Kinetics of oxidation of benzyl alcohols by the dication and radical cation of ABTS. Comparison with laccase-ABTS oxidations: An apparent paradox

Article abstract of DOI:10.1039/b504199f

Laccase, a blue copper oxidase, in view of its moderate redox potential can oxidise only phenolic compounds by electron-transfer. However, in the presence of ABTS (2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) as a redox mediator, laccase reacts with the more difficult to oxidise non-phenolic substrates, such as benzyl alcohols. The role of ABTS in these mediated oxidations is investigated. Redox interaction with laccase could produce in situ two reactive intermediates from ABTS, namely ABTS⁺⁺ or ABTS ^?+. These species have been independently generated by oxidation with Ce(IV) or Co(III) salts, respectively, and their efficiency as monoelectronic oxidants tested in a kinetic study towards a series of non-phenolic substrates; a Marcus treatment is provided in the case of ABTS ⁺⁺. On these grounds, intervention of ABTS⁺⁺ as a reactive intermediate in laccase-ABTS oxidations appears unlikely, because the experimental conditions under which ABTS⁺⁺ is unambiguously generated, and survives long enough to serve as a diffusible mediator, are too harsh (2 M H₂SO₄ solution) and incompatible with the operation of the enzyme. Likewise, ABTS^?+ seems an intermediate of limited importance in laccase-ABTS oxidations, because this weaker monoelectronic oxidant is unable to react directly with many of the non-phenolic substrates that laccase-ABTS can oxidise. To solve this paradox, it is alternatively suggested that degradation by-products of either ABTS ⁺⁺ or ABTS^?+ are formed in situ by hydrolysis during the laccase-ABTS reactions, and may be responsible for the observed oxidation of non-phenolics. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b504199f

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A R T I C L E
Kinetics of oxidation of benzyl alcohols by the dication and radical
cation of ABTS. Comparison with laccase–ABTS oxidations: an
apparent paradox
Barbara Branchi, Carlo Galli* and Patrizia Gentili*
Dipartimento di Chimica and IMC-CNR Sezione Meccanismi di Reazione, Universit a` ‘La
Sapienza’, P. le A. Moro 5, I-00185, Roma, Italy. E-mail: carlo.galli@uniroma1.it;
Fax: +39 06 490421
Received 23rd March 2005, Accepted 17th May 2005
First published as an Advance Article on the web 10th June 2005
Laccase, a blue copper oxidase, in view of its moderate redox potential can oxidise only phenolic compounds by
ꢀ
electron-transfer. However, in the presence of ABTS (2,2 -azinobis(3-ethylbenzthiazoline-6-sulfonate) as a redox
mediator, laccase reacts with the more difficult to oxidise non-phenolic substrates, such as benzyl alcohols. The role
of ABTS in these mediated oxidations is investigated. Redox interaction with laccase could produce in situ two
++
•+
reactive intermediates from ABTS, namely ABTS or ABTS . These species have been independently generated by
oxidation with Ce(IV) or Co(III) salts, respectively, and their efficiency as monoelectronic oxidants tested in a kinetic
++
study towards a series of non-phenolic substrates; a Marcus treatment is provided in the case of ABTS . On these
++
grounds, intervention of ABTS as a reactive intermediate in laccase–ABTS oxidations appears unlikely, because the
++
experimental conditions under which ABTS is unambiguously generated, and survives long enough to serve as a
diffusible mediator, are too harsh (2 M H SO solution) and incompatible with the operation of the enzyme.
2
4
•
+
Likewise, ABTS seems an intermediate of limited importance in laccase–ABTS oxidations, because this weaker
monoelectronic oxidant is unable to react directly with many of the non-phenolic substrates that laccase–ABTS can
++
•+
oxidise. To solve this paradox, it is alternatively suggested that degradation by-products of either ABTS or ABTS
are formed in situ by hydrolysis during the laccase–ABTS reactions, and may be responsible for the observed
oxidation of non-phenolics.
Introduction
difficult to oxidise but more abundant (80%) functional groups
14,20–24
of lignin, such as the benzyl alcohol subunits (Scheme 1).
The interaction of sterically demanding substrates with enzymes
may be unfeasible whenever access to the active site is impeded.
Under these circumstances some redox enzymes resort to
1
mediators. These small-sized compounds perform as electron
shuttles and enable communication between the enzyme and
2
a bulky substrate. For example, lignin peroxidase (LiP) is a
heme-enzyme endowed with a redox potential of 1.4 V vs.
Scheme 1 The catalytic cycle of a laccase–mediator oxidising system.
3,4
5
NHE, and its natural substrate is the biopolymer lignin; a
direct enzyme–substrate interaction is prevented by the small-
Because laccase is more readily available and easier to
manipulate than LiP or other delignifying enzymes, laccase–
4b,6
sized active site of LiP. Veratryl alcohol (3,4-dimethoxybenzyl
alcohol; VA), a secondary metabolite of the LiP-producing
mediator catalytic systems begin to find biotechnological ap-
7
25–27
Phanerochaete chrysosporium fungus, acts as a redox mediator.
plications in selective organic transformations,
in textile dye
28
29,30
VA is oxidised to radical cation by LiP, and then diffuses away
from the active site and concurs to the oxidative biodegradation
of lignin by enabling the transport of electrons between substrate
bleaching, in bioremediation of soils and water,
or also
for an environmentally-benign delignification of wood pulp for
2
0–22,31–34
paper manufacture.
8–12
ꢀ
and enzyme.
Other examples of redox mediators can be found in the case of
ABTS (i.e., 2,2 -azinobis(3-ethylbenzthiazoline-6-sulfonate)
was the first to be employed among the mediators of
13–15
1,2,20,21,24
redox catalysis of electrochemical reactions,
where suitable
laccase.
This water soluble compound undergoes one-
species are selectively oxidised at the electrode and then oxidise
electron oxidation to the relatively stable blue coloured radical
16,17
•+ 20,24,35–37
the desired substrate in solution.
A successful redox mediator
cation ABTS ,
at a redox potential (0.69 V vs. NHE) that
must have a redox potential compatible with the substrate it is
aimed at, but it also needs to be sufficiently stable in its oxidised
form to perform as an electron shuttle efficiently.
almost matches that of laccase (Scheme 2). Indeed, the standard
assay of the activity of laccase involves spectrophotometric
•
+
38
monitoring of the generation of ABTS with time. Further
•
+
++
Besides LiP, white-rot fungi also excrete laccase, a family of
one-electron oxidation of ABTS to dication ABTS occurs
24,35–37
‘
blue copper’ oxidases that catalyse the four-electron reduction
of O to 2H O by sequential one-electron uptake from a
suitable reducing substrate. Having a lower redox potential
electrochemically at higher potential (1.1 V vs. NHE),
but
2
2
this red-coloured species is less stable. Furthermore, oxidation
1
•+
++
of ABTS to ABTS is endoergonic for laccase.
