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P-phenol derivatives as enhancers of the chemiluminescent luminol-horseradish peroxidase-H2O2 reaction: substituent effects
JOURNAL
OF
LUMINESCENCE ELSEMER
Journal
of Luminescence
65 (1995) 33 39
P-phenol derivatives as enhancers of the chemiluminescent luminol-horseradish peroxidase-H,02 reaction : substituent effects F. Garcia Sanchez*, A. Navas Diaz, J.A. Gonzalez Garcia Department
of Analytical Chemistry, Received
Facul?/
of Sciences,Unirersit?, of
2 May 1994; revised 9 January
1995; accepted
Mdaga.
29071
9 January
Mdaga.
Spain
1995
Abstract We studied the substituent effects of ten p-phenol derivatives and aniline on the chemiluminescence from the luminol-horseradish peroxidaseeH,Oz system. The enhancer effects of some compounds (phenol, p-cresol, p-coumaric acid, p-hydroxybenzoic acid, p-hydroxymethylbenzoate, p-hydroxybenzaldehyde and aniline) were studied against pH and concentration. These compounds enhanced the chemiluminescent emission of luminol between 2 and 40 times. Phenols with activative substituents (in the para-position) of the benzene ring and phenol, produced a greater increase (at low concentration) in the chemiluminescence than those with deactivating substituents. Phenols with very active or very deactivating substituents of the benzene ring decreased the chemiluminescence of luminol. We found certain relations between the pKa values and the reducing character of these phenol derivatives with their enhancer or inhibition effects on the luminol chemiluminescence.
1. Introduction Certain benzothiazole [l-3], phenol [4], naphthol [S] and aromatic amine [6,7] derivatives enhance light emission from the horseradish peroxidase-catalyzed oxidation of cyclic diacylhydrazides such as luminol. The intense and prolonged light emission is easily measured, horseradish peroxidase (HRP) and HRP-conjugates can be assayed sensitively in seconds. The applicability of these enhanced chemiluminescent reactions to immunoassays have been demonstrated in several cases [S--lo]. Nevertheless, numerous benzothiazole, phenol, naphthol and aniline derivatives produce a non-enhancement of light emission
*Corresponding author. 0022-2313/95/$09.50 6.1 1995 SSDI 0022-23 13(95)00047-X
when incorporated into diacylhydrazide-peroxidase oxidation. Substituent effects have been studied and compared in several benzothiazoles and para-substituted halophenols [5], but the relation between structures of phenol derivatives and their enhancer characters is not yet clear. In this paper, we have studied the enhancer or non-enhancer effects of aniline and some p-phenol derivatives: p-nitrophenol, p-hydroxybenzoic acid, p-hydroxymethylbenzoate, p-hydroxybenzaldehyde, phenol, p-cresol, L-tyrosine, p-coumaric acid, hydroquinone and p-methoxyphenol from the chemiluminescence of the peroxidase-luminolH202 system. It may be emphasized that aniline, phenol, p-hydroxymethylbenzoate, p-hydroxybenzoic acid and p-hydroxybenzaldehyde have not been described as chemiluminescent enhancers of
Elsevier Science B.V. All rights reserved
34
F.G. Sanchez et al. /Journal
this system until now. The substituent influences on the reductor character and pK, of phenol derivatives were related with the chemiluminescent enhancements or inhibitions.
of Luminescence
65 (1995) 33-39
set to 700 V. The samples were placed in a quartz cuvette continuously stirred with a magnetic stirrer.
3. Methods 2. Experimental.
3.1. General procedure
2.1. Reagents
A quartz cuvette was filled with 1 ml of a trisHCl buffer solution (0.1 M, pH 8.5) 20 ul of luminol (0.01 M), 60 ul of hydrogen peroxide (0.1 M) and 20 ul of p-phenol derivative (1 mM), the cuvette was filled to 2950 ul with bidistilled water. Ten seconds after the spectrometer began to record, the chemiluminescence reaction was triggered by injecting horseradish peroxidase with a syringe, through a septum. And the kinetics of the light emission were recorded.
All stock solutions were prepared in bidistilled Luminol (5amino-2,3-dihydro-1,4water. phthalazinedione) (Sigma, St. Louis, MO, U.S.A) was prepared by dissolving 0.0913 g of luminol 97% in a few drops of NaOH and the volume was adjusted to 50 ml with tris-HCl buffer 0.1 M (pH 8.6). Horseradish peroxidase (Sigma) 73 U/ml was prepared in tris-HCl buffer (pH 8.5); hydrogen peroxide (Panreac Montplet and Esteban S.A., Barcelona, Spain) was prepared from hydrogen peroxide 6% w/v by diluting with water to 0.1 M. (Chydroxybenzalp-Hydroxybenzaldehyde dehyde), p-hydroxybenzoic acid (Chydroxybenzoic acid), p-coumaric acid (Chydroxycinnamic acid) and L-tyrosine (a-amino-4-hydroxyhydrocinnamic acid) from Sigma; p-cresol (Cmethylphenol) and p-methoxyphenol (Cmethoxyphenol) were purchased from Aldrich-Chemie, Steinheim and p-hydroxymethylbenzoate (4-hydroxymethylbenzoate) from Aldrich Chemical Company, Inc., Milwaukee, USA; phenol was supplied by E. Merck, Darmstadt, Germany; hydroquinone (4-hydroxyphenol) was obtained from Panreac, Montplet and Esteban S.L., Barcelona, Spain.; and aniline (phenylamine) from Probus S.A. Badalona, Spain. All stock solutions of these compounds were 0.01 M or 0.001 M in bidistilled water. 2.2. Instrumentation The chemiluminescence experiments were carried out in a Perkin-Elmer LS-50 (Beaconsfield, UK) luminescence spectrometer with the light source switched-off. The apparatus was set in the phosphorescence mode with 0.00 ms of delay time and 60 ms of gate time. The slit width of the emission monochromator was set at 20 nm with A,,,,= 425 nm and the photomultiplier voltage was
