Rabbit serum albumin hydrolyzes the carbamate carbaryl

Article abstract of DOI:10.1021/tx015577o

One of the main detoxification processes of the carbamate insecticides is the hydrolysis of the carbamic ester bond. Carboxylesterases seem to play important roles in the metabolization of carbamates. This study performs a biochemical characterization of the capabilities of rabbit serum albumin (RSA) to hydrolyze the carbamate carbaryl. Rabbit serum albumin was able to hydrolyze carbaryl with a K_cat of 7.1 × 10^-5 s^-1. The K_m for this hydrolysis reaction was 240 μM. Human, chicken, and bovine serum albumins were also able to hydrolyze carbaryl. The divalent cation Cu²⁺ at 1 mM concentration inhibited around 50% of the hydrolysis of carbaryl by RSA. Other mono- and divalent cations at 1 mM concentration and 5 mM EDTA exerted no significant effects on the hydrolysis of carbaryl by RSA. The inhibition of the carbaryl hydrolysis by sulfydril blocking agents suggests that a cysteine residue plays an important role in the active center of the catalytic activity. Both caprylic and palmitic acids were noncompetitive inhibitors of the carbaryl hydrolysis by RSA. The carboxyl ester p-nitrophenyl butyrate is a substrate of RSA and competitively inhibited the hydrolysis of carbaryl by this protein, suggesting that the hydrolysis of carbaryl and the hydrolysis of carboxyl esters occur in the same catalytic site and through a similar mechanism. This mechanism might be based on the carbamylation of a tyrosine residue of the RSA. Serum albumin is a protein universally present in nontarget species of insecticides; therefore, the capability of this protein to hydrolyze other carbamates must be studied because it might have important toxicological and ecotoxicological implications.

Full text of DOI:10.1021/tx015577o

5
20
Chem. Res. Toxicol. 2002, 15, 520-526
Ra bbit Ser u m Albu m in Hyd r olyzes th e Ca r ba m a te
Ca r ba r yl
Miguel A. Sogorb,* Victoria Carrera, M o´ nica Benabent, and Eugenio Vilanova
Divisi o´ n de Toxicolog ´ı a, Instituto de Bioingenier ´ı a, Universidad Miguel Hern a´ ndez,
Av. del Ferrocarril s/ n. 03202-ELCHE, Spain
Received October 19, 2001
One of the main detoxification processes of the carbamate insecticides is the hydrolysis of
the carbamic ester bond. Carboxylesterases seem to play important roles in the metabolization
of carbamates. This study performs a biochemical characterization of the capabilities of rabbit
serum albumin (RSA) to hydrolyze the carbamate carbaryl. Rabbit serum albumin was able
-
5
-1
to hydrolyze carbaryl with a Kcat of 7.1 × 10
m
s . The K for this hydrolysis reaction was 240
µM. Human, chicken, and bovine serum albumins were also able to hydrolyze carbaryl. The
divalent cation Cu2 at 1 mM concentration inhibited around 50% of the hydrolysis of carbaryl
by RSA. Other mono- and divalent cations at 1 mM concentration and 5 mM EDTA exerted no
significant effects on the hydrolysis of carbaryl by RSA. The inhibition of the carbaryl hydrolysis
by sulfydril blocking agents suggests that a cysteine residue plays an important role in the
active center of the catalytic activity. Both caprylic and palmitic acids were noncompetitive
inhibitors of the carbaryl hydrolysis by RSA. The carboxyl ester p-nitrophenyl butyrate is a
substrate of RSA and competitively inhibited the hydrolysis of carbaryl by this protein,
suggesting that the hydrolysis of carbaryl and the hydrolysis of carboxyl esters occur in the
same catalytic site and through a similar mechanism. This mechanism might be based on the
carbamylation of a tyrosine residue of the RSA. Serum albumin is a protein universally present
in nontarget species of insecticides; therefore, the capability of this protein to hydrolyze other
carbamates must be studied because it might have important toxicological and ecotoxicological
implications.
