminol luminescence catalyzed by peroxidase in an aqueous-DMSO mixed solvent. HzOz: 15 PM, luminol: 180 /..&I, peroxidase: 0.32 units ml-‘, DMSO content: 0% (), 10% (-. -L 20% (- - - -), 30% (..o..*. ). The mixed solvent was buffered at pH 7.0 with 0.1 M phosphate. Fig. 2b. Correlation between total luminescence and DMSO content. Data from Fig. 2a are replotted in terms of integrated overall luminescence with respect to DMSO content.
Effects of dimethyl sulfoxide on the chemiluminescent reaction of the luminol-H,O,-peroxidase system The promoting effects of DMSO were studied for further details in the presence of 15 PM hydrogen peroxide, 180 ,uM luminol and 27 nM HRP. When DMSO content was increased up to 30 vol.%, the total luminescence increased markedly, as shown in Fig. 2a. On the other hand, the slope of the luminescence increase gradually became smaller with increasing DMSO concentration. The slope of the luminescence increase reached a maximum when 10 vol.% DMSO was placed in the reaction medium, and decreased when a concentration of DMSO greater than 10% was added. This may reflect a deactivation of peroxidase at the higher DMSO concentrations. Figure 2b shows a linear semilogarithmic relationship between total luminescence and DMSO concentration. A similar logarithmic increase in total luminescence was observed when 150 PM HzOz, 1.8 PM luminol and 0.32 units ml-’ peroxidase were incubated in the same mixed solvent. In this substrate ratio, i.e. [luminol]/[H,O,] = 0.012, the luminescent reaction lasted over several hours, whereas in the former case, where [luminol]/[HzOz] was approximately 6.0, the photon-generating reaction terminated within 1 h in the mixed solvent (30 vol.% DMSO). Hemin is also a catalyst for the luminol-HzOz system, and this reaction was likewise affected by DMSO. The total luminescence increased exponentially with increasing concentration of DMSO in a similar way. However, the initial luminescence was accelerated with increasing DMSO concentration, probably due to a nonproteinic organometallic complex whose catalytic action is not inhibited at high DMSO concentrations. Luminescence spectrum The luminescence spectrum of the photon-generating reaction catalyzed by peroxidase, both in the presence and absence of DMSO, was studied with
44 an
optical multichannel analyzer. Luminol and hydrogen peroxide concentrations were increased to strengthen luminescence, since the diode array shows lower sensitivity than the photon counter, Figure 3 shows the luminescence spectra of the peroxidase-luminol-HzOz system in the presence or absence of DMSO. This result is consistent with the luminescence spectrum of the luminol-H202 system in a water-DMSO mixed solvent [ 11. The luminescence spectrum in aqueous solution has peak luminescence at 430 nm; however, in the presence of DMSO the spectrum shows two peaks, at 430 nm and at 480 nm.
p, ; :,-A I
:
I
t’
:
:
:
‘\
i
?
\
\
:
‘\
,’ #’
,,I’
400
WAVELENGTH
500
\
‘.
‘...
600
(Nfi)
Fig. 3. Luminescence spectra of the luminol-HOOD system in aqueous solution (-) and a mixed solvent containing 1:3 H20:DMS0 (- - - -). Every component was increased, to 50 mM (HzOz), 5 mM (luminol) and 40 units ml‘-” (peroxidase), to produce luminescence spectra measurable with the optical multichannel analyzer. The number of scans was 5000.
