Kinetics of Activation of Latent Mushroom(Agari cus bi sporus)

Tyrosinase by Benzyl Alcohol

Juan Carl os Esp n* and Harry J. Wi chers
Agrotechnol ogi cal Research I nsti tute (ATO-DLO), Bornsesteeg 59, P.O. Box 17,
6700 AA Wageni ngen, The Netherl ands
A l atent i soform of Agaricus bisporus tyrosi nase has been i sol ated and acti vated by benzyl al cohol ,
one of the major vol ati l e compounds i n mushrooms of thi s genus. The progress curve that descri bes
the acti vati on process reached the steady-state rate (V
ss
) after a l ag peri od (). The rate of acti ve
tyrosi nase formati on was cal cul ated by coupl i ng the oxi dati on of o-di phenol s to the acti vati on process.
V
ss
depended on benzyl al cohol , o-di phenol , and l atent tyrosi nase concentrati ons. The l ag peri od
depended on benzyl al cohol concentrati ons but not on o-di phenol and enzyme concentrati ons. The
si ze of the l atent mushroom tyrosi nase was 67 kDa, determi ned by SDS-PAGE and Western bl otti ng
assays. Thi s si ze was not modi fi ed after acti vati on by benzyl al cohol . The presence of a l ag peri od
and the l ack of change of the mol ecul ar mass of the protei n after acti vati on coul d i ndi cate a sl ow
conformati onal change of the protei n to render the fi nal acti ve form. The val ues of the ki neti c
constants V
max
and K
m
on the o-di phenol s 4-tert-butyl catechol , L-DOPA, and dopami ne were di fferent
between the l atent tyrosi nase acti vated by benzyl al cohol and the commerci al tyrosi nase. They mi ght
i ndi cate that a di fferent fi nal acti ve tyrosi nase, dependi ng on the acti vator used, coul d ari se.
Keywords: Agaricus; activation; benzyl alcohol; latent; mushroom; tyrosinase
I NTRODUCTI ON
Tyrosi nase or pol yphenol oxi dase (EC 1.14.18.1, PPO)
as present i n pl ant ti ssues pl ays an i mportant rol e i n
the qual i ty of frui t and vegetabl e processi ng and duri ng
storage of the processed foods. Preventi on of browni ng
i n foods, enzymati c or nonenzymati c, has l ong been the
concern of food sci enti sts (Dawl ey and Fl urkey, 1993;
Kahn and Zaki n, 1995; Esp n et al ., 1998a). Tyrosi nase
i s a copper-contai ni ng enzyme that, i n the presence of
mol ecul ar oxygen, catal yzes two di fferent reacti ons: the
hydroxyl ati on of monophenol s to o-di phenol s (monophe-
nol ase acti vi ty) and the oxi dati on of o-di phenol s to
o-qui nones (di phenol ase acti vi ty), whi ch, i n turn, non-
enzymati cal l y pol ymeri ze to render brown, bl ack, or red
pi gments (mel ani ns) (Prota, 1988; Mart nez and Whi -
taker, 1995).
The enzyme tyrosi nase can be found as ei ther l atent
or acti ve form (Whi taker, 1995). Latent tyrosi nase
represents >95% of total tyrosi nase acti vi ty i n mush-
rooms (Yamaguchi et al ., 1970; van Leeuwen and
Wi chers, 1999). Acti ve tyrosi nase i s the major factor
responsi bl e i n the enzymati c browni ng of mushrooms,
causi ng severe economi cal l osses to mushroom growers.
Latent tyrosi nase from many sources can be acti vated
by di fferent treatments or agents, whi ch i ncl ude deter-
gents (Moore and Fl urkey, 1990; Nel l ai appan and
Sugumaran, 1996), aci d shock (Kenten, 1957), fatty
aci ds (Sugumaran and Nel l ai appan, 1991), al cohol s
(Asada et al ., 1993), proteases (Ki ng and Fl urkey, 1987;
Robi nson and Dry, 1992; Chosa et al ., 1997), and
pathogen attack (Sol er-Ri vas et al ., 1997).
The ai m of the work presented here i s to study the
ki neti cs for the acti vati on of a l atent mushroom tyro-
si nase by benzyl al cohol , one of the most abundant
endogenous vol ati l e compounds i n Agaricus bisporus
mushroom.