1,18
(
0.6–0.8 V vs. NHE) than LiP, the Cu(II)-containing laccase
ABTS reportedly mediates laccase in the catalytic oxidation of
39
can oxidise uniquely the easily oxidisable phenolic subunits
of lignin (phenoloxidase activity), whose abundance is merely
benzyl alcohols by oxygen, according to Scheme 1. Table 1 gives
the yield of carbonylic products that we obtained in laccase–
19
24
2
0% among the functional groups of the biopolymer. Redox
ABTS oxidations of a few non-phenolic precursors.
mediators can expand the versatility of laccase as a delignifying
enzyme, because they enable the oxidation of other, more
The yields are moderate, but laccase would never oxidise these
substrates without the mediator. A precise assessment of the
2
6 0 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 0 4 – 2 6 1 4
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5

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Table 1 Yield of oxidation of benzylic alcohols by laccase from
For a more precise assessment of this point, it was deemed
a
•
Poliporus pinsitus and ABTS, under O
2
++
+
of first priority to generate both ABTS and ABTS in-
dependently and unambiguously, by resorting to appropriate
chemical oxidants, and to explore their reactivity per se in the
oxidation of a series of substrates spanning over a wide range of
redox potentials. This could enable a ‘redox potential boundary’
to be determined for the substrate, beyond which a genuine
b
Substrate
Yield of aldehyde or ketone (%)
3
7
•
+
++
ET oxidation is not possible by either ABTS or ABTS .
Subsequent comparison with the reactivity performances of
the laccase–ABTS system towards the same substrates could
disclose similarities between the oxidation patterns, and enable
the form of the Medox involved to be pinpointed.
c
2
2
PhCH
2
OH
2
5
The results herein reported, besides underlining the experi-
mental difficulties associated with an unambiguous generation
1
++
of ABTS , allow us to fix some points. Oxidation of many
++
non-phenolic substrates by ABTS is indeed possible, but
++
the generation of ABTS requires conditions unsuited to the
•
+
operation of an enzyme. In contrast, ABTS is unable to oxidise
many substrates that laccase–ABTS does instead oxidise. To
solve this paradox, it is suggested that degradation by-products
of the Medox species may be responsible for the oxidation of
non-phenolics by laccase–ABTS.
a
b
From ref 24. Reaction conditions: [Subst.] = 20 mM, [ABTS] = 6 mM,
−
1
◦
[
laccase] = 3 U ml , in buffered (pH 5) water solution at 25 C for
2
4 h; O initially purged in the solvent for 30 min. Product yields are
2
calculated on the molar amount of substrate, the rest of mass balance
c
being unreacted recovered substrate. Veratryl alcohol, VA.
Results and discussion
++
Generation of ABTS
The dication of ABTS (cf. Scheme 2) can be unambiguously
generated by an oxidant of appropriate strength in a two-electron
3
6
process. We employed the cerium(IV) salt Ce(SO
4
) (hereafter
2
0
43
Ce(IV); E 1.4 V vs. NHE) and, by using a 1 : 2 ABTS : Ce(IV)
molar ratio, observed a UV–vis spectrum (kmax = 520 nm, e =
4
−1
−1
3
.6 × 10 M cm ) (Figs. 1 and 2) complying with the literature
description of the red-coloured dication (kmax = 518 nm, e = 3.6 ×
0 M cm ) analogously generated by a Ce(IV) salt. The
4
−1
−1
36
1
++
absorption spectrum of ABTS is not stable, however, because
it suffers from hydrolytic cleavage.
36,44
Scheme 2 The two oxidation steps of ABTS.
•
+
structure of the intervening Medox form (whether ABTS , or
++
the stronger oxidant ABTS ) has not been reported yet. The
++
only indirect hint about ABTS being the actual Medox form
35
in laccase–ABTS reactions comes from a seminal study. The
++
ABTS species was generated electrochemically, and shown
able to oxidise the non-phenolic electron-rich veratryl alcohol
Fig. 1 [ABTS] 3.2 × 10− M, in 0.1 M citrate buffer at pH = 5.
5
0
11
•+
(
E 1.35 V vs. NHE), whereas the weaker oxidant ABTS ,
analogously generated at the electrode, could not do so. The
inference was then made that any observed oxidation of VA
by the laccase–ABTS system (cf. Table 1) would by necessity
++ 35
involve the intermediacy of ABTS . This inference was
subsequently ‘stretched’ to uphold the alleged monoelectronic
25
oxidation by laccase–ABTS of alkylbenzenes, even though
these are more difficult to oxidise than VA in electron-transfer
(
ET) routes, having redox potential in the 1.7–2.7 V vs. NHE
40
range. Consequently, although mediation of laccase by ABTS
undoubtedly occurs with suitable non-phenolic substrates that
the enzyme does not oxidise directly,
of other substrates (e.g. alkylbenzenes)
24,39
a comparable oxidation
seems decidedly un-
25,41,42
favourable from a redox standpoint. An insufficient knowledge
of the mediation phenomenon by ABTS towards laccase clearly
appears.
40
Fig. 2 [ABTS++] 4 × 10−5 M, in 2 M H
2 4
SO .
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 0 4 – 2 6 1 4
2 6 0 5

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The rate of hydrolysis considerably decreases (but does not
36
stop) in strongly acidic media. For example, by using a 2 M
++
H
2
SO
4
–MeCN 5 : 1 mixed solvent, the half-life of ABTS is
9
0 s. In contrast, in the pH 5 buffered water solution typical of
24
laccase–ABTS oxidations, no absorption band unambiguously
++
pertaining to ABTS could be detected on mixing Ce(IV) and
ABTS solutions, not even after the very short observation time
accessible to the stopped-flow spectrophotometer (15 ms). Then,
the 2 M H
2
+
SO
4
–MeCN 5 : 1 mixed solvent, where the half-life
+
of ABTS is sufficiently long, is the standard solvent in this
−
3
−1
study, and the related k(decay) of the dication (i.e., 8 × 10 s ,
◦
at 25 C) represents the background reactivity in this particular
medium. The kinetic determinations were run with the stopped-
++
flow device, and ABTS was generated by fast mixing [Ce(IV)]
−
4
−4
3
× 10 and [ABTS] 1.2 × 10 M solutions. Although the redox
data of ABTS (Scheme 2) were obtained in either acetate or
citrate buffer solutions at pH 5,
of ABTS (i.e., 1.1 V vs. NHE) is documented on increasing the
24,35,36
0
no major change in the E
++
36
++
acidity up to 1.5 M HClO
4
. We observe that in a previous study
Fig. 3 Values of kobs for the decay of ABTS as a function of [VA].