+
In tr od u ction
Chemical pesticides are actually widely used in agri-
ates after subneuropathic insults (organophosphates, 2,5-
hexanedione, bromophenylacetylurea, or even traumatic
axonopathy (3)), the subsequent neuropathy is promoted
or becomes more severe.
culture because they contribute to pest control in a very
effective way. The three main groups of chemical insec-
ticides are (a) organophosphorus compounds, (b) pyre-
throids, and (c) carbamates. Among all of these groups,
carbamates probably own the widest range of pesticide
activities because they are insecticides, fungicides, and
herbicides. Carbamates are widely used insecticides in
agriculture and in urban gardens. They are also effective
in controlling pest in buildings and on a wide range of
indoor plants (1).
The acute toxic effects of organophosphorus and car-
bamate pesticides are based on the inhibition of acetyl-
cholinesterase. Indeed, they are able to phosphorylate or
carbamylate a serine residue of acetylcholinesterase,
yielding an inhibited enzyme and originating the typical
cholinergic acute effects (lacrimation, salivation, miosis,
convulsions, and death) on the basis of acetylcholine
accumulation on synaptic terminals (1). However, while
the phosphorylation of the enzyme is irreversible, the
carbamylated enzyme-inhibitor complex can be hydro-
lyzed by a water molecule, yielding a free active enzyme
The selective toxicity of chemical insecticides is based
on the existence of effective detoxification enzymes in the
nontarget species (essentially mammals and birds).
Therefore, understanding the detoxification routes might
be extremely important in the risk assessment of the
carbamates and other chemical insecticides.
The two main routes for the detoxification of carbam-
ates are the oxidation and the hydrolysis of the carbamic
ester bond (1). The chemical structure of carbamate
insecticide renders these compounds susceptible to hy-
drolysis by carboxylesterases. In addition, the metabolites
resulting from the oxidation of parental carbamate still
conserve the intact carbamic bond; therefore, they can
be hydrolyzed by carboxylesterases. Thus, hydrolysis
seems to be a critical step in the detoxification route of
carbamates.
Several carboxylesterases with a capability to hydro-
lyze different carbamates have been studied in various
bacteria strains, like Blastobacter sp. (4), Arthrobacter
(5), Pseudomonas (6), Achromobacter sp. (7), and Micro-
(
1).
In addition to the described cholinergic effects, the
carbamates exhibit other important toxic effect called
promotion” (2). When animals are treated with carbam-
cocus sp. (8). However, to our knowledge, detailed toxi-
cological and biochemical studies about carboxylesterases
able to hydrolyze carbamates in vertebrates are not
available.
“
Because carbaryl (1-naphthalenylmethyl carbamate)
has a relatively simple structure, this compound is an
*
To whom correspondence should be addressed. Phone: 3496658506.
Fax: 34966658511. E-mail: msogorb@umh.es.
1
0.1021/tx015577o CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/06/2002

Hydrolysis of Carbaryl by Albumins
Chem. Res. Toxicol., Vol. 15, No. 4, 2002 521
appropriate model for toxicological and metabolic studies
of carbamate insecticides (1). We have performed a
partial characterization of the capability of rabbit serum
1
albumin (RSA) to hydrolyze carbaryl. Michaelis kinetic
parameters for the hydrolysis of the substrate and for
the inhibition of the hydrolysis by several compounds
were studied. The response of the activity to EDTA and
to several divalent cations was also characterized. All of
the collected data allowed for the proposition of a hy-
drolysis mechanism on the basis of the carbamylation of
a tyrosine residue of RSA.
Exp er im en ta l P r oced u r es
Biologica l Tissu es. All biological tissues were supplied by
Sigma-Aldrich Qu ´ı mica S.A. (Madrid, Spain). The employed
tissues were (a) rabbit serum albumin (RSA) (Sigma product
A-9438), (b) chicken serum albumin (CSA) (Sigma product
A-3686), (c) human serum albumin (HSA) (Sigma product
A-1887), (d) bovine serum albumin (BSA) (Sigma product
A-4503), and (e) rabbit serum (Sigma product R-4505). According
to the supplier catalog, the purity of the albumin samples ranged
from 96% to 99%, with RSA and HSA also being essentially fatty
acid-free.