MATERI ALS AND METHODS
Reagents. 4-tert-Butyl catechol (TBC), L-DOPA, dopami ne,
and tyrami ne were purchased from Si gma (St. Loui s, MO). Al l
other reagents were of anal yti cal grade and al so suppl i ed by
Si gma. Mi l l i -Q system (Mi l l i pore Corp., Bedford, MA) ul tra-
pure water was used throughout thi s research.
Preparation of Commercial Tyrosinase. Fl uka (1200
uni ts/mg) tyrosi nase was puri fi ed by usi ng an ani on exchange
col umn (DEAE-Sepharose Fast Fl ow, l ength )75 cm, di ameter
) 5 cm; Pharmaci a, Uppsal a, Sweden). The col umn was
equi l i brated wi th 20 mM Bi s-Tri s buffer (pH 6). A stepwi se
gradi ent of i ncreasi ng sodi um chl ori de (NaCl ) concentrati ons
was appl i ed (3 mL/mi n). One major i soform wi th an i soel ectri c
poi nt of 4.3 was i sol ated. Fracti ons wi th thi s i soform, di al yzed
and concentrated, were used as the source of commerci al
enzyme.
Preparation of aLatent MushroomTyrosinase. Boxes
wi th U1-spawned compost and casi ng soi l from the Mushroom
Experi mental Stati on (Horst, The Netherl ands) were trans-
ported after bud i ni ti ati on to a cl i mate room at ATO-DLO (18
C, 80% rel ati ve humi di ty). Mushrooms were harvested at
stage 5 and frozen i n l i qui d ni trogen i mmedi atel y after pi cki ng.
The devel opmental stage was determi ned accordi ng to the
procedure of Hammond and Ni chol s (1976). The freeze-dri ed
mushrooms were ground under l i qui d ni trogen to a fi ne powder
wi th a mortar and pestl e. The powder was rehydrated wi th
10 mM sodi um phosphate buffer (PB) contai ni ng 10 mM
ascorbi c aci d and mi xed thoroughl y on a vortex shaker. Thi s
homogenate was then extracted for 25 mi n on i ce and
centri fuged at 12000g for 10 mi n.
The supernatant was i mmedi atel y appl i ed to the same
col umn above-descri bed. The col umn was al so equi l i brated
wi th 20 mM Bi s-Tri s buffer (pH 6). A stepwi se gradi ent of
i ncreasi ng sodi um chl ori de (NaCl ) concentrati ons was appl i ed
(3 mL/mi n). The di fferent fracti ons were assayed wi th TBC i n
* Author to whom correspondence shoul d be addressed (fax
31-317-475347; e-mai l J.C.ESPI N@ato.dl o.nl ).
3503 J. Agric. Food Chem. 1999, 47, 35033508
10.1021/jf981334z CCC: $18.00 1999 American Chemical Society
Published on Web 08/17/1999
the absence and presence of SDS to di scri mi nate between
acti ve and l atent tyrosi nase i soforms. A major l atent i soform
wi th an i soel ectri c poi nt of 5.6 (as determi ned by anal yti cal
i soel ectri c focusi ng, resul ts not shown) was el uted at 50 mM
NaCl concentrati on. The proporti on of l atent tyrosi nase (100%)
was determi ned by compari ng the acti vi ti es i n the presence
and absence of SDS. Thi s l atent tyrosi nase i soform showed a
band of 67 kDa determi ned by SDS-PAGE and Western
bl otti ng anal ysi s (Fi gure 1). The exi stence of thi s 67 kDa l atent
mushroom tyrosi nase has not been previ ousl y reported. Thi s
fi ndi ng agrees wi th the previ ousl y reported putati ve tyrosi nase
cDNA cl one, whi ch encoded a protei n of 64 kDa (Wi chers et
al ., 1995; van Gel der et al ., 1997).
Electrophoresis.SDS-PAGE experi ments were performed
under denaturi ng condi ti ons i n 10% pol yacryl ami de gel s wi th
a mi ni gel Bi o-Rad system. Sampl es were di l uted wi th 50 mM
TCB (pH 7), contai ni ng 0.5 mM -mercaptoethanol , 2% SDS,
1% bromophenol bl ue, and 10% gl ycerol . El ectrophoresi s was
conducted at a constant vol tage of 200 V i n a buffer (pH 6.8)
contai ni ng 3 g/L Tri s base, 14.4 g/L gl yci ne, and 1 g/L SDS.