++
++
ABTS was reported to be generated by oxidation of ABTS
Inset: example of the decrease in A520 of ABTS (0.13 mM) in the
2
−
45
presence of VA (3.9 mM); the open circles are the experimental points,
with S
2
O
8
in 20% MeCN–H
2
O solution, isolated as a red-
and the line shows the computed first-order decay for kobs = 0.843 s−
1
.
brown brittle precipitate, and re-suspended in 20% MeCN for
++
oxidation experiments. In view of the fast hydrolysis of ABTS
in solutions that are not strongly acidic, the true nature of the
oxidising species in that investigation appears suspicious.
−1 −1
constant was obtained as 210 (± 6) M s . This value confirms
45
the previously determined rate constant of oxidation of VA
170 M s ) by the electrochemically generated ABTS . The
intercept in Fig. 3 is almost negligible, supporting the k
−
1
−1
++ 35
(
Kinetic study of the ET step. The monoelectronic oxidation
2
ꢁ k−1
of a series of substituted benzyl alcohols was kinetically inves-
•
+
++
[
ABTS ] assumption.
tigated by generating ABTS with Ce(IV) in the 2 M H
MeCN 5 : 1 mixed solvent, aiming at determining the electron-
transfer rate constants k (in Scheme 3). In fact, according to
the steady-state approximation,
2
SO –
4
The kinetic study was extended to a series of substituted
benzyl alcohols and ethers, presenting a range of oxidation
potential broad enough to appreciate the efficiency of electron-
1
++
abstraction by ABTS as a function of the electron-donor
propensity of the substrates. A few methoxy-substituted aro-
matic compounds were also investigated, in order to cover an
even wider range of redox potential of the substrate. The results
are reported in Table 2. Unfortunately, precise rate constants
could be determined only for a few compounds. In fact, the rate
+
+
v = −d[ABTS ]/dt
++
•+
=
k
1
k
2
[ABTS ][Subst.]/(k−1[ABTS ] + k
the kinetic system for Scheme 3 becomes:
2
)
+
+
•+
v = k
1
[ABTS ][Subst.] whenever k
2
ꢁ k−1 [ABTS ].
◦
++
The kinetic study was performed at 25 C with a stopped-
of oxidation of very electron-rich substrates by ABTS was too
flow apparatus where pre-mixing of ABTS and Ce(IV) (1 :
fast to determine with our stopped-flow device. On the contrary,
the ‘apparent’ rate constant for electron-poor substrates was so
slow to match the k(decay) of ABTS . Despite this, the reasonable
++
2
.5 molar ratio) quantitatively generated ABTS (at the initial
4
−
++
concentration of 1.2 × 10 M reported above), and then this
solution was quickly added to the chosen substrate at an initial
expectation that the ET rate (k ) is larger for the easier to oxidise
1
−
3
concentration ([Subst.] = 1.2 × 10 M or higher) that would
substrates is qualitatively met. A more quantitative relationship
between the rate of ET oxidation and thermodynamic driving
enable a pseudo-first-order treatment of the data. The decrease
++
++
in the A520 value of ABTS , during the one-electron reduction
force to the ET between donor substrates and acceptor ABTS
•
+
47
to ABTS by the substrate was time monitored.
was sought within the Marcus theory framework and ensuing
48
eqn. (1).
ꢂ
0ꢀ
2
DG = k/4 (1 + DG /k)
(1)
Unfortunately, the redox potentials of the substrates were
available in different solvents, so that a homogeneous assessment
47
of their redox power was largely hampered. More than
that, irreversible E values were often accessible because the
P
+
+
Scheme 3 Kinetic scheme of the oxidation by ABTS
.
radical cations of benzylic compounds deprotonate very fast
++
47,49–53
It must be emphasized that ABTS was quantitatively
generated from ABTS by using an almost stoichiometric amount
2.5 : 1 Ce(IV)–ABTS molar ratio) of the monoelectronic oxidant
a 2-electron process, with DE ca. 0.3 V), thereby avoiding any
excess of Ce(IV) that could oxidise the non-phenolic substrate
directly. This was not the case in previous studies, where ABTS
was generated by using a 100 fold amount of the oxidant
peroxodisulfate).
In order to check the reliability of our kinetic approach,
veratryl alcohol (VA) was investigated. The observed pseudo-
first-order rate constants of oxidation by ABTS (kobs = k [VA]),
with [VA] in the 1–4 × 10 M range, were 40–130 times faster
than the spontaneous decay of ABTS in the 2 M H SO
(k
2
in Scheme 3),
thereby preventing the determination of
0
reversibleE values withoutsophisticated techniques. Microelec-
(
(
trodes and fast sweep scans of the potential were unavailable to
0
54,55
us,
and therefore it was impossible to determine the missing
0
E data directly. We solved this problem by determining the
charge-transfer absorption band (hmCT) of the substrates.
+
+
56
45,46
0
(
Determination of hmCT and E data. Because the tendency of
a substrate to transfer an electron to an electrode is expected
to match the tendency to form an electron donor–acceptor
complex with a reference acceptor compound, the determination
++
−
3
0
of hmCT data may enable to circumvent the unavailability of E
redox potentials.
++
56–58
2
4
–
The charge-transfer (CT) absorption band
MeCN 5 : 1 mixed solvent, so to be unambiguously distinct.
(i.e., the hmCT band, obtained vs. tetracyanoethylene, TCNE) of
From the kobs vs. [VA] plot (Fig. 3), the second order rate
benzylic derivatives structurally comparable to our ones was
2
6 0 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 0 4 – 2 6 1 4

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+
+
◦
Table 2 Rate constants of oxidation (k
1
) of the substrates by ABTS at 25 C, in 2 M H
2
SO
4
–MeCN 5 : 1 mixed solvent. Literature redox potentials
11,51,57,59–61
0
(
V vs. NHE),
experimental hmCT data (eV vs. TCNE), and extrapolated E data (V, in MeCN and in H
2
O) of the substrates are reported.
++
24,35,36
0
Knowledge of the reduction potential of ABTS (1.1 V, in H
2
O)
enabled to calculate the values in the DE column, and correction for charges
0
ꢀ
solvation gave the values in the DG column (see text)
0
E exp/v vs. NHE
0
0
0
0ꢀ
−1
hmCT/eV in
E extrap/V vs.