Ch em ica ls. Ca u tion : The following chemicals are hazard-
ous and should be handled carefully. Carbaryl(1-naphthalenyl-
methyl carbamate) of 99.5% purity and permethrin ((3-phenoxy-
phenyl)methyl-3-(2,2-dichloroethenyl)-2,2-dimethyl cyclopro-
panecarboxylate) of 95.5% purity were supplied by Labor Dr
Ehrenstorfer-Sch a¨ fers (Augsburg, Germany). O-hexyl-O-2,5-
dichlorophenyl phosphoramidate (HDCP) (purity higher than
F igu r e 1. Time course of carbaryl hydrolysis by rabbit serum
(
top panel) and RSA (bottom panel). The concentration of
1
-naphthol is plotted against the incubation time. The total
hydrolysis (9) of carbaryl was assayed at 37 °C and pH 7.4 in
the presence of 350 µL of rabbit serum (top panel) or 3.5 mg of
RSA (bottom panel). Chemical hydrolysis (b) was monitored
under identical conditions in absence of biological tissues. In
all cases, the carbaryl concentration was 600 µM. Enzymatic
hydrolysis (O) was calculated at each time as the difference
between total hydrolysis and chemical hydrolysis.
9
9%) was supplied by Dr Nauman (Bayer Chemical Co.,
Germany). The carboxyl ester p-nitrophenyl butyrate (p-NPB)
and all other chemicals were supplied at analytical grade by
Sigma-Aldrich Qu ´ı mica S.A..
En zym a tic Assa ys. Carbaryl hydrolyzing activity (carbary-
lase) was assayed monitoring the release of hydrolysis product
(
1-naphthol) by a colorimetric method (9). Standard assays were
performed as follows: 50 µL of 12 mM carbaryl in dried acetone
was mixed with 500 µL of 200 mM Tris (pH 7.4). Tissue at the
appropriate concentration was added to make a final volume of
i
type of inhibition and the K was calculated fitting the results
to the Lineweaver-Burk equation by using SigmaPlot software.
1
mL. Typical standard assays were performed with 350 µL of
serum or 3.5 mg of albumin. This mixture was incubated at 37
C for 5 h. The reaction was stopped by adding 350 µL of 2%
Resu lts
°
H yd r olysis of Ca r b a r yl b y Biologica l Tissu es.
When carbaryl was incubated with rabbit serum, the
release of 1-naphthol was detected. The amount of
released 1-naphthol was time-dependent and was linear
from 1 to 5 h (Figure 1). The specific activity of rabbit
serum carbaryl hydrolyzing activity (carbarylase) is
displayed in Table 1.
The release of 1-naphthol is consistent with the hy-
drolysis of the carbamic ester bond of carbaryl or any of
its oxidized metabolites. The capability of serum albu-
mins to hydrolyze several carboxyl esters (10-12), phos-
phoric triesters (13), and phosphoramidates (13, 14) was
previously reported. Therefore, we decided to test the
capability of RSA to hydrolyze carbaryl. RSA was also
able to hydrolyze carbaryl in a time-dependent way
SDS/1.2 mM 4-aminoantipyrine in 50 mM Tris (pH 8.0). The
complex formed by the released 1-naphthol and 4-aminoantipy-
rine was further reduced by 175 µL of 30 mM potassium
hexacyanoferrate (III). The color of the reduced complex (ꢀ )
-
1
-1
7
100 ( 600 M
cm ) was determined at 510 nm. The
concentration of 1-naphthol was calculated by comparing the
results with a calibration curve (range between 5 and 100 µM).
All values were corrected for nonenzymatic hydrolysis with
samples assayed in identical conditions without biological tissue.
Kin etics. For the determination of Michaelis constants,
standard assays were performed employing several carbaryl
concentrations in the range of 60-1200 µM. The results were
fitted to the Lineweaver-Burk equation using SigmaPlot 5.0
software (Sigma-Aldrich Qu ´ı mica S. A.).
