Purification,
Characterization and Biological Activity of an L-Amino
Acid Oxidase from Trimeresurus mucrosquamatus Venom

WEI Ji-Fu^1,2,
WEI Qin¹, LU Qiu-Min¹, TAI Hong¹, JIN Yang¹,
WANG Wan-Yu¹, XIONG Yu-Liang^1*

( ¹Department of Animal
Toxicology, Kunming Institute of Zoology, the Chinese Academy of Sciences,
Kunming 650223, China;

²The Graduate School of
the Chinese Academy of Sciences, Beijing 100039, China )

Abstract An L-amino acid oxidase (TM-LAO)
from the venom of Hunan Trimeresurus mucrosquamatus was purified to
homogenicity by three steps including DEAE Sephadex A-50 ion-exchange
chromatography, Sephadex G-75 gel filtration and Resourse Q ion-exchange
chromatography. TM-LAO is composed of two identical subunits with a molecular
weight of 55 kD by SDS-polyacrylamide gel electrophoresis. The molecular weight
was different with that of LAO purified from the same species distributed in
Taiwan that was 70 kD. The 24 N-terminal animo acid sequence of TM-LAO is
ADNKNPLEECFRETNYEEFLEIAR, which shares high similarity with other Viperid snake
venom LAOs and has moderate similarity with Elapid snake venom LAOs. Further
studies found that TM-LAO inhibited the growth of E.coli, S.aurues
and B.dysenteriae. TM-LAO also showed cytotoxicity and platelet
aggregation activity. All the biological activities were eliminated by
catalase, a H2O2 scavenger. It shows that these
biological effects are possibly due to the formation of H2O2
produced by TM-LAO.

Key words L-amino acid oxidase; Trimeresurus
mucrosquamatus; antibacterial activity; cytotoxicity; platelet aggregation

L-amino
acid oxidase (LAO, EC1.4.3.2) is a dimeric flavoprotein containing
non-covalently bound FAD or FMN as cofactor, and is present in various
resources including snake venoms[1]. It catalyzes the oxidative deamination of L-amino
acids to produce the corresponding α-keto acids along with the production of
ammonia and hydrogen peroxide via an imino acid intermediate[2]. To date, many
LAOs were purified and studied from various snake venoms except sea snakes
venoms[3-9]. But their physiological role in snake envenomation is not well
understood. Recent studies indicate that LAOs purified from snake venoms induce
apoptosis in vascular endothelial cells and thus may contribute to prolonged
bleeding from the vessel walls damaged by snake bite[10, 11]. LAOs also
affected the function of platelet, Li et al.[12] reported that the LAO
purified from Ophiophagus hannah (king cobra) induced platelet aggregation
directly, but the controversial reports were from the LAOs from Agistrodon
halys Pallas, Agistrodon halys blomhoffi, and Naja kaouthia snake. These
enzymes inhibited the platelet aggregation induced by agonist such as ADP or
collagen [9, 13, 14]. Besides the apoptosis inducing and platelet aggregation
inhibiting/inducing activities, snake venom LAOs were reported to show other
activities such as antibacterial[15], edema formation[16], hemolysis[17] and
hemorrhage activities[8], and these biological effects are possibly or partly
due to the production of highly localized concentrations of H2O2.

Ueda et al.[3] first purified and studied the
enzymatic property of LAO from the venom of Trimeresurus mucrosquamatus
distributed in Taiwan, but the biological activities of this enzyme were not
reported. We purified LAO (designated as TM-LAO) from Trimeresurus
mucrosquamatus venom distributed in Hunan Province, China. TM-LAO had a
different molecular weight with LAO purified by Ueda et al.[3]. In
addition, some biological activities including platelet aggregation inducing,
antibacterial and cytotoxicity were also studied.

1 Materials and Methods

1.1  Materials

Trimeresurus mucrosquamatus venom was collected from Hunan Province.
Sephadex G-75, Sephadex G-150, DEAE Sephadex A-50, Resourse Q and protein
molecular weight markers were from Amersham Pharmacia Biotech, Uppsala, Sweden.
Catalase, arsenate, L-phenylalanine and EDTA were purchased from Sigma,
St. Louis, U.S.A. Other chemicals and reagents were of analytical grade.