3.2. pH studies The general reaction was repeated with 1 ml of one of these buffer solutions: potassium dihydrogen phosphate buffer (pH 6-7.5), tris(hydroxymethy1) aminomethane buffer (pH 7.5-8.5), borax buffer sodium buffer (pH 8.5-lo), bicarbonate (pH 10-11) disodium hydrogen phosphate buffer (pH 1l-l 1.5), and KCl/NaOH buffer (11.5-12.5). We carried out these pH studies with 20 ul of pcoumaric acid 1 mM, or with 100 ul of other enhancers 1 mM (phenol, p-cresol, p-hydroxybenzoic acid, p-hydroxymethylbenzoate, p-hydroxybenzaldehyde and aniline). Initially we measured the maximal chemiluminescence intensity, but the relative standard deviation (RSD) of blank (without phenol derivative) was greater than 50% at pH over 8.5; thus finally, we measured only the area under the emission curve in the chemiluminescent emission study at different pH values. 3.3. Concentration
studies
The general chemiluminescent reaction was repeated with tris-HCl buffer (pH 8.5) using these enhancers at different concentrations: p-coumaric acid 10 uM (5-500 ul), aniline 1 mM (2-1500 ul),
F.G. Sanchez et al. 1Journal of Luminescence 65 (1995) 33-39
p-hydroxymethylbenzoate 10 mM (lo-1870 pl), phydroxybenzaldehyde 10 mM (lo-2370 pl), p-hydroxybenzoic acid 10 mM (10-500 pl), phenol 1 mM (2-1000 ~1) and p-cresol 1 mM (2-200 ~1) in a quartz cuvette with a final volume of 3 ml. In this study we measured the area under the emission curve and the intensity at 300 s.
35
methoxyphenol decreased the chemiluminescent emission. The enhancement effect appears to be promoted by phenol derivatives with moderately activating substituents (substituents that have less electronwithdrawing power than hydrogen, or substituents with negative Hammett constant [ll]) in parapositions such as: p-coumaric acid, and p-cresol, and also by phenol derivatives with moderately deactivating substituents (substituents that have more electron-withdrawing power than hydrogen, or substituents with positive Hammett constant) in the para-position such as: p-hydroxybenzaldehyde, p-hydroxybenzoic acid and p-hydroxymethylbenzoate. On the other hand, an inhibition effect was favored by compounds with strong activating substituents (hydroquinone and p-methoxyphenol) or strong deactivating substituents (p-nitrophenol). Table 1 shows that the enhancement was dependent on the substituent in para-position. The enhancer effect order in these conditions was: pcoumaric acid > p-cresol > phenol > aniline > phydroxybenzoic acid > p-hydroxymethylbenzoate > p-hydroxybenzaldehyde > tyrosine > p-nitrophenol > hydroquinone > p-methoxyphenol.
4. Results and discussion Table 1 shows data for the p-phenol derivatives studied and aniline, including their normalized intensities (blank = 1, without enhancer) and normalized areas under emission curve (blank = 1, without enhancer) at pH 8.5. Table 1 contains also the pKa values and concentration ratios between their acidic forms (EH or ArOH) and basic forms (E- or ArO-) at pH 8.5 for some phenol derivatives. From this table, we observed that p-coumaric acid, p-cresol, phenol, aniline, p-hydroxybenzoic acid, p-hydroxymethylbenzoate and p-hydroxybenzaldehyde enhanced the chemiluminescence of the luminol-H,O,-horseradish peroxidase system whereas p-nitrophenol, hydroquinone and p-
Table. 1 Normalized relative emissions (Blank = 1, without phenol derivative nor aniline) of chemiluminescence peroxidase-Hi,Oz system following the addition of different phenol derivatives and aniline Compound None (blank) p-nitrophenol p-hydroxybenzoic
pKa
acid
p-hydroxymethylbenzoate p-hydroxybenzaldehyde Phenol p-cresol Tyrosine p-coumaric acid Hydroquinone p-methoxyphenol Aniline
7.15 4.4gd 9.32’
of the luminol-horseradish
“[Em]:[EH]
I”
A’
22:l
1.OOO+ 0.048 1.083 + 0.090 1.849 + 0.343
1.000 * 0.042 0.817 k 0.017 1.352 k 0.031
1:7
7.66 9.89 10.17
7:l 1:25 1145
10.35
1:71
2.008 1.341 3.020 4.520 1.028 34.236 0.728 0.261 1.800
k 1.463 k 0.069 f 1.938 f 0.007 kO.111 k 0.001 + 0.028 + 0.044 f 0.24
“Ratio between basic form (Em or Ara-) and acidic form (EH or Ar-OH) of phenol derivatives “Maximum intensity of chemiluminescence with relative standard deviation. ‘Area beneath the chemiluminescence emission curve with relative standard deviation. Experimental [H,O,] = 2 mM, [tris-HCI] = 33 mM pH = 8.5, [peroxidase] = 1.2 U/ml, b-phenol derivative] d and e are pK, of first and second acid-base reactions.