Effect of Ca tion s a n d Or ga n ic Com p ou n d s. Reaction
samples were incubated as was described previously in the
presence of the appropriate compound. The type of inhibition
was determined by incubating several inhibitor concentrations
in the presence of several substrate concentrations. Finally, the
(
2
Figure 1). The specific carbarylase activity of RSA was
3 nmol of 1-naphthol/5 h/mg of protein (Table 1).
Chicken, human, and bovine serum albumin also
displayed carbarylase activity (Table 1). However, among
all of the assayed tissues, RSA exhibited the highest level
of carbarylase activity; the lowest level was detected for
CSA (Table 1).
To obtain in the reaction media enough concentration
of carbaryl for estimating, with accuracy, the Michaelis
kinetic constants, the acetone concentration was in-
1
Abbreviations: BSA, bovine serum albumin; carbarylase, carbaryl
hydrolyzing activity; CSA, chicken serum albumin; DCC, N,N′-dicy-
clohexylcarbodiimide; DTNB, 5,5′-dithio-bis(2-nitrobenzoic acid); HDCP,
O-hexyl-O-2,5-dichlorophenyl phosphoramidate; Hg(OAc) , phenylm-
2
ercuric acetate; MVK, methyl vinyl ketone; N-EM, N-ethylmaleimide;
p-OHMB, p-hydroxymercuribenzoic acid; p-NPB, p-nitrophenyl bu-
tyrate; RSA, rabbit serum albumin.

5
22 Chem. Res. Toxicol., Vol. 15, No. 4, 2002
Sogorb et al.
Ta ble 1. Ca r ba r yl Hyd r olyzin g Activities of Ra bbit Ser u m a n d Ser u m Albu m in s fr om Va r iou s Sp eciesa
tissue
specific activityb
Kcat (s-1
)
Km (µM)
rabbit serum
serum albumin of
rabbit
chicken
human
1.5 ( 0.2 (n ) 12)
(7.1 ( 1.4) × 10- (n ) 4)
5
240 ( 60 (n ) 4)
23 ( 4 (n ) 17)
4.4 ( 0.7 (n ) 3)
16 ( 3 (n ) 3)
13 ( 2 (n ) 3)
bovine
a
In all cases, mean ( SD for n independent experiments is displayed. Specific activity was calculated using 600 µM substrate
concentration. Km and Kcat were obtained incubating different substrate concentration (over range of 60-1200 µM) with RSA at 37 °C
during 5 h at pH 7.4. Michaelis kinetic parameters were calculated according to the Lineweaver-Burk equation. Kcat was calculated
assuming a molecular weight of 64 300 for RSA, one unique active center in each molecule, and that the release of product is linear for
b
5
h of reaction (see Figure 1). nmol of 1-naphthol/5 h/mg of protein.
F igu r e 2. Effect of EDTA and different cations (panel A) and of different organic compounds (panel B) on hydrolysis of carbaryl by
RSA. Activity was assayed incubating 3.5 mg of RSA with 600 µM substrate at pH 7.4 during 5 h at 37 °C in the presence of cations,
EDTA, or the organic compound at the concentration displayed in each case. Plotted is the mean ( SD for at least three independent
experiments. Control exhibited values similar to those displayed in Table 1: (*) statistically different from control, p < 0.01; (**)
statistically different from control, p < 0.001). Abbreviations are as previously defined.
creased to 15%. Standard assays were performed with
5% acetone in the medium and several substrate
concentrations. In these conditions, RSA hydrolyzed
triester) were studied. Standard assays in the presence
of those compounds at the concentrations indicated in
Figure 2B were performed. Among all of the assayed
compounds, DTNB and iodoacetate (cysteine-blocking
reagents) and the phosphoramidate HDCP were able to
inhibit carbarylase activity at around 50% (Figure 2B).
Stronger inhibition was detected when caprylic, palmitic,
and p-NPB were used as inhibitors (Figure 2B).