1.2  Enzyme isolation

Lyophilized crude venom of T. mucrosquamatus (1000
mg) was dissolved in 5 mL of 50 mmol/L Tris-HCl buffer, pH 8.9, applied to a
DEAE Sephadex A-50 column (2.6 cm×120 cm), and then eluted with the same buffer
with a linear gradient of 0－0.5 mol/L NaCl. The fractions contained
LAO activity were lyophilized, dissolved in 5 mL of 50 mmol/L Tris-HCl buffer,
pH 8.5, and then applied to Sephadex G-75 (3.2 cm×150 cm) column equilibrated
with the same buffer. The column was eluted at a flow rate of 15 mL/h and
collected in 3 mL per tube. LAO containing fractions were pooled, desiccated,
dialyzed against 25 mmol/L Tris-HCl buffer, pH 8.5, then loaded on to a
Resourse Q column (1 mL) and eluted using a NaCl gradient (0－0.6
mol/L). The purified enzyme solution was stored at in 40% glycerol at 4 ℃ until
use according to Ueda et al.[3].

1.3  L-amino acid oxidase activity

L-amino
acid oxidase activity was determined by the method of Wellner et al.[18].
One unit of LAO activity was defined as the amount of enzyme required giving an
absorbance (A300) of 0.03 under the determined conditions.

1.4  SDS-PAGE and determination of molecular
weight

Electrophoresis was performed on a 15%
polyacrylaminde gel following the methods of Laemmli[19]. The gel was stained
with Coomassie blue R-250 (Sigma). Molecular weight of the purified enzyme was
estimated by SDS-PAGE.

1.5  Analytical gel filtration

This was carried out according to the method of
Andrews[20] with a Sephadex G-150 column (2 cm × 80 cm). Blue dextran 2000 was
used to measure the void volume (V0) of the column.

1.6  Determination of N-terminal amino acid
sequence

Sequence determination of the protein was performed
by Edman degradation with an Applied Biosystems model 476A sequencer.

1.7  Platelet aggregating activity

Platelet aggregation was measured by the
turbidimetric method of Born et al.[21], using a LNY-1 aggregometer
(Precie Group, Beijing, China). Rabbit platelet-rich plasma (PRP) was prepared
by centrifuging blood collected in 0.38 % sodium citrate at 135 g for 20 min at
room temperature. After removal of PRP, the remaining blood was centrifuged for
10 min at 1240 g and platelet-poor plasma (PPP) was obtained. The platelet
count was adjusted to 40×104 platelets per μL. PRP (0.175 mL) was preincubated
at 37 ℃ for 5 min. The aggregation was initiated by the addition of sample.
Platelet aggregation was monitored over 5 min.

1.8  Antibacterial assays

Bacteria were obtained from the Center for Medical
Culture Collection (Bacteria) (CMCCB), National Institute for the Control of
Pharmaceutical and Biological Products, Ministry of Public Health, Beijing,
China. Bacteria used in this study include E.coli (CMCCB44102), S.aurues
(CMCCB26003), B.pyocyaneus (CMCCB10104), B.megaterium (CMCCB11207) and
B.dysenteriae (CMCCB14103). A disc diffusion assay was used with the following
modifications[22]: bacteria [200 μL of a 0.1 A600 culture containing 1.75 × 109
colony forming units (CFU)/mL] were spread onto 15 mL nutrient agar plates (90
mm diameter). Sterile paper discs (7 mm diameter) were then placed onto the
agar surface and 15 μL of sample was added per disc. Plates were incubated at
37 ℃. After 18 h, the diameters of inhibition zones were recorded in mm minus
the disc diameter.

1.9  Edema assay

The method of
Vishwanath et al.[23] was followed. Groups of six mice were injected in
the right footpads with different doses of LAO in 20 μL of saline. The left
footpads received 20 μL of saline, which served as controls. The increase in
weight due to edema was calculated as the edema ratio. Minimum edema does is
the amount of protein required to cause an edema ratio of 120%.

1.10       Measurement
of cytotoxicity

C8166 (human
T-cell leukemia virus type I-transformed lymphoblastoid cell line) cells were
maintained in RPMI-1640 (Gibco, Grand Island, NY), supplemented with 15%
heated-inactivated fetal bovine serum, 100 u/mL penicillin, and 0.1 mg
streptomycin at 37 ℃ in a 5% CO2-humidified incubator.
LAO (1-10 mg/L) was added to the cells (106 /ml) in 96-well micro titer plates.
After different times, 50 μl MTT [3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyltetra-zolium bromide] stock solution (5 g/L in PBS) was added to the
well and the plate was incubated for 4 h. Following aspiraton of the medium,
150 μl of DMSO was added to solubilize the MTT-formazan product. After 30 min
at room temperature, the plate was read with a microplate reader at 570 nm. All
the measurements were performed in triplicate[24].

1.11       Protein
quantitation

Protein
concentration was determined by the method of Bradford[25].