1.298 1.261 2.184 2.409
k + k & 1.070+ 6.880 & 0.852 k 0.194 f 1.810 +
0.047 0.018 0.032 0.001 0.059 0.327 0.043 0.006 0.039
in tris-CIH conditions: = 6.7 PM.
buffer at pH 8.5. [luminol]
= 66.7 FM,
36
F.G. Sanchez et al. /Journal
A mechanism has been proposed [ 12-173 for the luminol-H,O,-horseradish peroxidase system to explain these enhancer and inhibition effects. The mechanism is not completely established, but kinetic data for reactivity of enhancers with the enzyme intermediates (HRP-I and HRP-II) [15-163 support that a pathway involving generation of the phenoxy radical enhancer, followed by reaction of the enhancer phenoxy radical with luminol is a major element in the process: HRP + H202 + (horseradish peroxidase)
(1)
HRP-I + HZ0 (enzyme intermediate-I) 1.a. Unenhanced HRP-I + LH- --*HRP-II + L.- + HzO, (luminol)
qf
Luminescence 65 (1995) 33-39
are the most highly-reducing phenols. These compounds inhibited chemiluminescence (Table 1) and this might be explained, if the enhancer radicals formed from the phenol derivatives in stages 4 and 5 are too weak to oxidize luminol to the luminol radical, and so prohibit stage 6 [ 171. Similar results are reported for caffeic acid, protocatechuic acid, gentisic acid, 2,3-dihydroxybenzoic acid, vanillic acid, ferulic acid, etc. [IS]. Fig. 1 shows the area under the emission curve between 0 and 300 s (these areas are not normalized, blank areas # 1, without enhancer) against pH of the p-phenols that showed enhancer effects and aniline. At pH > 8.6, chemiluminescence intensity maxima of the blank had a very high relative standard deviation (RSD), in some cases greater than 50%. In this case, we have preferred
(2)
(luminol radical)
HRP-II + LH- + HRP + L.-
I
,’
4’
60
(enzyme intermediate-II)
’
,A
(3)
/
1.b. Enhanced. HRP-I + EH + HRP-II + E. + HzO, (enhancer)
(4)
(enhancer radical)
HRP-II + EH + HRP + E.
(5)
E. + LH- +EH
(6)
+ L.-.
2. L.- + 02 +L + oz.-,
(7)
(Ion radical superoxide) L-- + oz.-
+ Lo*2-,
(8)
LOz2- + AP2-*,
(9)
(excited 3-aminophthalate
dianion)
AP2-* --, AP2- + hv(425 nm), (3-aminophthalate
10
6
(luminol endoperoxide)
(10)
dianion)
We could explain some of the enhancer or inhibition effects of the compounds studied here using this mechanism. The interpretation of other phenomena are not yet clear. Phenol derivatives with very activating substituents, such as hydroquinone and p-methoxyphenol,
12
PR Fig. 1. Normalized areas (blank = 1, without enhancer) beneath the emission curves of chemiluminescence between 0 and 300 s against pH, for the luminol-horseradish peroxidase-H,O, system. (0) Unenhanced (blank, without enhancer), relative standard deviation (RSD) in the range 157.8%; (V) p-hydroxymethylbenzoate, RSD in the range 0.46 to 11.5%; (A) phydroxybenzaidehide, RSD in the range 1.6 to 15%; (A). phydroxybenzoic acid, RSD in the range 1.7 to 13.6%; (V). p-coumaric acid, RSD in the range 0.09 to 4.3%; (0). Phenol, RSD in the range 2.1-16.4%; (m). p-Cresol, RSD in the range 2.7-37.6%; (0) Aniline, RSD 1.4% to 12.3%. Experimental conditions: [luminol] = 66.7 pM, [HZO,] = 2 mM, [buffer] = 33 mM, [peroxidase] = 1.2 U/ml, [p-coumaric acid] = 6.7 uM and [other p-phenol derivatives] = 33.3 pM.
F.G. Sanchez et al. 1 Journal cfLumine.wence
to measure the areas under the light emission curves because their maxima RSD were about 10%. Emission maxima were in the pH range 7.5 to 9.5; we chose pH 8.5 to study the chemiluminescence against enhancer concentration. Fig. 2 shows the normalized area (blank area = 1, without enhancer) beneath the emission curve and the normalized intensity at 300 s (blank intensity at 300 s = 1, without enhancer) against the enhancer concentration at pH 8.5. This figure shows that these new enhancers (phenol, p-hydroxybenzoic acid, p-hydroxymethylbenzoate, phydroxybenzaldehyde and aniline) increased between 2 and 40 times the blank areas between 0 and 300 s, or increased between 1.5 and 30 times the blank intensity at 300 s. We think these signals could be used in analytical determinations of phenol, aniline, p-hydroxybenzoic acid, p-coumaric acid, etc., and in chemiluminescent immunoassays [8&10], etc. Also inhibition effects could be used in analytical methods to determine the inhibitor [ 191, e.g., p-methoxyphenol. We observe in Figure 2 that compounds with deactivating substituents (p-hydroxybenzoic acid,
40
65 (1995) 33-39
31
p-hydroxybenzaldehyde and p-hydroxymethylbenzoate) had a lower enhancer effect at low concentration than phenol and p-cresol (with an activating substituent), but at higher concentrations, p-hydroxybenzoic acid, p-hydroxybenzaldehyde and phydroxymethylbenzoate were greater enhancers than phenol and p-cresol. Also, we observed that the enhancer effect is more important with phenol that with aniline at low concentration (concentration lesser than 3 x lo-’ M), but at higher concentration aniline produced more enhancement than phenol. It is important to emphasize that the concentration of p-coumaric acid (4-hydroxycinnamic acid) studied had a maximum of 1.7 x 10m6 M (in Fig. 2) because the high intensity of the signal that suddenly decreases, causes the photomultiplier to approach saturation at about 6.7 x 1O-6 M. Therefore, p-coumaric signal against concentration in Fig. 2 was only studied up to 1.7 x lo-’ M. This compound has the greater enhancer effect at low concentration, but at higher concentrations we have not studied it. The -NOz, -CHO and COOH groups are deactivating substituents whereas the group -CH3 is an
+i,
,‘> ~. /’
_~A ~- ..
P
20 / lo
+C_.-----, -cd _ __ __ _.._--A
0 1.; 0
200 [Phenol
10
400 deriv.]