To study the type of inhibition by fatty acids, several
substrate concentrations were incubated in the presence
of several caprylic (from 0.1 to 10 mM) or palmitic (from
6 to 300 µM) acid concentrations. In these experiments,
the acetone concentration in the medium was increased
to 15% for identical reasons as those outlined previously.
1
-
5
-1
carbaryl with a Kcat of 7.1 × 10
Table 1).
Effect of EDTA a n d Meta ls on Ca r ba r yl Hyd r o-
s
m
and a K of 240 µM
(
lyzin g Activity of RSA. To find evidence if carbarylase
activity of RSA is metal-dependent, the hydrolysis of
carbaryl was assayed in standard conditions in the
2
+
2+
2+
2+
presence of 5 mM EDTA or 1 mM Ca , Zn , Cu , Mg
,
2
+
+
+
Mn , Na , and K . Among all of the assayed metals,
Cu2 caused significant inhibition of carbarylase activity
+
(Figure 2A). All other cations and EDTA caused no
significant effect (neither inhibition nor activation) on
carbarylase activity (Figure 2A).
Effect of Sever a l Or ga n ic Com p ou n d s on Ca r -
ba r yl Hyd r olyzin g Activity of RSA. To perform a
biochemical characterization of the active center of car-
barylase activity, the effects of several organic compounds
When Vmax and K
concentration, we detected that K
m
were calculated for each inhibitor
was maintained
m
approximately constant while Vmax decreased when the
inhibitor concentration was increased. It suggested non-
competitive inhibition. The K
using Lineweaver-Burk plots (Table 2). In the case of
caprylic, a K of 480 µM was estimated. However, the K
was around 3.7 times lower for the inhibition of palmitic
i
constants were calculated
(nine selective amino acid blocking chemicals, one short-
and one long-chain fatty acid, one carboxyl ester; one
pyrethroid, one phosphoramidate, and one phosphoric
i
i

Hydrolysis of Carbaryl by Albumins
Chem. Res. Toxicol., Vol. 15, No. 4, 2002 523
Ta ble 2. In h ibitor y Effect of Som e Or ga n ic Com p ou n d s
on Ca r ba r yl Hyd r olyzin g Activity of RSAa
compound
caprylic acid
palmitic acid
p-nitrophenyl butyrate
Ki (µM)
type of inhibition
480
130
45
noncompetitive
noncompetitive
competitive
a
Different substrate concentrations were incubated with dif-
ferent inhibitor concentrations at 37 °C during 5 h at pH 7.4. After
that, color was developed as in standard protocol. Type of inhibi-
tion and Ki were determined according to the Lineweaver-Burk
equation.
Ta ble 3. Ad d itive In h ibitor y Effect of Cu 2 a n d
+
p-Nitr op h en yl Bu tyr a te on Ca r ba r yl Hyd r olyzin g
a
Activity of RSA
condition
activityb
% inhibition
control
19
11
17
8.4
0
42
11
56
5
0
5
0 µM p-nitrophenyl butyrate
2
+
.5 mM Cu
0 µM p-nitrophenyl butyrate +
2
+
0
.5 mM Cu
a
Carbaryl hydrolyzing activity was assayed in standard condi-
tions (3.5 mg of RSA, pH 7.4, 600 µM substrate, and 5 h of
incubation at 37 °C) in absence of inhibitors (control) or in the
2
+
presence of p-nitrophenyl butyrate, Cu , for both compounds.
b
nmol of 1-naphthol/5 h/mg of protein.
acid (Table 2).
The carboxyl ester p-NPB displayed a strong inhibitory
potency toward RSA carbarylase activity (Figure 2B).