2   Results

2.1  Purification and molecular weight of
TM-LAO

Three chromatographic steps were employed for
purification of this enzyme. The purification scheme was summarized in Fig.1.
The purified enzyme was named TM-LAO. SDS-PAGE conducted in the reduced and
non-reduced conditions both gave molecular weight of 55 kD [Fig. 2(A)].
Meanwhile, the apparent molecular weight of this enzyme estimated by analytical
gel filtration was 110 kD [Fig. 2(B)]. These results suggested this enzyme was
composed of two non-covalent subunits.

Fig.1 Purification scheme of TM-LAO

(A) Chromatography of crude venom of Trimeresurus
mucrosquamatus on DEAE Sephadex A-50 ion-exchange column; (B) Isolation of
LAO containing fractions from DEAE Sephadex A-50 ion-exchange column on a
Sephadex G-75 column; (C) Purification of TM-LAO in Resourse Q column. The
peaks contained LAO activity were shown by arrow.

Fig.2 Molecular weight determination of TM-LAO by
SDS-PAGE and gel filtration on a Sephadex G-150 column

(A) SDS-PAGE of TM-LAO. 1, markers; 2,
TM-LAO in reduced conditions; 3, TM-LAO in non-reduced conditions. (B) Gel
filtration on a Sephadex G-150 column. Protein standards and their molecular
weights are: 1, γ-globulin (160 kD); 2, bovine serum albumin (66 kD); 3, chicken
egg ovalbumin (43 kD); 4, bovine trypsinogen (24 kD).

2.2 N-terminal
sequence

The N-terminal 24 amino acid sequence was determined
and compared with other snake venom LAOs (Table 1). The N-terminal sequence of
TM-LAO has a close similarity (identities between 75% to 87%) to LAOs from
Viper snake venoms, a moderate similarity (67% identity) to LAO from Naja
kaouthia. Sequence comparison showed that 13 of 24 amino acids were
conservative in those LAOs.

Table 1Comparison of N-terminal amino
acid sequence of TM-LAO with other venom LAOs

Completely
conserved residues in all sequences are marked by italic.

2.3 Platelet
aggregation-inducing activities

TM-LAO induced aggregation of rabbit platelets in
platelet rich plasma. And the aggregation rate increased with the increasing of
TM-LAO concentration [Fig.3(A)]. In addition, the platelet aggregation induced
by the enzyme (50 mg/L) was inhibited by catalase (200 u/mL) [Fig.3(B)].

Fig.3 Rabbit platelet aggregation induced by TM-LAO

(A) Concentration-dependent platelet
aggregation induced by TM-LAO; (B) Inhibition of catalase (200 u/mL) on
platelet aggregation induced by TM-LAO (50 mg/L).

2.4
Antibacterial proprieties

As shown in Fig.4, TM-LAO inhibited the growth of
E.coli, S.aurues and B.dysenteriae. The antibacterial effect was eliminated
with catalase (data not shown). Meanwhile, antibacterial effect on B.pyocyaneus
and B.megaterium was undetectable.

Fig.4 Antibacterial effect of TM-LAO

The values represent an inhibition zone
in mm, minus the 7 mm diameter of the disc, after 18 h incubation. Bacteria
inoculums per plate contained 1.75 × 109 colony-forming units, which were
spread onto agar plates. Sterile paper discs (7 mm diameter) were then placed
onto the agar surface and 15 μL of sample was added per disc.

2.5
Edama-inducing activity

TM-LAO induced edema in paw pads of mice and caused a
rapid and substantial increase in the paw volume with a minimum edema does
(MED) of 8 μg/paw. A does of less than 8 μg TM-LAO produced an edema that
peaked 1 h after the injection and the level sustained for up to 6 h. At the
doses of 15 μg, edema sustained for more than 24 h.

2.6
Cytotoxicity of TM-LAO

TM-LAO killed the C8166 cells in a does-dependent
manner (Fig.5). Treatment of TM-LAO with repeated freezing and thawing
abolished both LAO and cytotoxic activities (data not shown), suggesting the
involvement of the LAO activity in TM-LAO-inducing cytotoxicity. Catalase, an
H2O2 scavenger, completely inhibited the cytotoxicity of TM-LAO when the
concentration was 20 g/L (data not shown). It is suggesting that H2O2, one of
the products of LAO reaction, mediates TM-LAO-inducing cytotoxicity.

Fig.5 Cytotoxicity of TM-LAO

TM-LAO induced C8166 cells death in a
concentration-dependent manner. Cell viability is expressed as the percent of
ratio of A570 of oxidized formazan MTT of LAO-treated cells with the one of
control cells.

