+
+
6620 J. Am. Chem. Soc., Vol. 118, No. 28, 1996
Easton et al.
8.5 contained sodium perborate (3 mmol dm-3), luminol (0.5 mmol
dm-3), and enhancer (0.1 mmol dm-3) added from a 10 or 100 mmol
dm-3 stock in dimethyl sulfoxide. These solutions were placed in
Microfluor “B” black, flat-bottom, 96-well microtiter plates (Dynatech),
and reaction was initiated by the addition of 10 µL of a 5 nmol dm-3
solution of HRP.
The oxidation of luminol was also monitored spectrophotometrically,
using a Hewlett-Packard 8452A diode-array spectrophotometer fitted
with 1-cm quartz cells stirred magnetically. The reaction was carried
out under the same conditions as the luminescence experiments, except
for the peroxidase concentration, which was 15 times higher to
compensate for the inferior sensitivity of this detection method, relative
to the luminometry. The increase of absorbance at 520 nm was
monitored taking readings at 1 s intervals for up to 100 s. The initial
rate was determined by fitting a straight line to the initial points.
The rates of reaction of peroxidase compounds I and II with the
luminescence enhancers were determined using a stopped-flow spec-
trophotometer, Model 1705 from Applied Photophysics, by the method
described previously.9 In brief, solutions of horseradish peroxidase
compound I were prepared by mixing native (ferric) enzyme (2 µmol
dm-3) and hydrogen peroxide (2 mmol dm-3) in Tris-HCl buffer (0.05
mol dm-3) and NaCl (0.1 mol dm-3) at pH 8.5. These solutions were
rapidly mixed in the reaction cell with solutions of the phenolic
compound (20-400 µmol dm-3), and the formation of compound II
via reaction 2 was monitored by the buildup of the absorbance of
compound II at 426 nm, the isobestic point of the spectra of ferric
enzyme and compound I. The concentration of the peroxidase was
chosen to give an easily detectable absorption, and the excess of
phenolic compound assured pseudo-first-order conditions and comple-
tion of the reaction within the time scale accessible to the stopped-
flow spectrophotometer. The observed rate of formation of compound
II was proportional to the concentration of the phenolic compound,
and the rate constant for reaction 2 (kcpd-I) was determined from the
slope of the linear plots of observed rate against concentration.
Figure 1. Structures and abbreviations of the phenolic compounds
investigated in this study.
than the reaction with compound II.8,9 In view of this kinetic
behavior, it has been suggested that the enhancers act by reaction
with compound II, thereby accelerating the turnover of the
enzyme.9,10 However, this does not fully explain the observed
enhancement, and it has also been suggested that the enhancer
may act as a redox mediator between the enzyme and luminol,
i.e., it acts as the enzyme substrate and the resulting enhancer
radical reacts with luminol in an electron-transfer reaction
generating the luminol radical.2,11
In the present study, we have used pulse radiolysis to generate
the radicals of a series of phenolic compounds that acted as
chemiluminescence enhancers (Figure 1) and to monitor the
electron-transfer reactions between those phenoxyl radicals and
luminol. Those reactions were found to be reversible, and the
corresponding equilibrium constants could be determined, which
enabled the reduction potential of the phenoxyl radicals to be
calculated. In addition, the rate constants of the peroxidase
compounds I and II with the enhancers were measured by
stopped-flow spectrophotometry. On the basis of the experi-
mental results, a simple model of enhancement of chemilumi-
nescence is proposed that enables a quantitative estimation of
the efficiency of an enhancer on the basis of its redox properties.
For the determination of the rate of reaction of compound II with
the phenolic compounds, similar experiments were performed but with
the concentration of hydrogen peroxide only 2 µmol dm-3 in the
reaction mixture. Upon mixture with the phenolic compound (10-
200 µmol dm-3 in the reaction mixture), compound II was formed,
and its subsequent decay was monitored by the absorbance at 426 nm.
This rate was also proportional to the concentration of the phenolic
compound, and the kcpd-II was determined from the slope of the linear
regression.
Pulse radiolysis was performed with a 4 MeV van de Graaff
accelerator as described previously.13 Pulses of 10 ns were used which
delivered doses of ca. 1 Gy, as determined by thiocyanate dosimetry.14
The solutions were prepared in 10 mmol dm-3 phosphate buffer at pH
8.5 and contained 0.05 mol dm-3 sodium azide and variable concentra-
tions (up to 1 mmol dm-3) of luminol and/or the phenolic compound.
Before irradiation, these solutions were saturated with oxygen-free
nitrous oxide (from British Oxygen Company). All radiolysis experi-
ments were performed at room temperature (22 ( 2 °C). Under these
Experimental Section
•
conditions, the pulse of electrons generates the azidyl radical (N3 ) in
Horseradish peroxidase (HRP-4B) was purchased from Biozyme.
4-Hydroxy-3-[3-(4-hydroxyphenyl)-1-oxo-2-propenyl]-2H-1-benzopy-
ran-2-one (HHBP) was prepared according to the method of Oyama et
al.12 Other phenolic compounds were purchased from Aldrich or
Sigma, and further chemicals and buffers were from BDH.
less than 1 µs and with a radiation chemical yield of ca. 0.6 µmol J-1
(refs 15 and 16).
Results
The determination of the chemiluminescence enhancement activity
of the phenolic compounds was performed in an Amerlite plate reader
from Amersham International, set at a dwell time of 0.2 s/well. The
reaction mixture (90 µL) prepared in 0.1 mol dm-3 borate buffer at pH
(i) Enhancement of Chemiluminescence. The phenolic
compounds investigated in the present study increased the
intensity of the chemiluminescence resulting from the HRP-
catalyzed oxidation of luminol. The enhancement efficiency
(f) was measured by the maximum intensity relative to the
system without enhancer, as listed in Table 1.
(8) Cormier, M. J.; Prichard, P. M. J. Biol. Chem. 1968, 243, 4706-
4714.
(9) Vlasenko, S. B.; Arefyev, A. A.; Klimov, A. D.; Kim, B. B.; Gorovits,
E. L.; Osipov, A. P.; Gavrilova, E. M.; Yegorov, A. M. J. Biolumin.
Chemilumin. 1989, 4, 164-176.
(13) Candeias, L. P.; Everett, S. A.; Wardman, P. Free Radical Biol.
Med. 1993, 15, 385-394.
(14) Tabata, Y. Pulse Radiolysis; Tabata, Y., Ed.; CRC Press: Boca
Raton, 1990.
(15) Alfassi, Z. B.; Schuler, R. H. J. Phys. Chem. 1985, 89, 3359-3363.
(16) Alfassi, Z. B.; Harriman, A.; Huie, R. E.; Mosseri, S.; Neta, P. J.
Phys. Chem. 1987, 91, 2120-2122.
(10) Hodgson, M.; Jones, P. J. Biolumin. Chemilumin. 1989, 3, 21-25.
(11) Thorpe, G. H. G.; Kricka, L. J. In Bioluminescence and Chemi-
luminescencesNew PerspectiVes; Scholmerich, J., Andreesen, R., Kapp,
A., Ernst, M., Woods, W. G., Eds.; John Wiley: Chichester, U.K., 1987;
pp 199-208.
(12) Oyama, Y.; Hosaka, S.; Makino, T. US Patent No. 5,206,149, 1993.