When kinetic parameters were calculated in the presence
of 15% acetone and several p-NPB concentrations, we
detected that Vmax maintained constant. However, K
increased with the inhibitor concentration. These data
are consistent with competitive inhibition. The K dis-
exhibited by fatty
m
F igu r e 3. Effect of iodoacetate (panel A) and DTNB (panel B)
on the hydrolysis of carbaryl by RSA. The effect of sulfydril
blocking agents on carbaryl hydrolyzing activity was tested
assaying the activity using 600 µM substrate, 5 h of incubation
at 37 °C and pH 7.4, and different concentrations of iodoacetate
and DTNB. Control exhibited values similar to those displayed
in Table 1.
i
played by p-NPB was lower than the K
i
acids (Table 2).
Copper at a 1 mM concentration was able to inhibit
the hydrolysis of carbaryl by RSA at around 50% (Figure
2
+
2
). It is not possible to reach higher Cu concentrations
suggest that p-NPB, HDCP, and carbaryl might be
hydrolyzed at the same active center.
at pH 7.4; therefore, it was not possible to reach enough
inhibition for a detailed analysis of the inhibition.
However, we detected additional inhibitory effects of Cu2
+
The solubility of the phosphoramidate HDCP is ex-
tremely low. It does not allow for the study of the kinetics
of the inhibition of carbarylase activity through standard
analysis procedures. To determine if carbaryl and HDCP
are hydrolyzed by RSA in the same active center, we
incubated a mixture containing 160 µM RSA and 40 µM
HDCP at 37 °C for 15 min. We established that 90% of
HDCP present in the mixture had been hydrolyzed by
RSA (data not shown) in these conditions. Afterward, the
mixture was diluted 5 times, and the incubation at 37
and p-NPB (Table 3), suggesting that the carboxyl ester
and the cation exerted their respective actions in different
sites of the enzyme.
Iodoacetate and DTNB displayed the capability to
inhibit the carbarylase activity of RSA. However, because
it was not possible to obtain high inhibition percentages
at the maximum inhibitor concentration and because
they are nonreversible blocking agents, it was not pos-
sible to perform a Michaelis analysis of the inhibition.
We detected that both reagents inhibited carbarylase
activity in a concentration-dependent way (Figure 3). The
maximum inhibition percentage was, in both cases,
around 50%. However, DTNB is the most powerful
inhibitor because this maximum inhibition was reached
at a lower concentration of DTNB than for iodoacetate.
°
C was prolonged. Aliquots of the diluted mixture were
extracted at different times, then carbaryl was added,
and carbarylase activity was assayed according to the
standard procedure. The results were compared with
control samples initially incubated in absence of HDCP.
Immediately after dilution, the sample incubated with
HDCP displayed around 50% activity of the control
Rea ctiva tion of RSA Ca r ba r yla se Activity In h ib-
ited by HDCP . The phosphoramidate HDCP was previ-
ously described for CSA as an inhibitor of p-NPB hydro-
lyzing activity (14). Both substrates are hydrolyzed by
CSA in the same active center by a similar mechanism
(Figure 4). The percentage of activity was increased when
the incubation at 37 °C was prolonged, reaching 90% of
control activity for 2 h of incubation.
Discu ssion
(14). HDCP and p-NPB have also demonstrated the
capability to inhibit the hydrolysis of carbaryl by RSA
This study is (to our knowledge) the first detailed
biochemical report on the capability of RSA to hydrolyze
the carbamate carbaryl.
(Figure 2B). In addition, RSA is able to hydrolyze HDCP
(13), carbaryl (Table 1), and p-NPB. All these data

5
24 Chem. Res. Toxicol., Vol. 15, No. 4, 2002
Sogorb et al.
additive (Table 3). It suggests that Cu2 exerts its effects
+
at a site other than the active center. It is previously
2
+
reported that albumin posseses binding sites for Cu
(
2
+
15). These data suggest that the binding of Cu to RSA
might alter the conformation of the active center, gen-
erating an enzyme molecule that is still able to hydrolyze
carbaryl but with less efficacy than an enzyme without
Cu2 . This effect is in contrast with the activation by Cu
+
2+
of the HDCP hydrolyzing activity found in CSA (17). It
might be explained if we consider that the changes
2
+
originating in the enzyme by the binding of Cu origi-
nate slight modifications on the active center that make
the enzyme more effective for the hydrolysis of HDCP
but less effective for the hydrolysis of carbaryl.