E extrap/V vs. DE /V DG /kcal mol
−
1
−1
Substrate
k
1
/M
s
in H
1.36
2
O
in MeCN MeCN vs. TCNE NHE in MeCN NHE in H
2
O
in H
2
O
in H
2
O
2
1
7
6
10
—
—
—
—
2.16
2.19
2.19
2.02
1.60
1.63
1.63
1.48
—
−0.26
−0.26
−0.26
−0.23
6.3
4
1.36
—
6.3
6.3
5.6
7
(ca. 1.4)
1.36
—
6
1.33
—
—
1.39
1.38
1.42
—
—
—
2.68
2.22
2.58
—
—
—
—
−0.29
−0.28
−0.32
7.0
6.8
7.7
1.66
1.97
1
1
29
3
(ca. 1.4)
(ca. 1.1)
—
—
2.48
1.74
1.89
1.39
−0.29
7.1
4
b
(
ꢁ 10 )
ca. 1.2
ca. 1.3
ca.
ca. 5
−
0.2
5
4
—
—
—
1.98
2.68
2.49
3.45
—
1.39
1.49
1.35
−0.29
−0.39
−0.25
7.0
9.3
6.1
a
—
—
1
7
(ca. 1.54) 2.06
1.52
4
(
ꢁ 10 )
—
—
—
1.61
1.61
1.11
—
1.30
1.30
−0.2
−0.2
4.9
4.9
—
1.05
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 0 4 – 2 6 1 4
2 6 0 7

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Table 2 (cont.)
0
E exp/v vs. NHE
0
0
0
0ꢀ
−1
hmCT/eV in
E extrap/V vs.
E extrap/V vs. DE /V DG /kcal mol
−
1
−1
Substrate
k
1
/M
s
in H
—
2
O
in MeCN MeCN vs. TCNE NHE in MeCN NHE in H
2
O
in H
2
O
in H
2
O
4
(
ꢁ 10 )
1.36
1.91
—
1.33
−0.23
5.6
a
—
—
—
—
1.58
1.70
2.09
2.24
—
—
1.35
1.37
−0.25
−0.27
6.1
6.5
a
—
—
2.00
2.59
—
1.40
−0.30
7.3
a
The observed pseudo-first-order decay matched the value of kdecay of ABTS++, i.e. 8 × 10−3
−1
b
s
.
Solubility problems.
0
already found to correlate linearly with the reversible E redox
57
potentials (eqn. (2)).
0
E = 0.862 × hmCT (eV) − 0.012
(2)
We have extended these measurements to our substrates vs.
TCNE in MeCN solution, because experimental problems
prevented the use of the 2 M H
2
SO –MeCN 5 : 1 mixed solvent.
4
The obtained hmCT data (as hmCT = hc/kmax; data in Table 2) were
0
plotted vs. the E values available in MeCN for some of the
11,51,57,59
substrates,
and provided the experimental relationship
(
eqn. (3); plot in Fig. 4):
0
(
E )MeCN = 0.881 × hmCT (eV) − 0.299
(3)
0
From it, the missing E potentials for other substrates of the
study could be extrapolated in MeCN (values given in Table 2).
0
Fig. 5 Correlation of E (in H
2
O solution) vs. hmCT for benzyl alcohols.
4
7,48
Marcus treatment
. The electrochemical driving force to
++
the ET between acceptor ABTS and each donor substrate,
calculated as DE (in V; see Table 2) from the individual E
0
0
0
−1
values in H
2
O, was converted into DG (1 V = 23.06 kcal mol )
data. From the relationship:
0
ꢀ
0
2
DG = DG + (Z
1
Z
2
)/(Dr12)e f
(5)
0
ꢀ
the final DG data were obtained (given in Table 2). The contri-
2
bution from the electrostatic interaction term (Z
1
Z )/(Dr12)e f
2
−
1
59
(overall value: +0.3 kcal mol ) was reckoned as follows.
Z
1
is the charge of the reduced oxidant (i.e., a radical anion; see
+
37
charge of ABTS. in Scheme 2),
Z is the charge of the oxidised
2
substrate (i.e., a radical cation), the dielectric constant D was
0
taken equal to 78, while a value of −23 was used for the ratio
Fig. 4 Correlation of E (in MeCN solution) vs. hmCT for benzyl alcohols
2
and polymethoxybenzenes.
e f /r12, in keeping with the assumptions (i.e., f = 0.57, r12
=
˚
A) adopted in the monoelectronic oxidation of structurally
59
7
0
0ꢀ
The hmCT data were also plotted vs. E values available in H
for some of the compounds,
2
O
comparable ArCH Z derivatives. The DG data were finally
2
60,61
providing another experimental
used to numerically solve the Marcus equation (eqn. (1)), on the
relationship (eqn. (4); plot in Fig. 5):
basis of an appropriate value for the reorganisation barrier (k) of
the reaction, obtained as follows. Being the intrinsic barrier kox
0
(
E )
H
O
= 0.102 × hmCT (eV) + 1.14
(4)
•
2
++
+
−1 36
for the ABTS /ABTS couple reported as 27 kcal mol , and
0
•+
Once again, missing E potentials in H
from this correlation line. In the end, two sets of extrapolated
E data, one in MeCN and the other in H
were gathered. Since the E of ABTS is obtained in water
2
O could be extrapolated
being kred for a ArCH
2
Z –ArCH
2
Z couple available as 55 kcal
−
1
59
−1
mol , a mean value of 41 kcal mol , as (kox + kred)/2, gave the
0
47,48
ꢂ
2
O (see Table 2),
k value of the reaction.
The DG data accordingly calculated
0
++
0ꢀ
from (eqn. (1)) are plotted as a solid line vs. DG (Fig. 6). On
2
0,24,36
solution,
and the mixed solvent of the kinetic study
this plot, the experimental rate constant values (k
1
, in Table 2),
0
ꢂ=
approximates water better than MeCN, the E data in H
experimental and extrapolated ones) were preferred for use in
the Marcus treatment.
2
O (both
suitably converted into DG data according to the relationship
1
1
ꢂ=
k = 6 × 10 exp(-DG /RT), are represented as black circles
(᭹). These circles are indeed interpolated by the curve calculated
2
6 0 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 0 4 – 2 6 1 4

View Article Online
Scheme 4 Mechanism of the oxidation reaction.
water (Scheme 4), a 50% yield indicates quantitative conversion;
this is reasonably well verified (Table 3). It is concluded that
++
ABTS truly converts appropriate substrates into oxidation
products. Even in the presence of a ‘moderately endoergonic’ (≤
0
.3 V) ET step, the subsequent and irreversible deprotonation
+
•
of Subst drives the thermodynamically unfavourable oxidation
forward. However, this does not hold for PhCH OH, which is
2
recovered unreacted because its redox potential makes the ET
step too endoergonic.
Oxidation by ABTS•
+
•
+
Oxidation of phenols by ABTS had already been investigated
64
kinetically, but no analogous study was available for non-
phenolics (Scheme 5). By using a stoichiometric amount of
the monoelectronic oxidant potassium 12-tungstocobaltate(III)
Fig. 6 Marcus plot for the electron-transfer oxidation of non-phenolic
++
substrates by ABTS
.