20
30
600 x 10 ’
M
200 [Phenol
400 deriv.]
600
800
1000
x 10 5 M
Fig. 2. Normalized areas (blank = I, without enhancer) under chemiluminescence emission curves, between 0 and 300 s against enhancer concentration (left); and Intensity at 300 s against enhancer concentration (right); for the luminol-peroxidase-H,0, system at pH = 8.5: (0) p-hydroxybenzaldehide; (0) p-hydroxymethylbenzoate; (V) p-hydroxybenzoic acid; (A) aniline; (V phenol; (0) p-cresol; (W) p-coumaric acid. Experimental conditions: [luminol] = 66.7 FM, [H,O,] = 2 mM, [tris-HCI buffer] = 33 mM at pH = 8.5, [peroxidase] = I.2 UlmL.
38
F.G. Sanchez ei al. /Journal
activating substituent of the benzene ring. Table 1 and Fig. 2 show that according to the pKa values of phenols the concentration of acidic forms (Ar-OH) in tris-HCl buffer at pH 8.5 increased in p-nitrophenol < p-hydroxybenzalorder: the acid < phenol < pdehyde < p-hydroxybenzoic cresol. At this pH, the enhancer effect increased in the same order. Table 1 and Figure 2 show that phenol and p-cresol had greater enhancer effects (at concentration < 6.7 x lO-‘j M) than p-hydroxybenzoic acid, p-hydroxymethylbenzoate and p-hydroxybenzaldehyde. The ratios between the acidic and basic forms of phenol and p-cresol at pH 8.5 were 25:l and 47:l respectively, whereas of p-nitrophenol, p-hydroxybenzaldehyde and p-hydroxybenzoic acid were 1:22, 1:7 and 7:l respectively. The enhancer effect increased with the pKa value because the concentrations of phenol derivatives in acidic form (Ar-OH) increased. This could be explained because horseradish peroxidase reacts only with the acidic form (Ar-OH) to give a phenoxy radical, in the stages 4 and 5, and not with the basic form (Ar-O-) and so this fact obstructs stage 6. Fig. 2 shows that p-cresol had greater synergy than phenol when the enhancer concentration increased, and the optimal p-cresol concentrations (enhancer concentration with maximal enhancement) was less than the optimal phenol concentration. Similar results are reported for o-cresolphthalein against phenolphthalein, o-tolidine against benzidine and cresol red against phenol red [20]. Also, it is known that the benzil radicals (Ph-CH2.) are very stable, and that horseradish peroxidase catalyzed the hydroxylation of benzil methyl groups [S]. We suggest that this might be explained by supposing that there are interactions between the enhancer radicals (Ar-0.) or hydroxyl radicals (OH.) formed, and the -CH3 groups of these enhancers, these interactions could destroy the phenoxy radicals (CH,-Ph-0.) formed. This could explain the non-enhancement effect of Ltyrosine in Table 1. Chemiluminescent emission from the unenhanced luminol-H,O,-horseradish peroxidase oxidation had a emission maximum around 425 nm. The chemiluminescence spectra were remarkably similar to the unenhanced reaction (with
of Luminescence
65 (1995) 33-39
maxima around 425 nm), although it was not possible to determine chemiluminescence emission from the enhancer alone. This is in accordance with the hypothesis that aminophthalate is the emitter and not the enhancer [S].
5. Conclusions
Phenol, p-cresol, p-hydroxybenzoic acid, p-hydroxymethylbenzoate, p-hydroxybenzaldehyde and aniline are found to be enhancers of the luminolhorseradish peroxidase-HzOz system. We observed certain relationships between the kind of substituent, character reductor and pKa values of phenol derivatives and the enhancer or inhibition effect on the luminol chemiluminescence. We think these enhancer and inhibition effects could be used in chemiluminescent immunoassays and in analytical determinations of p-coumaric acid, phenol, pcresol, aniline, p-methoxyphenol, etc. _
Acknowledgements
We thank the Comision Interministerial de Ciencia y Tecnologia (Project PB93-1006) for financial support.
References Cl] G.H.G. Thorpe, L.J. Kricka, E. Gillespie, S. Moseley, R. Amess, N. Baggett, T.P. Whitehead, Anal. Biochem. 145 (1985) 96. [2] G.H.G. Thorpe, S.B. Moseley, L.J. Kricka, R.A. Stott, T.P. Whitehead, Anal. Chim. Acta. 170 (1985) 101. [3] T.P. Whitehead, G.H.G. Thorpe, T.J.N. Carter, C. Groucutt, L.J. Kricka, Nature 305 (1983) 158. [4] G.H.G. Thorpe, L.J. Kricka, S.B. Moseley, T.P. Whitehead, Clin. Chem. 31 (1985) 1335. [S] L.J. Kricka, R.A.W. Stott, G.H.G. Thorpe, in: Luminescence Techniques in Chemical and Biochemical Analysis, eds. W.R.G. Baeyens, D.D. Keukeleir, K. Korkidis (Dekker, New York, 1991) p. 599. [6] L.J. Kricka, A.M. O’Toole, G.H.G. Thorpe, T.P. Whitehead, U.S. Patent (1988) 4,729,950. [7] D.S. Milbrath, Eur. Patent Application (1987) 219352. [8] D.C. Ireland, D. Samuel, .J. Biolumin. Chemilumin. 2 (1988) 215.