Iodoacetate and DTNB are able to inhibit the hydroly-
sis of carbaryl by RSA (Figure 2B). However, the maxi-
mum inhibition is around 50% (Figure 4). This suggests
that there are some cysteine residues in the protein that
are necessary for the integrity of the enzyme but not for
the catalytic process itself. The maximum percentage of
inhibition by thiol blocking agents is similar to the
inhibition of HDCP hydrolyzing activity detected in CSA
by those agents (17).
We previously reported that CSA is able to hydrolyze
HDCP and p-NPB by a similar mechanism on the basis
of the phosphorylation-dephosphorylation or acetyla-
tion-deacetylation of a tyrosine residue in the active
center (14). The dephosphorylation process was found to
be extremely slow (14). Carbarylase activity of RSA was
inhibited by p-NPB in a competitive way (Table 2),
demonstrating that p-NPB and carbaryl are hydrolyzed
in the same active center and suggesting that this site
might be the same as that where HDCP is hydrolyzed.
RSA carbarylase activity was also inhibited by HDCP
(Figure 2B). The time-dependent reactivation of carbary-
lase activity inhibited by HDCP (Figure 4) suggests that
carbaryl and HDCP are hydrolyzed in the same active
center by a comparable mechanism. Indeed, immediately
after the incubation of RSA with HDCP, the active
centers of most of the RSA molecules are phosphorylated
by HDCP, causing an important inhibition of carbarylase
activity. However, the enzyme is dephosphorylated slowly
with time, and the carbarylase activity is recovered
(Figure 4).
The amino acid sequence Arg-Tyr-Thr-Arg was identi-
fied as responsible for the specific labeling of BSA by the
organophosphorus insecticide diisopropyl fluorophos-
phate (15). When the albumin amino acid sequence was
known, the residue Tyr410 for BSA or Tyr411 for HSA
was recognized as the residue acetylated during the
hydrolysis of p-nitrophenyl acetate by these proteins.
RSA displays the amino acid sequence Arg-Tyr-Thr-Lys
(14). This sequence differs from BSA and HSA sequences
only in the last arginine, which is substituted by lysine.
Both arginine and lysine are basic amino acids and
probably could play similar roles in a hypothetical
hydrolysis of esters. Therefore, the sequence Arg-Tyr-Thr-
Lys might be the target of the acetylation during p-NPB
hydrolysis and the target of carbamylation during car-
baryl hydrolysis.
F igu r e 4. Reactivation of the carbaryl hydrolyzing activity in
RSA after inhibition by HDCP. A mixture with 160 µM RSA
plus 40 µM HDCP was incubated at 37 °C during 15 min.
Afterward, the mixture was diluted 5 times, and the incubation
at 37 °C was prolonged. Finally, carbaryl was added at different
times, and carbaryl hydrolyzing activity was assayed in stan-
dard conditions. Results were compared with control samples
treated in identical conditions in absence of HDCP. There were
no significant differences among control samples incubated
during different times. Controls exhibited values similar to
displayed in Table 1.
The hydrolysis of carbaryl by RSA was inhibited by
fatty acids in a noncompetitive way (Table 2), suggesting
that fatty acids bind to the enzyme in a site not related
to the active center. Fatty acids also have been described
as inhibitors of several carboxylesterase (10-11, 14) and
phosphotriesterase activities (14) of serum albumins. It
is well-known that albumin has at least six different
binding sites for fatty acids, with three of them being
unknown (15). We conclude that the fatty acid does not
bind in the catalytic site involved in the carbaryl hy-
drolysis. The inhibition might be explained if we assume
that the binding of fatty acids to RSA might alter the
conformation of the enzyme, resulting in reduced hydro-
lytic activity.
Serum albumin accounts for around 50% of the total
serum proteins (16). If we use these data to extrapolate
the specific carbarylase activity of the RSA contained in
our rabbit serum sample, we conclude that RSA present
in serum displays around 8 times lower activity than the
sample of purified RSA. The explanation for this phe-
nomena can be attributed to inhibition by fatty acids.