0
57
•+
(
viz., Co(III)W; E 1.1 V), we generated ABTS quantitatively
as confirmed by the absorption spectrum (kmax = 420 nm, e =
.5 × 10 M cm ; Fig. 7).
according to the k = 41 kcal mol− barrier. For comparison, two
1
4
−1
−1
36,37
3
other profiles are given in Fig. 6, being calculated from a higher
−
1
−1
(
ꢀ
i.e., 60 kcal mol ; ᭺ symbols) and lower (i.e., 20 kcal mol ;
symbols) k value; it clearly appears that not any curve will
fit the experimental data correctly. The much better agreement
−
1
with the k = 41 kcal mol curve confirms the appropriateness
of the chosen Marcus parameters.
A few considerations are possible on the basis of the solid plot
of Fig. 6. For the substrates represented as black circles, rate-
•
+
++
Scheme 5 Kinetic scheme of the oxidation by ABTS .
determining monoelectronic oxidation by ABTS is viable, even
if slightly endoergonic, and the k values are in Table 2; these
substrates have E in the 1.3–1.4 V range. Substrates endowed
1
0
0
ꢂ
−1
with E < 1.3 V would have DG values > −10 kcal mol
4
−1 −1
and k > 10 M s , therefore too fast for our experimental
1
technique, as confirmed by the tetramethoxy- or trimethoxy-
derivatives in Table 2. On the other extreme, substrates endowed
0
ꢂ
−1
with E > 1.4 V would have DG values < −16 kcal mol and
< 1 M s , thereby slower than the decay rate constant of
−
1
−1
k
1
++
ABTS (cf. PhCH OH in Table 2). Monoelectronic oxidation of
2
++
such electron-poor substrates by ABTS is therefore doubtful.
This is an important point, because the laccase–ABTS oxidation
of benzyl derivatives and polycyclic aromatic hydrocarbons,
similarly endowed with redox potential well above 1.6 V, was
instead assumed to take place by ET through the ABTS
intermediate.
25
41
40
++
+
+
•
+
−5
Product analyses with ABTS as the oxidant. These were
carried out under kinetic conditions with six significant sub-
strates (see Experimental section for details).
Fig. 7 [ABTS ] 3 × 10 M, in 0.1 M citrate buffer at pH = 5.
ABTS• survives in MeCN or in buffered (pH 5) water
+
−
4
−1
Because the formation of the product requires a second oxida-
tion of the intermediate benzyl radical to a cation that reacts with
solutions much longer (half-life of 90 min; kdecay 1.3 × 10 s )
++
than ABTS did.
++
Table 3 Product analysis in the oxidations with ABTS , in 2 M H
ABTS] = 0.4, [Ce(IV)] = 1 mM
2
SO
4
–MeCN 5 : 1 mixed solvent at room temperature. Conditions: [Subst.] = 4,
[
a
Substrate
Oxidation product
Yield (%)
3
,4-Dimethoxybenzyl alcohol (VA)
Veratryl aldehyde
Veratryl aldehyde
50
36
b
Methyl ether of VA
4
3
1
-Methoxybenzyl alcohol
,4,5-Trimethoxybenzyl alcohol
,2,4-Trimethoxybenzene
4-Methoxybenzaldehyde
3,4,5-Trimethoxybenzaldehyde
Quinone(s)
40
46
n.d.
0
c
Benzyl alcohol
—
a
++
b
c
Calculated vs. the mmol of ABTS
. Minor amounts of the corresponding methyl ester are also formed (see, ref. 62). This substrate, lacking
benzylic C–H bonds, gave a mixture of products, most likely quinones (according to GC–MS inference; see ref. 63), which was not further investigated.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 0 4 – 2 6 1 4
2 6 0 9

View Article Online
Once again, VA was the first substrate examined under the
The kinetic data come out uniformly slower than those with
−
5
−5
++
conditions: [ABTS] 7 × 10 , [Co(III)W] 9 × 10 , [VA] 7 ×
ABTS by at least two powers of ten, but no simple relationship
−
◦
3
−2
0
1
2
0
− 7 × 10 M, in buffered water solution (pH 5) at
emerges between k
1
and E data. Product analysis with the
•+
5 C. Bleaching of ABTS in the presence of VA was monitored
electron-rich compounds (Table 4) supports oxidation to an
acceptable extent. In the case of less electron-rich substrates,
•
+
in the 690–730 nm range, where the spectrum of ABTS presents
a significant shoulder and there is minimal interference from
spurious absorption bands. The drop of ABTS absorption
was slower than with the stronger oxidant ABTS , so that a
conventional and not stopped-flow spectrophotometer could be
used. However, no simple pseudo-first-order kinetic behaviour
was observed, in spite of a 100 : 1 or even 1000 : 1 ratio in the
•
+
though, the drop in ABTS absorption vs. time was as slow
•
+
as the spontaneous decay of this oxidant, thus precluding
++
determination of meaningful k
1
values. Consistently, product
analysis gave no evidence of oxidation of 4-methoxybenzyl
•
+
alcohol, nor of dimethoxytoluene or benzyl alcohol by ABTS
(not given in Table 4).
•
+
[
VA] : [ABTS ] initial concentrations.
In kinetic studies of consecutive reactions (as in Scheme 5),
where the ET step is analogously endoergonic, it is necessary
Oxidations with the laccase–ABTS system. Recent X-ray
studies provide important structural details on laccase from
to take into account the full equation resulting from the steady
66a,b
Trametes versicolor,
and even for an adduct between ABTS
59,65
state approximation:
66c
and laccase from Bacillus subtilis. The substrate binding
site is a negatively charged depression on the surface of the
enzyme near the cavity where copper T1, the primary electron
•
+
•+
v = −d[ABTS ]/dt = k
1
k
2
[ABTS ][Subst.]/(k−1[ABTS] + k
2
)
1,2,66,67
By following the mathematical analysis provided by Kochi
acceptor, lies.
The electron removed from the substrate
65
•+
•+
et al., and by a non linear fit of the x = [ABTS ]/[ABTS ]
vs. t data, according to the complex biparametric equation:
0
is shuttled from T1 to the T2/T3 trinuclear Cu(II) cluster, the
site of oxygen binding and reduction, in order to regenerate
Cu(II) at T1, and enable the oxidation of a second molecule
of substrate. The negative charge in the substrate binding site
−
logx − a(1 − x) = bt
where a = (k−1C)/(k−1C + k ); b = k [Subst.]/(k−1C + k
C = [ABTS ] + [ABTS]
and k−1/k could be attained (Table 4), even
66a
2
1
k
2
2
)
is expected to stabilise the product of oxidation. This may
1
−
•
+
be a crucial point. We have determined kcat (510 s ) and
0
0
−
5
−1
−1
K
M
(1.1 × 10
M
s ) for laccase from Poliporus pinsitus
the values of k
1
2
and ABTS (see Experimental); the data confirm a substantial
enzyme–mediator affinity, and indeed laccase-generated ABTS
is clearly detectable by spectrophotometry. However, production
•
+
though this was not possible for all the substrates tested in
Table 2.