F.G. Sanchez et al. /Journal
[9] G.H.G. Thorpe, E. Gillespie, R. Haggart, L.J. Kricka, T.P. Whitehead, in: Analytical Applications of Bioluminescence and Chemiluminescence, eds. L.J. Kricka, P.E. Stanley, G.H.G. Thorpe, T.P. Whitehead (Academic Press, London 1984) p. 243. [IO] G.H.G. Thorpe, L.A. Willians, L.J. Kricka, T.P. Whitehead, H. Evans, D.R. Stanworth, J. Immunol. Meth. 79 (1985) 57. [II] E.J. King, in: Acid-Base Equilibria (Pergamon Press, Oxford, 1965) p. 178. [I21 M.J. Cormier, P.M. Prichard, J. Biol. Chem. 243 (1968) 4706. [13] A. Lundin, L.O.B. Hallander, in: New Perspectives, eds. J. Scholmerich, R. Andreesen, A. Kapp, M. Ernst, W.G. Wood (Wiley, Chichester, 1987) p. 555.
of Luminescence
[I41
65 (1995) 33-39
39
G.H.G. Thorpe, L.J. Kricka, in: New Perspectives, eds. J. Scholmerich, R. Andreesen, A. Kapp, M. Ernst. W.G. Wood (Wiley, Chichester, 1987) p. 199. [15] M. Hodgson, P. Jones, J. Biolum. Chemilum. 3 (1989) 21. [I63 S.B. Vlasenko, A.A. Arefyev, A.D. Klimov, B.B. Kim, E.L. Gorovits, A.P. Osipov, E.M. Gavrilova. A.M. Yehorov. J. Biolum. Chemilum. 4 (1989) 164. [I73 T.E.G. Candy, P. Jones, J. Biolumin. Chemilum. 6 (1991) 239. [ I83 A. Navas Diaz, F. Garcia Sanchez, J.A. Gonzalez Garcia, J. Biolumin. Chemilumin., in press. [I91 Y.L. Huang, J.M. Kim, R.D. Schmid, Anal. Chim. Acta. 266 (1992) 317. [20] F. Garcia Sanchez, A. Navas Diaz, J.A. Gonzalez Garcia, J. Photochem. Photobiol., in press.
OF
LUMINESCENCE ELSEMER
Journal
of Luminescence
65 (1995) 33 39
P-phenol derivatives as enhancers of the chemiluminescent luminol-horseradish peroxidase-H,02 reaction : substituent effects F. Garcia Sanchez*, A. Navas Diaz, J.A. Gonzalez Garcia Department
of Analytical Chemistry, Received
Facul?/
of Sciences,Unirersit?, of
2 May 1994; revised 9 January
1995; accepted
Mdaga.
29071
9 January
Mdaga.
Spain
1995
Abstract We studied the substituent effects of ten p-phenol derivatives and aniline on the chemiluminescence from the luminol-horseradish peroxidaseeH,Oz system. The enhancer effects of some compounds (phenol, p-cresol, p-coumaric acid, p-hydroxybenzoic acid, p-hydroxymethylbenzoate, p-hydroxybenzaldehyde and aniline) were studied against pH and concentration. These compounds enhanced the chemiluminescent emission of luminol between 2 and 40 times. Phenols with activative substituents (in the para-position) of the benzene ring and phenol, produced a greater increase (at low concentration) in the chemiluminescence than those with deactivating substituents. Phenols with very active or very deactivating substituents of the benzene ring decreased the chemiluminescence of luminol. We found certain relations between the pKa values and the reducing character of these phenol derivatives with their enhancer or inhibition effects on the luminol chemiluminescence.
1. Introduction Certain benzothiazole [l-3], phenol [4], naphthol [S] and aromatic amine [6,7] derivatives enhance light emission from the horseradish peroxidase-catalyzed oxidation of cyclic diacylhydrazides such as luminol. The intense and prolonged light emission is easily measured, horseradish peroxidase (HRP) and HRP-conjugates can be assayed sensitively in seconds. The applicability of these enhanced chemiluminescent reactions to immunoassays have been demonstrated in several cases [S--lo]. Nevertheless, numerous benzothiazole, phenol, naphthol and aniline derivatives produce a non-enhancement of light emission
*Corresponding author. 0022-2313/95/$09.50 6.1 1995 SSDI 0022-23 13(95)00047-X
when incorporated into diacylhydrazide-peroxidase oxidation. Substituent effects have been studied and compared in several benzothiazoles and para-substituted halophenols [5], but the relation between structures of phenol derivatives and their enhancer characters is not yet clear. In this paper, we have studied the enhancer or non-enhancer effects of aniline and some p-phenol derivatives: p-nitrophenol, p-hydroxybenzoic acid, p-hydroxymethylbenzoate, p-hydroxybenzaldehyde, phenol, p-cresol, L-tyrosine, p-coumaric acid, hydroquinone and p-methoxyphenol from the chemiluminescence of the peroxidase-luminolH202 system. It may be emphasized that aniline, phenol, p-hydroxymethylbenzoate, p-hydroxybenzoic acid and p-hydroxybenzaldehyde have not been described as chemiluminescent enhancers of
Elsevier Science B.V. All rights reserved
34
F.G. Sanchez et al. /Journal
this system until now. The substituent influences on the reductor character and pK, of phenol derivatives were related with the chemiluminescent enhancements or inhibitions.
of Luminescence
65 (1995) 33-39
set to 700 V. The samples were placed in a quartz cuvette continuously stirred with a magnetic stirrer.
3. Methods 2. Experimental.
3.1. General procedure
2.1. Reagents
A quartz cuvette was filled with 1 ml of a trisHCl buffer solution (0.1 M, pH 8.5) 20 ul of luminol (0.01 M), 60 ul of hydrogen peroxide (0.1 M) and 20 ul of p-phenol derivative (1 mM), the cuvette was filled to 2950 ul with bidistilled water. Ten seconds after the spectrometer began to record, the chemiluminescence reaction was triggered by injecting horseradish peroxidase with a syringe, through a septum. And the kinetics of the light emission were recorded.