Effects of LIMSO on the initiuL rate of the ~u~~neseent reaction The initial rate of the luminol reaction catalyzed by HRP was spectrophotometrically studied in medium containing 2.90 mM HzOz, 240 PM luminol and 0.28 units ml-’ peroxidase. The effect of DMSO on the initial increase in absorbance at 410 nm was studied by increasing DMSO concentration from 0 to 30 vol.%. Figure 4 presents the relation of absorbance change at 410 nm to the solvent concentration. The rate of aminophthalate formation decreased markedly when DMSO concentration increased. A similar result was obtained when the slope of luminescence increase was plotted against the DMSO concentration, as illustrated in Fig. 5. The initial change in luminescence intensity corresponds to the rate of aminophth~a~ formation, as shown in Fig. 4. Active role of dissolved oxygen and its reactive species in the luminol reaction The role of dissolved oxygen in the luminol-H,Oz system was studied with a medium which contained 1.46 mM HzO, and 180 ,uM luminol. The reactions were carried out at 20 “C with 0.026 units ml-“ peroxidase in 0.1 M phosphate buffer (pH 7.0) in the presence of 10 vol.% DMSO. The
45
\
0
\ %,
Ob 0
5 10 15 DMSO CONTENT(Z)
M
0
10
20
JJ
4J
DNSO CONTENT (X,v/v)
Fig. 4. Effect of DMSO content on the initial rate of reaction in the luminol-HZOZperoxidase system. Absorbance change at 410 nm is plotted us. DMSO content. The reaction medium was buffered at pH 7.0 with 0.1 M phosphate. HzOz: 2.90 mM, luminol: 240 /_&I,peroxidase: 0.28 units ml-‘. Fig. 5. Effect of DMSO concentration on the initial slope of the luminescent reaction. Initial change in luminescence intensity was plotted against DMSO content. Hz02: 6 PM, luminol: 370 /.&I, peroxidase: 0.012 units ml-‘.
concentrations of substrates and enzyme were adjusted in order to perform the experiments under the most suitable conditions, i.e. so that the slope of the luminescence increase could be clearly observed. Molecular oxygen dissolved in the medium was removed by nitrogen gas bubbling. Further removal of dissolved oxygen was performed by nitrogen gas bubbling in the presence of an oxygen-consuming glucose-glucose oxidase system. In the presence of dissolved oxygen, the luminescence intensity increased gradually after the onset of the reaction, as shown in Fig. 6, whereas in the absence of oxygen, i.e. in the medium in which dissolved oxygen was purged with nitrogen gas, the intensity was smaller. This finding led us inevitably to perform experiments where thorough removal of the oxygen formed from the decomposition of HzOz during the redox reaction between luminol and H,02 was required. Addition of the oxygen-consuming glucoseglucose oxidase system to the nitrogen gas-treated medium resulted in an intensity much smaller than that of the former two systems. The change in intensity is also shown in Fig. 6. These findings suggest that oxygen plays an essential part in the luminescent reaction of the luminol-H202 system. Disappearance of the luminescence was also observed in the hemincatalyzed luminescent reaction. In this reaction, however, the removal of oxygen resulted in an extremely small luminescence. The role of oxygen dissolved in the luminol-H,O, system was confirmed by the demonstration that oxygen is a key substance which accelerates the luminescent reaction. Oxygen may be reduced to either the superoxide radical or hydrogen peroxide in the course of the luminescent reaction. The existence of the superoxide radical potentially generated during the luminol reaction can be
(A)
/._-------
.NH .’
,’
_--
(B).- /-*---
/.I ,,..=0
2
4 Time
6
8
IO
( min1
Fig. 6. Effect of dissolved oxygen on the peroxidase-catalyzed luminescent reaction. HzOz: 1.46 mM, luminol: 180 PM, peroxidase: 0.026 units ml-‘, DMSO content: 10%. (A) No treatment to remove dissolved oxygen was performed. (B) The reaction medium was bubbled with nitrogen gas before reaction. (C) The reaction medium was bubbled with nitrogen gas before reaction in the presence of oxygen-consuming glucose (1.64 mM)-glucose oxidase (7 7 units ml-’ ) system.
enzymatically prevented by annihilating the radical with superoxide dismutase. The luminescent reaction was then studied in a medium where 15 PM H202, 180 PM luminol and 0.32 units ml-’ of peroxidase were mixed with various concentrations of superoxide dismutase. Table 1 shows that the total luminescence decreased upon increasing the concentration of superoxide dismutase from 0 to 360 units ml-‘. Enzymatic annihilation of the superoxide radical did not result in complete disappearance of luminescence. However, superoxide is easily dismutated to O2 and H,Oz in the presence of the dismutase, both of which in turn are involved in the HRP-catalyzed luminescent reaction. This is why complete disappearance of luminescence was not observed upon addition of superoxide dismu tase .