For mol ecul ar mass determi nati on, the cal i brati on ki t of SDS-
PAGE standards (l ow range of M
r) from Bi o-Rad was used.
After el ectrophoresi s, the gel s were equi l i brated for 30 mi n at
4 C i n transfer buffer (TB) contai ni ng 3 g/L Tri s base, 14.4
g/L gl yci ne, and 20%methanol (v/v). The gel s were then bl otted
onto a PVDF membrane for 1 h at a constant vol tage of 100 V
i n a Mi ni Trans-Bl ot el ectrophoreti c transfer cel l (Bi o-Rad).
The ECL protocol (Amersham I nt., Engl and) was fol l owed to
devel op tyrosi nase bands on the gel . Thi s method i s based on
l i ght emi ssi on for detecti on of i mmobi l i zed speci fi c anti gens.
After el ectrobl otti ng, the membrane was ri nsed i n TBS and
i ncubated i n the bl ock sol uti on (l ow-fat dri ed mi l k) for 1 h.
After i ncubati on, the membrane was washed i n TTBS for 25
mi n and i ncubated wi th the fi rst anti body (anti -AbPPO)
di l uted i n TTBS sol uti on for 2 h. After i ncubati on wi th anti -
AbPPO, the membrane was washed i n TTBS for 25 mi n and
then i ncubated wi th the secondary anti body (HRP-Ab) di l uted
i n TTBS sol uti on for 1 h. After i ncubati on wi th HRP-Ab, the
membrane was washed i n TTBS for 35 mi n. To detect the
bands, the reagents of the ki t were mi xed (1:1) accordi ng to
the manufacturers i nstructi ons. After 1 mi n of i ncubati on, the
membrane was i mmedi atel y exposed to a photographi c fi l m
for 1 mi n.
Enzymatic Assays. Tyrosi nase acti vi ty on TBC was de-
termi ned at 400 nm by measuri ng the accumul ati on of 4-(tert-
butyl )benzo-1,2-qui none (TBQ). Thi s o-qui none was hi ghl y
stabl e at every pH assayed and for a l onger peri od than those
used i n the acti vi ty measurements (Wai te, 1976; Ros et al .,
1994a). The fi nal vol ume of assay was 1 mL. One uni t of acti ve
form of tyrosi nase was defi ned as the amount of the enzyme
that produces 1 mol of TBQ per mi nute.
Tyrosi nase acti vi ti es on L-DOPA and dopami ne were de-
termi ned by measuri ng dopachrome and dopami nechrome
accumul ati ons at 475 and 480 nm, respecti vel y (Ros et al .,
1994b).
N,N-Di methyl formami de (DMF) (1%, v/v) was added to the
assay medi um to enhance the sol ubi l i ty of benzyl al cohol (BA).
Thi s proporti on of DMF di d not al ter ki neti c properti es of
tyrosi nase (Esp n et al ., 1995a, 1997a, 1998b).
The spectrophotometri c assays were recorded i n an ul tra-
violet-visible Perkin-Elmer Lambda-2 spectrophotometer (U ber-
l i ngen, Germany), on-l i ne i nterfaced to a Penti um-100 mi cro-
computer (Ede, The Netherl ands). Temperature was control l ed
at 25 C wi th a ci rcul ati ng bath wi th a heater/cool er and
checked usi ng a preci si on of (0.1 C.
Kinetic Data Analysis.The val ues of Km and Vmax were
cal cul ated from tri pl i cate measurements of the steady-state
rate, Vss, for each i ni ti al substrate concentrati on ([S]0). The
reci procal s of the vari ances of Vss were used as wei ghti ng
factors to the nonl i near regressi on fi tti ng of Vss versus [S]0 to
the Mi chael i s equati on (Endrenyi , 1981). The fi tti ng was
carri ed out by usi ng a Gauss-Newton al gori thm (Marquardt,
1963) i mpl emented i n the Si gma Pl ot 2.01 program for
Wi ndows (Jandel Sci enti fi c, 1994).
Other Methods. Protei n content was determi ned by usi ng
the method of Bradford (1976) usi ng bovi ne serum al bumi n
as standard.
RESULTS AND DI SCUSSI ON
The acti vati on of l atent tyrosi nase by some al cohol s
has been previ ousl y reported (Asada et al ., 1993).
However, the acti vati on of l atent mushroom tyrosi nase
by vol ati l e compounds such as BA has not been previ -
ousl y publ i shed.