All stock solutions were prepared in bidistilled Luminol (5amino-2,3-dihydro-1,4water. phthalazinedione) (Sigma, St. Louis, MO, U.S.A) was prepared by dissolving 0.0913 g of luminol 97% in a few drops of NaOH and the volume was adjusted to 50 ml with tris-HCl buffer 0.1 M (pH 8.6). Horseradish peroxidase (Sigma) 73 U/ml was prepared in tris-HCl buffer (pH 8.5); hydrogen peroxide (Panreac Montplet and Esteban S.A., Barcelona, Spain) was prepared from hydrogen peroxide 6% w/v by diluting with water to 0.1 M. (Chydroxybenzalp-Hydroxybenzaldehyde dehyde), p-hydroxybenzoic acid (Chydroxybenzoic acid), p-coumaric acid (Chydroxycinnamic acid) and L-tyrosine (a-amino-4-hydroxyhydrocinnamic acid) from Sigma; p-cresol (Cmethylphenol) and p-methoxyphenol (Cmethoxyphenol) were purchased from Aldrich-Chemie, Steinheim and p-hydroxymethylbenzoate (4-hydroxymethylbenzoate) from Aldrich Chemical Company, Inc., Milwaukee, USA; phenol was supplied by E. Merck, Darmstadt, Germany; hydroquinone (4-hydroxyphenol) was obtained from Panreac, Montplet and Esteban S.L., Barcelona, Spain.; and aniline (phenylamine) from Probus S.A. Badalona, Spain. All stock solutions of these compounds were 0.01 M or 0.001 M in bidistilled water. 2.2. Instrumentation The chemiluminescence experiments were carried out in a Perkin-Elmer LS-50 (Beaconsfield, UK) luminescence spectrometer with the light source switched-off. The apparatus was set in the phosphorescence mode with 0.00 ms of delay time and 60 ms of gate time. The slit width of the emission monochromator was set at 20 nm with A,,,,= 425 nm and the photomultiplier voltage was
3.2. pH studies The general reaction was repeated with 1 ml of one of these buffer solutions: potassium dihydrogen phosphate buffer (pH 6-7.5), tris(hydroxymethy1) aminomethane buffer (pH 7.5-8.5), borax buffer sodium buffer (pH 8.5-lo), bicarbonate (pH 10-11) disodium hydrogen phosphate buffer (pH 1l-l 1.5), and KCl/NaOH buffer (11.5-12.5). We carried out these pH studies with 20 ul of pcoumaric acid 1 mM, or with 100 ul of other enhancers 1 mM (phenol, p-cresol, p-hydroxybenzoic acid, p-hydroxymethylbenzoate, p-hydroxybenzaldehyde and aniline). Initially we measured the maximal chemiluminescence intensity, but the relative standard deviation (RSD) of blank (without phenol derivative) was greater than 50% at pH over 8.5; thus finally, we measured only the area under the emission curve in the chemiluminescent emission study at different pH values. 3.3. Concentration
studies
The general chemiluminescent reaction was repeated with tris-HCl buffer (pH 8.5) using these enhancers at different concentrations: p-coumaric acid 10 uM (5-500 ul), aniline 1 mM (2-1500 ul),
F.G. Sanchez et al. 1Journal of Luminescence 65 (1995) 33-39
p-hydroxymethylbenzoate 10 mM (lo-1870 pl), phydroxybenzaldehyde 10 mM (lo-2370 pl), p-hydroxybenzoic acid 10 mM (10-500 pl), phenol 1 mM (2-1000 ~1) and p-cresol 1 mM (2-200 ~1) in a quartz cuvette with a final volume of 3 ml. In this study we measured the area under the emission curve and the intensity at 300 s.
35
methoxyphenol decreased the chemiluminescent emission. The enhancement effect appears to be promoted by phenol derivatives with moderately activating substituents (substituents that have less electronwithdrawing power than hydrogen, or substituents with negative Hammett constant [ll]) in parapositions such as: p-coumaric acid, and p-cresol, and also by phenol derivatives with moderately deactivating substituents (substituents that have more electron-withdrawing power than hydrogen, or substituents with positive Hammett constant) in the para-position such as: p-hydroxybenzaldehyde, p-hydroxybenzoic acid and p-hydroxymethylbenzoate. On the other hand, an inhibition effect was favored by compounds with strong activating substituents (hydroquinone and p-methoxyphenol) or strong deactivating substituents (p-nitrophenol). Table 1 shows that the enhancement was dependent on the substituent in para-position. The enhancer effect order in these conditions was: pcoumaric acid > p-cresol > phenol > aniline > phydroxybenzoic acid > p-hydroxymethylbenzoate > p-hydroxybenzaldehyde > tyrosine > p-nitrophenol > hydroquinone > p-methoxyphenol.