TABLE 1 Effect of superoxide dismutase on the luminescent reaction. The reaction was performed in the presence of HzOz (15 /.&I), luminol (180 PM), peroxidase (0.32 units ml-‘) and superoxide dismutase Superoxide dismutase (u ml-’ ) Luminescence (%)
0 100
90 87
180 81
270 78
360 77
These results indicate that the luminescent reaction proceeds via two pathways, i.e., catalyst-initiated reaction and delayed reaction. The enzyme-catalyzed luminescent reaction is not initiated until HzOz is added to the reaction medium. However, whether HzOz is involved in delayed
47
luminescence has not been fully clarified; this may be studied by the use of an annihilating enzyme of H202, catalase. Therefore, after the luminescence intensity reached its maximum, catalase (4.7 units) was added to the reaction medium (0.5 ml) containing 12 FM H,O,, 350 PM luminol and 6.0 nM (0.073 units ml-‘) HRP. The catalase addition caused ~s~n~eous disappearance of the luminescence from the luminol-H,O,-peroxidase system, which clearly proves the indispensable role of H20, in the delayed luminescence. The degree of luminescence disappearance could be correlated to a change in the amount of catalase.
Discussion Several schemes have been proposed concerning luminol luminescence [9 - 151; however, they lack clear evidence due to unsuccessful efforts to identify the reactive intermediates. In our previous report [16], the fact that nonenzymatic luminescence was created in the HRP-initiated luminescent reaction of the luminolHzO, system was clearly shown by applying immobilized HRP to the luminescent reaction. Since in this work the oxygen molecule and its radical as well as HzOz were shown to be essential reactive species, causing a noncatalytic luminescent reaction, thus the scheme illustrated in Fig. 7 was postulated, wherein molecular oxygen is required to oxygenate the luminol radical anion. The enzyme directly generates energized species such as the luminol radical (LH’ ), which has the potential to emit light and to generate 02; from oxygen. The 0,: radical can oxidize a luminol radical nonenzymatic~ly, with the resultant formation of 0 22- (H202). The luminol radical is oxygenated, which might be followed by the generation of an oxygenated luminol anion as illustrated in Fig. 7. The luminol radical anion is not oxygenated provided that oxygen is removed from the reaction medium. Therefore, the complete removal of oxygen molecules by the oxygen-consum~g glucoseglucose oxidase system led to a considerable disappearance of luminescence. DMSO displayed disparate effects on the rate of the luminescence reaction, mainly due to the effect of solvent on stabilization of such intermediates as the luminol radical anion and the oxygen radical anion generated by the catalysts described. Both radicals are species essential to the luminescence reaction, and their accessability to substrates is assumed to be increased by increasing the concentration of DMSO. Although we have not yet fully succeeded in identifying the reactive intermediates of the luminol reaction, our new findings that oxygen, oxygen radicals and hydrogen peroxide play quite important roles in the luminol luminescence, especially in a water-DMSO mixed solvent system, very strongly indicate the postulated nonca~ytic process of the luminescence reaction.
iH2
6
NH2 0
NonenzymaticProcess
Chemiluminescent Process Fig. 7. Postulated scheme for a nonenzymatic luminescent pathway where the essential involvements of oxygen molecule, superoxide radical and hydrogen peroxide are shown with respect to luminol and its radical species. The luminol radical is also produced in enzyme-catalyzed process.
References 1 2 3 4 5 6 7 8 9 10 11 12’ 13 14 15 16
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