The acti vati on of l atent mushroom tyrosi nase by BA
was characteri zed by the presence of a l ag peri od ()
pri or to the attai nment of the steady-state rate (V
ss
)
(Fi gure 2A). The mol ecul ar wei ght of the l atent tyrosi -
nase (67 kDa) di d not change after acti vati on by BA
(Fi gure 1). The presence of the transi ent phase i n the
acti vati on process suggested that the acti vati on coul d
take pl ace through a sl ow conformati onal change of the
enzyme to render the acti ve tyrosi nase. Moreover, V
ss
depended on BA concentrati ons wi th a si gmoi d pattern
(Fi gure 2B), whi ch al so happens i n the acti vati on of
other l atent tyrosi nases by SDS (Moore and Fl urkey,
1990; Nel l ai appan and Sugumaran, 1996). Thi s coul d
i ndi cate a possi bl e detergent-l i ke character of BA, whi ch
coul d expl ai n the acti vati on of the l atent enzyme by
means of a conformati onal change of the protei n, si mi l ar
to that caused by SDS. The l ag peri od of the acti vati on
process di mi ni shed i n a si gmoi d way wi th i ncreasi ng
BA concentrati ons. decreased to zero at certai n BA
concentrati ons, but a concomi tant decrease of V
ss
was
obtai ned (Fi gure 2A,B).
The opti mum BA concentrati on ([BA]
opt
]) to acti vate
the l atent enzyme vari ed i n a si gmoi d manner wi th pH.
The [BA]
opt
was maxi mum, 0.3 M (3% of BA v/v), at pH
>6.5 (Fi gure 3) because protonati on of the l atent
enzyme by aci d shock coul d faci l i tate the acti vati on
process at l ow pH. V
ss
changed i n a si gmoi d way and
i ncreased wi th pH (Fi gure 4). The profi l e of the opti mum
pH curve was equal to that previ ousl y observed for
commerci al tyrosi nase (Esp n et al ., 1997b).
Figure1. I denti fi cati on of mushroom tyrosi nase by Western
bl otti ng on SDS-PAGE: l ane 1, markers; l ane 2, l atent
mushroom tyrosi nase (10 g/mL); l ane 3, mushroom tyrosi nase
(10 g/mL) acti vated by BA (0.3 M); l ane 4, commerci al (Fl uka)
mushroom tyrosi nase (1 g/mL). See Materi al s and Methods
for detai l s.
3504 J. Agric. Food Chem., Vol. 47, No. 9, 1999 Esp n and Wichers
V
ss
was l i nearl y dependent and remai ned constant
wi th varyi ng l atent enzyme concentrati ons (Fi gure 5).
Furthermore, V
ss
was hyperbol i cal l y dependent and
remai ned constant on o-di phenol concentrati on (Fi gure
6).
K
m
val ues changed i n a hyperbol i c way wi th pH,
whereas V
max
val ues were constant (Fi gure 7; Tabl e 1).
Thi s i s al so the characteri sti c behavi or of commerci al
tyrosi nase, whi ch meant that the protonati on of BA-
acti vated tyrosi nase at l ow pH resul ted i n hi gher K
m
val ues toward i ts substrates. Thi s fi ts to a previ ousl y
proposed reacti on mechani sm for tyrosi nases from many
sources (mushroom, frog epi dermi s, grape, appl e, pear,
avocado, arti choke, and strawberry) (Rodr guez-Lopez
et al ., 1992; Ros et al ., 1994b; Esp n et al ., 1995a,b,
1997a-f, 1998b-e).
The val ues of the ki neti c constants V
max
and K
m
were
di fferent between the BA-acti vated and commerci al
tyrosi nases (Tabl e 1). The observati ons suggest that the
fi nal acti ve tyrosi nase has di fferent ki neti c properti es
dependi ng on the acti vator. For i nstance, the ful l y acti ve
commerci al tyrosi nase has a mol ecul ar mass of 43 kDa,
l ower than that for the BA-acti vated tyrosi nase (67
kDa). BA coul d i nduce a conformati onal change i n the
l atent tyrosi nase to render the acti ve tyrosi nase. There-
fore, the mechani sm of acti vati on i s obvi ousl y di fferent
for both mushroom tyrosi nases. Thi s suggests that
di fferent acti vators render acti ve tyrosi nases wi th di f-
ferent catal yti c and affi ni ty properti es toward thei r
substrates (Tabl e 1). For i nstance, K
m
val ues for TBC
were hi gher for commerci al tyrosi nase (3 mM at pH 6.8)
than for BA-acti vated tyrosi nase (2 mM at pH 6.8).