The successful substrates follow a limit kinetic behaviour
++
of ABTS by laccase would require that the initially formed
+
•
where deprotonation of the radical cation (k
the slow step, being the back-ET step faster (i.e., k−1 [ABTS] ꢁ
) and close to the diffusion limit, owing to the unfavourable
2
in Scheme 5) is
ABTS remains in the active site, in order to undergo the
second monoelectronic oxidation, instead of being turned out in
solution to enable the oxidation of a new molecule of ABTS. In
59
k
2
++
thermodynamic driving force (DE > 0.7 V) of the ET step.
Product analyses under kinetic conditions were also carried out,
and the yields are given in Table 4.
fact, enzymatic generation of ABTS from the tiny amounts of
+
•
ABTS produced in the catalytic cycle and released in solution,
•
+
seems improbable. Charge stabilisation might tie ABTS in the
a
b
•+
◦
1
Table 4 Rate constants (k ) and preparative yields for the oxidation of the substrates by ABTS , at 25 C in buffered water solution (pH 5)
−
1
−1
−1
0
c
d
Substrate
k
1
/M
s
k−1/k
2
/M
E /V vs. NHE in H
2
O
Yields (%)
7
0
5
1
.6
2 × 10
1.36
1.36
1.39
50
11
26
.2 × 10−
.3 × 10−
3
3.4 × 10
4.2 × 10
5
2
5
7
7
5
2.2 × 10
ca. 1.3
40
0
8
7
0
1
.13
15
1.8 × 10
1.39
1.33
e
5.7 × 10
quinones
a
−5
−5
−3
−2
b
Conditions: [ABTS] 7 × 10 , [Co(III)W] 9 × 10 , [Subst.] 7 × 10 − 7 × 10 M in buffered (pH 5) water solution. See Scheme 5. Conditions:
c
[
Subst.] = 2.5, [ABTS] = 0.25, [Co(III)W] = 0.30 mM in buffered water solution containing 4% MeCN for solubility reasons. From Table 2;
d
•+
e
extrapolated values in italics. Of carbonylic product vs. the molar amount of ABTS (viz. ABTS ). Qualitative result by GC-MS.
2
6 1 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 0 4 – 2 6 1 4

View Article Online
Table 5 Yields in the oxidation by laccase–ABTS, in 3 mL 0.1 M citrate buffer (at pH 5) containing 4% MeCN. Conditions: [Subst.] = 5 mM,
−
1
[
ABTS] = 0.5 mM, laccase 3 U ml , for 24 h at rt, under O
2
a
Substrate
Oxidation product
Yield (%)
3
4
,4-Dimethoxybenzyl alcohol (VA)
-Methoxybenzyl alcohol
Veratryl aldehyde
4-Methoxybenzaldehyde
—
300
70
0
Methyl ether of VA
,4,5-Trimethoxybenzyl alcohol
3
3,4,5-Trimethoxybenzaldehyde
65
a
The yields are calculated on the amount of ABTS.
++
active site, enabling the second step of oxidation to ABTS
within the enzyme. On the whole, ABTS has the charge of a
do react with some benzylic probe substrates that are chemical
69
models of lignin, yielding oxidation products consistent with
•
+
69a
dianion at pH 4–5, whereas ABTS is equivalent to a radical
the operation of an ET route: no oxidation of these particular
++
•+ 69b
•+
anion and ABTS is electrostatically neutral (cf. Scheme 2),
thus supporting an increasing affinity of these oxidised states
for the negatively charged enzymatic active site.
substrates occurs with preformed ABTS . Proposing ABTS
as the Medox species has therefore no consistent experimental
support.
Whereas on mixing suitable amounts of laccase and ABTS
Another point can be stressed. The yields of oxidation in
Table 1 were reckoned on the molar amount of substrate, whose
initial concentration is three times that of the mediator. Scheme 1
points out that the Medox species is continuously regenerated by
++
we detect no absorption bands pertaining to ABTS , the two-
++
electron oxidation of ABTS to ABTS is instead accomplished
by using a two-equivalent amount of the monoelectronic oxidant
35
Ce(IV) (or by anodic oxidation). However, this occurs only in
laccase–O
2
. In fact, yields in excess of 100% are obtained if
++
very acidic solution (2 M H
2
SO ), where ABTS survives with
4
calculated vs. the molar amount of mediator, thereby implying
24,40,69a
an half-life of 90 s. In buffered water solution at pH 5, instead, no
an oxidation process with turnover.
This is supported by
++
bands of ABTS are detected; consequently, its half-life ought
to be shorter than 15 ms, otherwise it had to be detected on
mixing Ce(IV) and ABTS in a 2 : 1 molar ratio in the stopped-
flow spectrophotometer.
the experiments reported in Table 5, where a 10 : 1 ratio of
[Subst.] : [ABTS] was employed, and the amount of laccase was
stoichiometric with respect to ABTS.
In Table 1 the yields of oxidation of a few benzylic alcohols
24
by laccase–ABTS were given. The feasibility of a monoelec-
tronic oxidation of substrates endowed with E < 1.4 V (i.e.,
Conclusions
0
excluding PhCH
2
OH) is confirmed by the present kinetic study
The phenoloxidase enzyme laccase becomes able to oxidise
non-phenolic substrates, such as benzylic alcohols, in the
presence of ABTS. Oxidation products (aldehydes or ketones)
are accordingly obtained. What is the mediating role of ABTS
(
Table 2). Therefore it would seem plausible to suggest that, in
++
the oxidations with the laccase–ABTS system, ABTS is the
reactive intermediate (Medox). We could assume that, following
the preliminary interaction with laccase, ABTS is kept within
the enzyme and further oxidised to ABTS . Electrostatic
interactions with amino acidic residues in the binding site could
stabilise both ABTS and particularly ABTS . This interaction
would protect the latter from hydrolysis, and possibly extend its
lifetime long enough to enable the monoelectronic oxidation
of electron-rich non-phenolic substrates, even though ABTS
would never accumulate in solution to extents spectrophotomet-
rically detectable (cf. Experimental). A similar protection offered
by the enzyme has been invoked by Aust et al., to explain the
•
+
•+
in these oxidations? Is it initially oxidised by laccase to ABTS ,
++
++
or else to ABTS , and subsequently either one of these reactive
intermediates carries out the non-enzymatic oxidation of the
non-phenolic substrate? In this study we have (i) underlined
the experimental difficulties associated with an unambiguous
•
+
++
++
generation of ABTS , (ii) generated it independently and
unambiguously by a chemical oxidant (Ce(IV)), and (iii) shown
it to be able to monoelectronically oxidise substrates endowed
with redox potential <1.4 V. Among these substrates is VA,
which is indeed oxidised by the laccase–ABTS system. However,
++
68
++
longer lifetime of the radical cation of mediator VA, whenever
associated to LiP, vs. the shorter lifetime of free VA in solution.