Fatty acids circulate in serum at a total concentration of
just under 1 mM, and only less than 0.1% of them
circulate in free form in plasma, with the rest being
bound to albumin (15). According to that and to the data
shown in Figure 2B, RSA present in rabbit serum must
be inhibited by bound fatty acids. However, according to
supplier information, the preparation of pure RSA is fatty
acid-free; therefore, its activity is not inhibited by these
acids.
EDTA had no significant effect on carbarylase activity,
indicating that there are no metals in the active center
of RSA (Figure 2A). This is supported by the observation
that none of the tested cations activated the carbarylase
activity. The phosphotriesterase activity found in RSA
is also EDTA-resistant, while other phosphotriesterase
activities found in rabbit serum lipoproteins fraction are
All discussed data are consistent with a mechanism of
hydrolysis on the basis of the transient carbamylation
of the Tyr411 residue of RSA. This mechanism will be
similar to the mechanism proposed for the hydrolysis of
carboxyl esters (10) and HDCP (14) by serum albumins.
The carbamylation of tyrosine will originate the release
2
+
Ca -dependent and, therefore, EDTA-sensitive (13).
Copper and p-NPB exerted an inhibitory effect on RSA
carbarylase activity (Figure 2). These effects seem to be

Hydrolysis of Carbaryl by Albumins
Chem. Res. Toxicol., Vol. 15, No. 4, 2002 525
probably is the most important organ involved in the
hydrolysis of carbamates via hydrolysis by albumins.
The promotion phenomenon is subject of maximum
concern because we might be on the verge of elucidating
the process of neurodegenerative diseases of as yet
unknown ethiology. The necessity to develop studies
about enzymes involved in the detoxification of promoters
(carbamates) seems clear because the action of these
enzymes might significantly reduce the amount of the
promoter before it reaches the target of promotion in
nervous systems.
Finally, it is remarkable that the capability of albumins
to hydrolyze carbamates has strong ecotoxicological
implications. Indeed, because albumin is universally
present in all vertebrate species, it will be possible to
design carbamate structures more susceptible to hydroly-
sis by albumins. In this way, the resistance of nontarget
species (vertebrates) to the carbamate insecticides will
be increased. This increase of resistance might be spe-
cially important in species lacking other disposition and
metabolic systems.
F igu r e 5. Proposed hydrolysis mechanism for the hydrolysis
of carbaryl by RSA. In the first step, carbaryl reacts with a
tyrosine residue of RSA, yielding free 1-naphthol and carbamy-
lated RSA. In the second step, a water molecule attacks the
carbamylated complex, releasing carbamic acid and free enzyme.
The free enzyme is ready for a new catalytic cycle while
2
carbamic acid quickly decomposes into CO and methylamine.
Ack n ow led gm en t. This study was supported by
Grant FIS 00/1100. M.A.S. holds a position inside the
Researcher Incorporation Program of the Spanish Min-
isterio de Ciencia y Tecnolog ´ı a. The authors thank Miss
Luc ´ı a Rojo for her technical assistance.
of 1-naphthol, yielding a carbamylated RSA. The car-
bamoyl moiety will be further released by the attack of
a water molecule. The released carbamic acid instanta-
2
neously decomposes into CO and methylamine. The
proposed mechanism is displayed in Figure 5. Neverthe-
less, our data cannot exclude the possibility that another
active center in albumin can hydrolyze carbaryl at lower
rate. This mechanism is similar to the mechanism
described for serine esterases (like acetylcholinesterase)
but with tyrosine instead of serine playing the most
important role.
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(
1) Alvares, A. P. (1993) Pharmacology and toxicology of carbamates.
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A very low Kcat (7.1 × 10 s-1) was observed for the
-5
hydrolysis of carbaryl by RSA (Table 1). This low turn-
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carbamylation took place.
As was demonstrated previously the carbarylase activ-
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(
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7
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(
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