However, K
m
val ues for both L-DOPA and dopami ne
were l ower for commerci al tyrosi nase (0.7 and 1.8 mM,
respecti vel y, at pH 6.8) than for BA-acti vated tyrosi nase
(4.7 and 5.5, respecti vel y, at pH 6.8) (Tabl e 1). Thi s coul d
be due to the presence of di fferent charges i n the acti ve
si te of the enzymes. Moreover, the sequence of V
max
Figure2. (A) Spectrophotometri c recordi ngs for the acti vati on
of a l atent mushroom tyrosi nase i soform by BA. Condi ti ons
were as fol l ows: 50 mM PB (pH 6.8), 2.5 mM TBC, 0.07 g/
mL l atent tyrosi nase, and BA (a) 0.1 M, (b) 0.2 M, (c) 0.25 M,
(d) 0.3 M, and (e) 0.35 M. (B) Dependence of V
ss (b) and (2)
on [BA]0. Condi ti ons were as i n Fi gure 2A.
Figure3. Dependence of opti mum BA concentrati on ([BA]opt)
on pH. Condi ti ons were as fol l ows: 50 mM AB (pH 5 and 5.5),
50 mM PB (pH 5.75-7.25), 2.5 mM TBC, and 0.035 g/mL
l atent mushroom tyrosi nase.
Figure 4. Dependence of Vss (b) and (2) on pH i n the
acti vati on of a l atent mushroom tyrosi nase by BA. Condi ti ons
were as fol l ows: 50 mM AB (pH 4-5.5), 50 mM PB (pH 5.75-
7.25), 2.5 mM TBC, [BA]opt at every pH, and 0.035 g/mL l atent
tyrosi nase.
Figure5. Dependence of Vss (b) and (2) on l atent tyrosi nase
concentrati on i n the acti vati on of l atent tyrosi nase by BA.
Condi ti ons were as fol l ows: 50 mM PB (pH 6.8), 2.5 mM TBC,
0.3 M BA, and 0.035-0.23 g/mL l atent tyrosi nase.
Activation of Latent Tyrosinase by Benzyl Alcohol J. Agric. Food Chem., Vol. 47, No. 9, 1999 3505
val ues (TBC > dopami ne > L-DOPA) was al ways the
same at every pH and for both tyrosi nase i soforms. Thi s
sequence coul d be expl ai ned on the basi s of a quanti ta-
ti ve effect of the ri ng substi tuent on the rate of phenol i c
compound oxi dati on catal yzed by several tyrosi nases
previ ousl y reported (Esp n et al ., 1998b-e). Thi s effect
i s rel ated to the capaci ty of the si de chai n of the di fferent
phenol i c compounds to donate el ectrons toward the
aromati c ri ng as wel l as to the mol ecul ar si ze of thi s
si de chai n (Esp n et al ., 1998b-e).
When l atent tyrosi nase was acti vated wi th hi gher or
l ower BA concentrati on than the opti mum, the ki neti c
constants V
max
and K
m
on TBC were di fferent. V
max
)
9.5 M/mi n and K
m
) 3.2 mM when the l atent enzyme
was acti vated wi th 0.35 M BA, V
max
) 1.5 M/mi n and
K
m
) 5.2 mM when acti vated wi th 0.1 M BA, and V
max
) 11.8 M/mi n and K
m
) 2 mM at the opti mum BA
concentrati on (0.3 M). Thi s i ndi cates that i f hi gher
benzyl concentrati on i s used for the acti vati on, the
unfol di ng of the enzyme i s very fast (no l ag peri od) but
the catal yti c effi ci ency of the fi nal acti ve form i s worse
(l ower V
max
and hi gher K
m
val ues) than when the
opti mum BA concentrati on i s used. At l ower BA con-
centrati on (l ong l ag peri od), the acti ve si te of the enzyme
i s al so somehow di fferent and l ess avai l abl e to the
substrate, and then the acti ve enzyme al so shows l ower
V
max
and hi gher K
m
val ues.