4. Results and discussion Table 1 shows data for the p-phenol derivatives studied and aniline, including their normalized intensities (blank = 1, without enhancer) and normalized areas under emission curve (blank = 1, without enhancer) at pH 8.5. Table 1 contains also the pKa values and concentration ratios between their acidic forms (EH or ArOH) and basic forms (E- or ArO-) at pH 8.5 for some phenol derivatives. From this table, we observed that p-coumaric acid, p-cresol, phenol, aniline, p-hydroxybenzoic acid, p-hydroxymethylbenzoate and p-hydroxybenzaldehyde enhanced the chemiluminescence of the luminol-H,O,-horseradish peroxidase system whereas p-nitrophenol, hydroquinone and p-
Table. 1 Normalized relative emissions (Blank = 1, without phenol derivative nor aniline) of chemiluminescence peroxidase-Hi,Oz system following the addition of different phenol derivatives and aniline Compound None (blank) p-nitrophenol p-hydroxybenzoic
pKa
acid
p-hydroxymethylbenzoate p-hydroxybenzaldehyde Phenol p-cresol Tyrosine p-coumaric acid Hydroquinone p-methoxyphenol Aniline
7.15 4.4gd 9.32’
of the luminol-horseradish
“[Em]:[EH]
I”
A’
22:l
1.OOO+ 0.048 1.083 + 0.090 1.849 + 0.343
1.000 * 0.042 0.817 k 0.017 1.352 k 0.031
1:7
7.66 9.89 10.17
7:l 1:25 1145
10.35
1:71
2.008 1.341 3.020 4.520 1.028 34.236 0.728 0.261 1.800
k 1.463 k 0.069 f 1.938 f 0.007 kO.111 k 0.001 + 0.028 + 0.044 f 0.24
“Ratio between basic form (Em or Ara-) and acidic form (EH or Ar-OH) of phenol derivatives “Maximum intensity of chemiluminescence with relative standard deviation. ‘Area beneath the chemiluminescence emission curve with relative standard deviation. Experimental [H,O,] = 2 mM, [tris-HCI] = 33 mM pH = 8.5, [peroxidase] = 1.2 U/ml, b-phenol derivative] d and e are pK, of first and second acid-base reactions.
1.298 1.261 2.184 2.409
k + k & 1.070+ 6.880 & 0.852 k 0.194 f 1.810 +
0.047 0.018 0.032 0.001 0.059 0.327 0.043 0.006 0.039
in tris-CIH conditions: = 6.7 PM.
buffer at pH 8.5. [luminol]
= 66.7 FM,
36
F.G. Sanchez et al. /Journal
A mechanism has been proposed [ 12-173 for the luminol-H,O,-horseradish peroxidase system to explain these enhancer and inhibition effects. The mechanism is not completely established, but kinetic data for reactivity of enhancers with the enzyme intermediates (HRP-I and HRP-II) [15-163 support that a pathway involving generation of the phenoxy radical enhancer, followed by reaction of the enhancer phenoxy radical with luminol is a major element in the process: HRP + H202 + (horseradish peroxidase)
(1)
HRP-I + HZ0 (enzyme intermediate-I) 1.a. Unenhanced HRP-I + LH- --*HRP-II + L.- + HzO, (luminol)
qf
Luminescence 65 (1995) 33-39
are the most highly-reducing phenols. These compounds inhibited chemiluminescence (Table 1) and this might be explained, if the enhancer radicals formed from the phenol derivatives in stages 4 and 5 are too weak to oxidize luminol to the luminol radical, and so prohibit stage 6 [ 171. Similar results are reported for caffeic acid, protocatechuic acid, gentisic acid, 2,3-dihydroxybenzoic acid, vanillic acid, ferulic acid, etc. [IS]. Fig. 1 shows the area under the emission curve between 0 and 300 s (these areas are not normalized, blank areas # 1, without enhancer) against pH of the p-phenols that showed enhancer effects and aniline. At pH > 8.6, chemiluminescence intensity maxima of the blank had a very high relative standard deviation (RSD), in some cases greater than 50%. In this case, we have preferred
(2)
(luminol radical)
HRP-II + LH- + HRP + L.-
I
,’
4’
60
(enzyme intermediate-II)
’
,A
(3)
/
1.b. Enhanced. HRP-I + EH + HRP-II + E. + HzO, (enhancer)
(4)
(enhancer radical)
HRP-II + EH + HRP + E.
(5)
E. + LH- +EH
(6)
+ L.-.
2. L.- + 02 +L + oz.-,
(7)
(Ion radical superoxide) L-- + oz.-
+ Lo*2-,
(8)
LOz2- + AP2-*,
(9)
(excited 3-aminophthalate
dianion)
AP2-* --, AP2- + hv(425 nm), (3-aminophthalate
10
6
(luminol endoperoxide)
(10)
dianion)
We could explain some of the enhancer or inhibition effects of the compounds studied here using this mechanism. The interpretation of other phenomena are not yet clear. Phenol derivatives with very activating substituents, such as hydroquinone and p-methoxyphenol,
12
PR Fig. 1. Normalized areas (blank = 1, without enhancer) beneath the emission curves of chemiluminescence between 0 and 300 s against pH, for the luminol-horseradish peroxidase-H,O, system. (0) Unenhanced (blank, without enhancer), relative standard deviation (RSD) in the range 157.8%; (V) p-hydroxymethylbenzoate, RSD in the range 0.46 to 11.5%; (A) phydroxybenzaidehide, RSD in the range 1.6 to 15%; (A). phydroxybenzoic acid, RSD in the range 1.7 to 13.6%; (V). p-coumaric acid, RSD in the range 0.09 to 4.3%; (0). Phenol, RSD in the range 2.1-16.4%; (m). p-Cresol, RSD in the range 2.7-37.6%; (0) Aniline, RSD 1.4% to 12.3%. Experimental conditions: [luminol] = 66.7 pM, [HZO,] = 2 mM, [buffer] = 33 mM, [peroxidase] = 1.2 U/ml, [p-coumaric acid] = 6.7 uM and [other p-phenol derivatives] = 33.3 pM.