3 Discussion

An L-amino acid oxidase named as TM-LAO from
the venom of Trimeresurus mucrosquamatus (distributed in Hunan Province,
China) was purified to homogeneity. It showed single band with molecular weight
of 55 kD when determined by SDS-PAGE in the absence and presence of
2-mercaptoethanol [Fig. 2(A)]. The apparent molecular weight of TM-LAO was 110
kD when estimated by gel filtration [Fig. 2(B)]. The molecular weight of LAO
from the same specie but distributed in Taiwan was reported to show 140 kD by
analytical gel filtration and 70 kD by SDS-PAGE[3]. This variation may be due
to different composition or differed glycosylation. In fact, variation in snake
venom composition is a ubiquitous phenomenon at all taxonomic levels[26].
Recent studies showed that the composition and some main components such as
PLA2[27] were different between the Trimeresurus mucrosquamatus venom
from Hunan Province (in Chinese mainland) and Taiwan Province. The variation of
molecular weight of LAOs from the Trimeresurus mucrosquamatus venom in
Hunan and Taiwan confirmed this founding. In a recent study on intraspecific
venom variation in Calloselasma rhodostma, Daltry et al.[28]
demonstrated a significant relationship between geographic variation in diet
and geographic variation in venom composition might reflect natural selection
for the greater efficiency in killing and/or digesting different prey types in
different regions. The intraspecific venom variation in Trimeresurus
mucrosquamatus may be explained by this hypothesis.

The N-terminal sequence of TM-LAO (Table 1) has a
close similarity (identities between 75% to 87%) to LAOs from Viperid snake
venoms and a moderate similarity (67% identity) to LAO from cobra (Naja
kaouthia). The determined structure of LAO from the Calloselasma rhodostoma
revealed that the residues 5-25 constituted one part of substrate-binding
domain[29]. From the comparison of the N-terminal sequence of LAOs, at least 13
amino acids of 24 were found highly conserved, suggesting that these conserved
amino acids may play an important role in the binding of substrate.

TM-LAO induced rabbit platelet aggregation and
catalase inhibited the aggregation (Fig.3). That agrees with the LAO from
Ophiophagus hannah venom, which induced human platelet aggregation. Since catalase
completely abolished aggregation effect, the authors premised that the
aggregation activity was related to H2O2[12]. On the contrary, L-amino
acid oxidases from Agistrodon halys Pallas, Agistrodon halys blomhoffi, Naja
kaouthia venoms inhibited platelet aggregation. Catalase neutralized the
anti-platelet effects of the enzyme. So the anti-platelet effects were also
related to H2O2[9, 13, 14]. Takatsuka et al.[13] thought these
conflicting results were likely due to the difference in the experimental procedure
or preparation of blood samples. However, it has been controversial whether
H2O2 itself induces or inhibits platelet aggregation. H2O2 in mmol/L
concentrations may change the shape of human platelet, enhancing both
aggregation and subsequent desegregation of platelets when added simultaneously
with ADP, whereas H2O2 in mmol/L concentrations may fuse platelets, act as an
agonist to platelet aggregation[30, 31]. Thus, Sakurai et al.[１４]
thought that the conflicting effect on the platelet was due to the H2O2 amounts
produced by LAOs.

TM-LAO inhibited the growth of E.coli, S.aurues and
B.dysenteriae. Among the bacteria tested, S.aurues was most sensitive to TM-LAO
(Fig.4). In addition, the antibacterial effect was neutralized by catalase
(data not shown), suggesting H2O2 produced by L-amino acid oxidation is
involved in the antibacterial effect. These results are in accordance with the
effects of two LAOs from the venom of Pseudechis austrlis[15].

L-amino acid oxidases from many snake venoms were reported to show
cytotoxicity by inducing cells apoptosis or necrosis[4, 7-11]. Apoxin-I, an LAO
isolated from western diamondback rattlesnake (C.atrox) venom, induced
apoptosis mediated by H2O2[11]. However, Suhr et al.[32] demonstrated
that LAO-inducing apoptotic mechanism has been clearly distinguishable from the
one stimulated directly by exogenous H2O2, suggesting that the LAO-inducing
apoptosis was not solely to H2O2 produced by enzymatic reaction. The molecular
details of which intracellular components were specifically associated with
LAO-inducing apoptosis were still unknown. TM-LAO, like Apoxin-I, induced
cytotoxicity mainly due to the H2O2 produced by enzymatic reaction. It was
suggested that the mechanism for LAO-inducing cytotoxicity might be related to
the venoms from which LAO was purified.

Though the LAOs’ physiological role in snake
envenomation is not well understood, LAOs were reported to share many
biological effects. Former studies and our present study demonstrated that
these effects were mainly due to the H2O2 produced by enzymatic reaction. But
the exact molecular mechanism for these effects was still in progress.

Acknowledgements We
thank Professor ZHENG Yong-Tang and Dr. WANG Jian-Hua for the measurement of
cytotoxicity.

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Received: October 16, 2002     Accepted:
November 11, 2002

*Corresponding author:
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