To our knowl edge there are no previ ous studi es i n
whi ch thi s ki neti c compari son has been carri ed out upon
acti vati on by al cohol s. However, i t i s known that there
are di fferent behavi ors of tyrosi nase i n the absence and
presence of SDS regardi ng i nhi bi tory sensi ti vi ty, ther-
mal stabi l i ty, opti mum pH, etc. (Moore and Fl urkey,
1990).
I t can be suggested that vol ati l e compounds that
natural l y occur i n mushrooms, such as BA, pl ay a rol e
i n the acti vati on of l atent mushroom tyrosi nase. How-
ever, the physi ol ogi cal si gni fi cance of the experi ments
presented here i s not readi l y apparent. Neverthel ess,
these experi ments have been conducted i n the hope that
the resul ts wi l l provi de some i nsi ghts i nto the mecha-
ni sm of the acti vati on phenomenon. Experi ments i n
modi fi ed atmospheres (resul ts not shown) coul d support
the physi ol ogi cal si gni fi cance of thi s process. I n these
experi ments, mushrooms i n BA gas atmosphere turned
brown much more qui ckl y than those wi th other vol ati l e
compounds such as acetone, methanol , or ethanol . Thi s
suggests a correl ati on of the acti vati on of l atent tyro-
si nase by BA (Fi gures 2-7) and the degree of browni ng
i n mushrooms. The hi gh BA concentrati on used i n the
i n vi tro assays (0.3 M, 3%v/v) was necessary to achi eve
rel i abl e ki neti c assay condi ti ons to descri be the acti va-
ti on process, because wi th much l ower BA concentra-
ti ons (experi ments i n modi fi ed atmospheres) i t took 2
days to detect si gni fi cant acti vati on. Assumi ng that the
formati on of BA i s correl ated to senescence, tyrosi nase
acti vati on mi ght be an event that i s i nseparabl y associ -
ated wi th senescence.
Figure6. Dependence of Vss (b) and (2) on TBC concentra-
ti on i n the acti vati on of l atent tyrosi nase by BA. Condi ti ons
were as fol l ows: 50 mM PB (pH 6.8), 0.3 M BA, 0.07 g/mL
l atent tyrosi nase, and 0.2-5 mM TBC.
Figure 7. Dependence of Vmax (b) and Km (2) on pH for the
BA-acti vated tyrosi nase i soform. Condi ti ons were as fol l ows:
50 mM AB (pH 5, 5.5), 50 mM PB (pH 5.75-7.25), 2.5 mM
TBC, [BA]opt at every pH, and 0.035 g/mL l atent tyrosi nase.
Table 1. Values of the Kinetic Constants Vmax and Km for
BA-Activated and Commercial MushroomTyrosinase
Isoforms
a
BA-acti vated i soform commerci al tyrosi nase
substrate pH
Vmax
(M/mi n) Km (mM)
Vmax
(M/mi n) Km (mM)
TBC
b
5 11.8 ( 0.81 4.7 ( 0.31 7.9 ( 0.45 5.6 ( 0.43
L-DOPA
c
1.4 ( 0.05 8.3 ( 0.48 1.8 ( 0.09 2.7 ( 0.20
dopami ne
c
5.5 ( 0.21 8.4 ( 0.45 7.2 ( 0.41 3.8 ( 0.38
TBC 5.5 12.1 ( 0.75 4.5 ( 0.26 8.0 ( 0.40 5.4 ( 0.41
L-DOPA 1.6 ( 0.05 8.1 ( 0.40 1.9 ( 0.10 2.5 ( 0.28
dopami ne 5.8 ( 0.30 7.9 ( 0.40 7.4 ( 0.51 3.6 ( 0.31
TBC 5.75 12.0 ( 0.79 3.5 ( 0.25 8.3 ( 0.41 5.1 ( 0.31
L-DOPA 1.5 ( 0.06 6.0 ( 0.30 1.7 ( 0.09 2.2 ( 0.21
dopami ne 5.5 ( 0.24 6.5 ( 0.42 7.3 ( 0.41 3.3 ( 0.25
TBC 6 11.8 ( 0.69 2.6 ( 0.21 8.0 ( 0.50 3.8 ( 0.41