F.G. Sanchez et al. 1 Journal cfLumine.wence
to measure the areas under the light emission curves because their maxima RSD were about 10%. Emission maxima were in the pH range 7.5 to 9.5; we chose pH 8.5 to study the chemiluminescence against enhancer concentration. Fig. 2 shows the normalized area (blank area = 1, without enhancer) beneath the emission curve and the normalized intensity at 300 s (blank intensity at 300 s = 1, without enhancer) against the enhancer concentration at pH 8.5. This figure shows that these new enhancers (phenol, p-hydroxybenzoic acid, p-hydroxymethylbenzoate, phydroxybenzaldehyde and aniline) increased between 2 and 40 times the blank areas between 0 and 300 s, or increased between 1.5 and 30 times the blank intensity at 300 s. We think these signals could be used in analytical determinations of phenol, aniline, p-hydroxybenzoic acid, p-coumaric acid, etc., and in chemiluminescent immunoassays [8&10], etc. Also inhibition effects could be used in analytical methods to determine the inhibitor [ 191, e.g., p-methoxyphenol. We observe in Figure 2 that compounds with deactivating substituents (p-hydroxybenzoic acid,
40
65 (1995) 33-39
31
p-hydroxybenzaldehyde and p-hydroxymethylbenzoate) had a lower enhancer effect at low concentration than phenol and p-cresol (with an activating substituent), but at higher concentrations, p-hydroxybenzoic acid, p-hydroxybenzaldehyde and phydroxymethylbenzoate were greater enhancers than phenol and p-cresol. Also, we observed that the enhancer effect is more important with phenol that with aniline at low concentration (concentration lesser than 3 x lo-’ M), but at higher concentration aniline produced more enhancement than phenol. It is important to emphasize that the concentration of p-coumaric acid (4-hydroxycinnamic acid) studied had a maximum of 1.7 x 10m6 M (in Fig. 2) because the high intensity of the signal that suddenly decreases, causes the photomultiplier to approach saturation at about 6.7 x 1O-6 M. Therefore, p-coumaric signal against concentration in Fig. 2 was only studied up to 1.7 x lo-’ M. This compound has the greater enhancer effect at low concentration, but at higher concentrations we have not studied it. The -NOz, -CHO and COOH groups are deactivating substituents whereas the group -CH3 is an
+i,
,‘> ~. /’
_~A ~- ..
P
20 / lo
+C_.-----, -cd _ __ __ _.._--A
0 1.; 0
200 [Phenol
10
400 deriv.]
20
30
600 x 10 ’
M
200 [Phenol
400 deriv.]
600
800
1000
x 10 5 M
Fig. 2. Normalized areas (blank = I, without enhancer) under chemiluminescence emission curves, between 0 and 300 s against enhancer concentration (left); and Intensity at 300 s against enhancer concentration (right); for the luminol-peroxidase-H,0, system at pH = 8.5: (0) p-hydroxybenzaldehide; (0) p-hydroxymethylbenzoate; (V) p-hydroxybenzoic acid; (A) aniline; (V phenol; (0) p-cresol; (W) p-coumaric acid. Experimental conditions: [luminol] = 66.7 FM, [H,O,] = 2 mM, [tris-HCI buffer] = 33 mM at pH = 8.5, [peroxidase] = I.2 UlmL.
38
F.G. Sanchez ei al. /Journal
activating substituent of the benzene ring. Table 1 and Fig. 2 show that according to the pKa values of phenols the concentration of acidic forms (Ar-OH) in tris-HCl buffer at pH 8.5 increased in p-nitrophenol < p-hydroxybenzalorder: the acid < phenol < pdehyde < p-hydroxybenzoic cresol. At this pH, the enhancer effect increased in the same order. Table 1 and Figure 2 show that phenol and p-cresol had greater enhancer effects (at concentration < 6.7 x lO-‘j M) than p-hydroxybenzoic acid, p-hydroxymethylbenzoate and p-hydroxybenzaldehyde. The ratios between the acidic and basic forms of phenol and p-cresol at pH 8.5 were 25:l and 47:l respectively, whereas of p-nitrophenol, p-hydroxybenzaldehyde and p-hydroxybenzoic acid were 1:22, 1:7 and 7:l respectively. The enhancer effect increased with the pKa value because the concentrations of phenol derivatives in acidic form (Ar-OH) increased. This could be explained because horseradish peroxidase reacts only with the acidic form (Ar-OH) to give a phenoxy radical, in the stages 4 and 5, and not with the basic form (Ar-O-) and so this fact obstructs stage 6. Fig. 2 shows that p-cresol had greater synergy than phenol when the enhancer concentration increased, and the optimal p-cresol concentrations (enhancer concentration with maximal enhancement) was less than the optimal phenol concentration. Similar results are reported for o-cresolphthalein against phenolphthalein, o-tolidine against benzidine and cresol red against phenol red [20]. Also, it is known that the benzil radicals (Ph-CH2.) are very stable, and that horseradish peroxidase catalyzed the hydroxylation of benzil methyl groups [S]. We suggest that this might be explained by supposing that there are interactions between the enhancer radicals (Ar-0.) or hydroxyl radicals (OH.) formed, and the -CH3 groups of these enhancers, these interactions could destroy the phenoxy radicals (CH,-Ph-0.) formed. This could explain the non-enhancement effect of Ltyrosine in Table 1. Chemiluminescent emission from the unenhanced luminol-H,O,-horseradish peroxidase oxidation had a emission maximum around 425 nm. The chemiluminescence spectra were remarkably similar to the unenhanced reaction (with
of Luminescence
65 (1995) 33-39
maxima around 425 nm), although it was not possible to determine chemiluminescence emission from the enhancer alone. This is in accordance with the hypothesis that aminophthalate is the emitter and not the enhancer [S].
5. Conclusions
Phenol, p-cresol, p-hydroxybenzoic acid, p-hydroxymethylbenzoate, p-hydroxybenzaldehyde and aniline are found to be enhancers of the luminolhorseradish peroxidase-HzOz system. We observed certain relationships between the kind of substituent, character reductor and pKa values of phenol derivatives and the enhancer or inhibition effect on the luminol chemiluminescence. We think these enhancer and inhibition effects could be used in chemiluminescent immunoassays and in analytical determinations of p-coumaric acid, phenol, pcresol, aniline, p-methoxyphenol, etc. _
Acknowledgements
We thank the Comision Interministerial de Ciencia y Tecnologia (Project PB93-1006) for financial support.
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