We do confirm this sort of enzymatic shielding for ABTS ,
whose spontaneous decay in buffered water solution increases
either ABTS is too short-lived a species to serve as a
diffusible mediator, or the experimental conditions under which
it survives longer are too harsh (2 M H SO as the solvent)
•
+
•
+
2
4
and incompatible with the normal operation of the enzyme. The
−
4
−1
++
from k(decay) = 1.3 × 10
s
(half-life of 90 min) when generated
(half-life of 96 h) when generated
intermediacy of ABTS appears unlikely therefore in laccase–
−
6
−1
•+
by Co(III)W, to 2 × 10
s
ABTS oxidations. In contrast, ABTS survives easily at the
by laccase (see Experimental). The relevance of this shielding,
however, implies that the oxidised mediator remains associated
with the enzyme and does not diffuse freely in solution, thereby
disrupting the very idea that ABTS performs as a diffusible
natural pH of laccase (i.e., 4–5). However, our kinetic study
•
+
with ABTS (generated by Co(III)W) shows that this weaker
ET oxidant is unable to react with substrates that laccase–
ABTS does oxidise. Binding in the active site of laccase does
not affect the redox properties of ABTS, as we establish from
20
mediator of laccase. In contrast to the measurable change in
•
+
++
•+
lifetime value of ABTS , no change in the redox potential values
of ABTS is detected on running the cyclic voltammetry in the
presence of laccase at saturation conditions (see Experimental).
Therefore interaction with the enzyme binding site has no
appreciable effect upon the propensity of ABTS to lose electrons.
an electrochemical experiment. If neither ABTS nor ABTS
can be regarded as reliable responsibles for the results obtained
with laccase–ABTS, then there would be no apparent role for
mediation! A shielding effect by the enzyme has been suggested
as being capable of protecting either one of the oxidised
derivatives of ABTS from hydrolysis, thereby extending their
lifetime and making their intervention more likely. However,
this implies that the oxidised mediator remains associated with
the enzyme, and does not operate freely in solution, thereby
•
+
When ABTS is independently generated by means of
Co(III)W, it is kinetically unable to oxidise substrates that
++
both preformed ABTS and the laccase–ABTS system do
oxidise. For example, Table 1 shows that PhCH OH is oxidised
by laccase–ABTS, even though for a meagre 2%, while 4-
methoxybenzyl alcohol reacts better (22%). There is no signif-
2
20
disrupting the concept of a diffusible mediator.
An explanation for this paradox could be that degradation
•
+
++
•+
icant oxidation of these two substrates by preformed ABTS
by-products of either ABTS or ABTS are formed in situ, and
++
++
(
cf. Table 4). Furthermore, both ABTS and laccase–ABTS
then react with non-phenolics. For example, ABTS is reported
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 0 4 – 2 6 1 4
2 6 1 1

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+
to give products of hydrolysis endowed with a redox potential >
ABTS• determined at 420 nm. A linear Lineweaver–Burke plot
44
++
−1
1
.2 V (Scheme 6), i.e., even higher than that of ABTS (1.1 V).
was thereby obtained (not shown), and the kcat (510 s ) and
−
5
−1 −1
Consequently, ‘moderately reluctant’ non-phenolics could be
K
M
(1.1 × 10
and slope, under the assumption of simple Michaelis–Menten
kinetics. Values of K clustering in a comparable range have
been reported, as an index of the affinity of laccase for other
M
s ) parameters reckoned from intercept
oxidised via an ET mechanism through the intervention of
++ 40
hydrolytic by-products of ABTS .
M
71
mediators or substrates. In contrast, it was verified that no
++
absorption bands unambiguously pertaining to ABTS can
be detected on mixing suitable amounts of laccase and ABTS
solutions, for observation times longer than 0.1 s.
Enzymatic reactions
++
Scheme 6 Products of hydrolysis of ABTS , from ref. 44.
The oxidation reactions were performed at room temperature
in stirred water solution (3 mL), buffered at pH 5 (0.1 M in
sodium citrate), containing 4% MeCN, and purged with O
Additionally, other degradation fragments of the Medox
species could be radicals, and one cannot exclude the contribu-
tion of radical oxidation routes towards very recalcitrant non-
2
24
for 30 min prior to the addition of the reagents. The initial
concentrations were: substrate (5 mM), ABTS (0.5 mM) and
40,69a,c
phenolics;
the efficiency of a radical route is in fact not
24,69a
−1
strongly affected by redox features of the substrate.
an amount of laccase (3 U ml ) almost stoichiometric with
In conclusion, ABTS-mediated oxidations with laccase, de-
spite being known and quoted as examples of the mediation
phenomenon, represent a very complex and still ambiguous case
of reactivity. No clear-cut evidence about the nature or structure
of the reactive intermediate(s) generated from interaction of the
enzyme with ABTS could be unambiguously assessed. It is likely
that, depending on the electron richness of the non-phenolic
substrate, more that one reactive intermediate and/or more than
one oxidation mechanism operate. Further studies are necessary
and in progress.
ABTS was used. After 24 h reaction time, GC analyses were
performed with respect to an internal standard (acetophenone
or p-methoxyacetophenone), suitable response factors being
determined from authentic products. The yields of oxidation
(Table 5) were reckoned vs. the molar amount of ABTS.
++
Kinetic study with ABTS
With reactive substrates the determinations were done with the
stopped-flow device, interfaced to a diode-array spectropho-
++
tometer. The ABTS species was generated by fast mixing a
−
4
3
1
× 10 M solution of Ce(SO
4
)
2
(viz. Ce(IV)) and a 1.2 ×
Experimental
−4
0
M solution of ABTS. Mixtures of H
2
SO and of MeCN
4
Instrumentation
in different proportions, or also mixtures of different solvents,
had been preliminarily tested in order to single out the medium
where the spontaneous decay of ABTS was long enough to
The HI-TECH SFA-12 stopped flow instrument was interfaced
to a HP 8453 diode array spectrophotometer; a conventional
UV–vis spectrophotometer (Perkin Elmer Lambda 18) was
alternatively used. Quartz cells of 1 cm optical path were
employed. A VARIAN 3400 Star gaschromatograph, fitted with
a 25 m × 0.25 mm methyl silicone (OV5) capillary column, was
employed in GC analyses. The identity of the reaction products
was confirmed by GC-MS analyses, run on a HP 5892 GC
equipped with a 15 m × 0.2 mm methyl silicone gum capillary
column, and coupled to a HP 5972 MSD instrument, operating
in electron impact at 70 eV.