L-DOPA 1.6 ( 0.06 5.1 ( 0.35 1.8 ( 0.08 1.2 ( 0.08
dopami ne 5.5 ( 0.25 5.8 ( 0.40 7.2 ( 0.51 2.3 ( 0.19
TBC 6.5 11.9 ( 0.89 2.1 ( 0.19 8.2 ( 0.40 3.3 ( 0.21
L-DOPA 1.4 ( 0.05 4.7 ( 0.25 1.8 ( 0.09 0.8 ( 0.41
dopami ne 5.5 ( 0.36 5.4 ( 0.35 7.1 ( 0.50 1.8 ( 0.15
TBC 6.8 11.9 ( 0.79 2.0 ( 0.17 8.0 ( 0.41 3.0 ( 0.21
L-DOPA 1.6 ( 0.05 4.7 ( 0.25 1.9 ( 0.12 0.7 ( 0.06
dopami ne 5.3 ( 0.31 5.5 ( 0.35 7.2 ( 0.41 1.8 ( 0.14
TBC 7.25 12.1 ( 0.91 1.9 ( 0.13 7.9 ( 0.42 2.9 ( 0.18
L-DOPA 1.5 ( 0.06 4.8 ( 0.31 1.8 ( 0.13 0.7 ( 0.04
dopami ne 5.4 ( 0.32 5.3 ( 0.30 7.1 ( 0.39 1.9 ( 0.15
a
(BA-acti vated enzyme): 50 mM PB (pH 6.8), [BA]opt at every
pH, 0.03 g/mL l atent tyrosi nase i soform. (Commerci al tyrosi -
nase): 50 mM PB (pH 6.8), 0.05 g/mL commerci al tyrosi nase.
b

) 400 nm (Wai te, 1976).
c
L-dopa ) 475 nm, dopami ne ) 480 nm
(Ros et al ., 1994b).
3506 J. Agric. Food Chem., Vol. 47, No. 9, 1999 Esp n and Wichers
The acti vati on of the l atent enzyme by BA mi ght occur
through a sl ow unfol di ng of the protei n to expose the
acti ve si te. Thi s i s consi stent wi th the constant mol ec-
ul ar wei ght after acti vati on and the presence of a l ag
peri od pri or to the attai nment of the steady-state rate.
ABBREVI ATI ONS USED
AB, sodi um acetate buffer; anti -AbPPO, fi rst anti body
(pol ycl onal anti body anti -tyrosi nase devel oped i n mouse);
BA, benzyl al cohol ; [BA]
0
, i ni ti al benzyl al cohol concen-
trati on; [BA]
opt
, opti mum benzyl al cohol concentrati on;
Bi s-Tri s, bi s[2-hydroxyethyl ]i mi notri s[hydroxymethyl ]-
methane; DMF, N,N-dimethylformamide; L-DOPA, L-3,4-
di hydroxyphenyl al ani ne; dopami ne, 3,4-di hydroxyphen-
ethyl ami ne; HRP-Ab, secondary anti body (anti -mouse
i mmunogl obul i n G conjugated wi th horseradi sh peroxi -
dase, devel oped i n goat); K
m
, Mi chael i s constant of acti ve
tyrosi nase toward TBC; M
r
, mol ecul ar mass; PB, sodi um
phosphate buffer; PVDF, I mmobi l on-P transfer mem-
brane, pore si ze ) 0.45 m (Mi l l i pore); SDS, sodi um
dodecyl sul fate; SDS-PAGE, sodi um dodecyl sul fate-
pol yacryl ami de gel el ectrophoresi s; , l ag peri od; TB,
transfer buffer, pH 8.3; TBC, 4-tert-butyl catechol ; TBQ,
4-(tert-butyl )benzo-1,2-qui none; TCB, tri s[hydroxy-
methyl ]ami nomethane chl orhi dri c buffer; Tri s, tri s-
[hydroxymethyl ]ami nomethane; TTBS, TCB sal i ne (pH
7.5) wi th 0.05% Tween 20; V
ss
, steady-state rate; V
max
,
maxi mum steady-state rate.
ACKNOWLEDGMENT
We are grateful to Dr. Cri sti na Sol er-Ri vas from ATO-
DLO for her assi stance i n Western bl ot assays.
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Recei ved for revi ew December 7, 1998. Revi sed manuscri pt
recei ved June 9, 1999. Accepted June 10, 1999. J.C.E. i s hol der
of Postdoctoral Grant FAI R/CT97-5004 from the European
Commi ssi on under the framework of the Agri cul ture, Agro-
I ndustry and Fi sheri es (FAI R) program.
JF981334Z
3508 J. Agric. Food Chem., Vol. 47, No. 9, 1999 Esp n and Wichers