++
run the kinetic study conveniently. The 2 M H
2
SO –MeCN 5 :
4
1
mixed solvent was the one selected for the study. In this
++
−3
−1
medium, the kdecay of ABTS was 8 × 10
s
(t1/2 = 90 s)
◦
at 25 C; it followed a kinetic first-order law, possible due to
44
cleavage of the dication molecule on interaction with water. In
solvent mixtures of lower acidity the spontaneous decay of the
dication was increasingly faster, and/or the absorption spectrum
++
of ABTS could not be attained; on the contrary, increasing the
acidity above 2 M H
2
SO
4
did not give additional advantages. In
the kinetic study, pre-mixing of the ABTS and Ce(IV) (1 : 2.5
++
molar ratio) solutions generated ABTS quantitatively (at the
General
−4
initial concentration of 1.2 × 10 M), and then this was added
Substrates and products were high purity commercial samples
to the solution of the substrate by the stopped-flow instrument.
++
(
Aldrich) and were used without further purification. Other
The bleaching of ABTS was time-recorded at 520 nm. The
3
−
precursors and products were available in the laboratory from
initial concentration of the substrate (1.2 × 10 M or higher)
enabled a pseudo-first-order kinetic treatment of the data. With
less reactive substrates, a conventional spectrophotometer was
used, the mixed solvent and the initial concentration of the
reactants being equal.
40,69
previous work.
Samples of Ce(SO
viz., Co(III)W) were employed as in previous cases.
ABTS was recrystallised from ethanol–water.
4 2
)
(viz. Ce(IV)) and of 12-tungstocobaltate(III)
24,69
(
MeCN
was HPLC grade from Carlo Erba. Buffers were prepared using
ultrapure water obtained with a MilliQ apparatus.
Kinetic study with ABTS•
+
Enzyme preparation
•
+
Due to the lower reactivity of ABTS with non-phenolic sub-
Laccase from Poliporus pinsitus was kindly donated by Novo
Nordisk Biotech; it was purified by ion-exchange chromatogra-
phy on Q-Sepharose by elution with phosphate buffer; laccase
fractions having an absorption ratio A280/A610 of 20–30 were
strates, a conventional spectrophotometer was used. The absorp-
•
+
tion spectrum of ABTS was quantitatively generated in 0.1 M
citrate buffer (pH 5) containing 4% MeCN (for solubility rea-
−
5
−5
sons) by mixing ABTS (7 × 10 M) and Co(III)W (9 × 10 M)
70
•+
considered sufficiently pure. The collected fractions were
concentrated by dialysis in cellulose membrane tubing (Sigma)
solutions. The half-life of ABTS is 90 min under these
conditions. On rapid addition of the appropriate amount of
−
1
−3
−2
against poly(ethylene glycol) to a final activity of 9000 U mL ,
as determined spectrophotometrically by the standard assay
with ABTS, which requires the determination of the initial,
the solution of substrate (in the 7 × 10 − 7 × 10 M range)
•
+
to preformed ABTS , the drop of the absorption was time-
monitored in the 690–730 nm range. No simple pseudo-first-
order kinetic behaviour was obtained, at a 100 : 1 or even 1000 :
•
+
linear increase of absorbance of ABTS at 420 nm per unit
−
1
38
•+
time per mL of purified enzyme (AU s ). In addition, this
laccase-catalysed oxidation was carried out upon varying the
concentration of ABTS, and the initial rate of appearance of
1 ratio of the [Subst.] : [ABTS ] initial concentrations, and the
kinetic treatment described in the text was followed, in order to
extract the kinetic data reported in Table 4.
2
6 1 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 0 4 – 2 6 1 4

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Determination of the spontaneous decay, and of the redox
potential of ABTS , in the presence of laccase
References
•
+
1
A. Messerschmidt, Multi-Copper Oxidases, World Scientific, Singa-
pore, 1997.
On the basis of the activity value of the purified laccase,
appropriate dilution with the 0.1 M citrate buffer enabled
to add an amount of laccase stoichiometric with 83 lmol
of ABTS in a cuvette containing 2.35 mL citrate buffer and
2 R. ten Have and P. J. M. Teunissen, Chem. Rev., 2001, 101, 3397–3413.
3
4
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84; (b) M. F. Gerini, D. Roccatano, E. Baciocchi and A. Di Nola,
0
.1 mL MeCN; the solvent mixture had been gently purged
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−
5
with O
2
. The concentration of ABTS was 3.3 × 10 M, and
5
J. K. Glenn, M. A. Morgan, M. B. Mayfield, M. Kuwahara and M. H.
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spectrophotometric observation began. After 5 min, the A420
•
+
value of ABTS , expected for that concentration and e value,
had been fully developed. Decrease of that A420 value was time-
recorded, and the kdecay value determined from curve fitting as
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3
7
8
1
−
6
−1
2
× 10 s . This is almost 70 fold slower that the spontaneous
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8
Cyclic voltammetry was carried out in acetate buffer as
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24
previously described, at a 2 mM initial concentration of
ABTS. An amount of laccase was added to the ABTS solution
that allowed full saturation: in fact, the solution turned blue.
By cyclic voltammetry we then reduced the laccase-generated
1
6860–16869.
1
1
1
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•
+
•+
ABTS to ABTS, and then re-oxidised it to ABTS and
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++
further on to ABTS . No difference in the value of the redox
potentials of ABTS could be determined in the presence of the
enzyme, with respect to the values obtained without the enzyme.
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++
Analysis of the oxidation products by ABTS , under exper-
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SO solution,
4
suitable amounts of the Ce(IV) salt (4.6 mmol) and of ABTS
++
(
2 mmol) were mixed, and the red colour of ABTS developed
4
22–426.
immediately. A MeCN solution (1 mL; 20 mmol) of the
substrate was quickly added by syringe, and the resulting
mixture kept under stirring at room temperature for 3 min,
2
3 F. Xu, J. J. Kulys, K. Duke, K. L. K. Krikstopaitis, H.-J. W. Deussen,
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++
2
2
2
4 M. Fabbrini, C. Galli and P. Gentili, J. Mol. Catal. B: Enzym., 2002,
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1
6, 231–240.
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5
27 M. Fabbrini, C. Galli, P. Gentili and D. Macchitella, Tetrahedron
•
+
ABTS were run analogously, under the following conditions.
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2
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3
3
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Determination of the hmCT data
Solutions (in MeCN) of the electron-donor substrate (5 ×
−
2
−3
34 A. M. Barreca, B. Sj o¨ gren, M. Fabbrini, C. Galli and P. Gentili,
10
M) and of the electron-acceptor TCNE (5 × 10 M)
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3
5 R. Bourbonnais, D. Leech and M. G. Paice, Biochim. Biophys. Acta,
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5
1
59
charge-transfer band of the donor-acceptor complex. The hmCT
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2 In a recent paper, duplication of the results in ref. 25 was attempted,
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support (grant QLK5-CT-1999-01277) to the OXYDELIGN
project.
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