Characterization of a New
Bradykinin-potentiating Peptide (TmF) from Trimeresurus mucrosquamatus

JIA Yong-Hong^{1, 2},
LI Dong-Sheng¹, ZHU Shao-Wen¹, ZHANG Li-Yue¹, DING
Li-Sheng³, WANG Wan-Yu¹, XIONG Yu-Liang¹*

(
¹Department of Animal Toxinology, Kunming Institute of Zoology, the
Chinese Academy of Sciences, Kunming 650223, China; ²Graduate School
of the Chinese Academy of Sciences, Beijing 100009, China; ³Chengdu
Institute of Biology, the Chinese Academy of Sciences, Chengdu 610041, China )

Abstract       A
novel bradykinin-potentiating peptide (BPP), designated as TmF, has been
purified to homogeneity from the venom of Trimeresurus mucrosquamatus by 70%
cold methanol extraction, Sephadex G-15 gel filtration and reverse-phase high
performance liquid chromatography (RP-HPLC). The amino acid sequence of TmF was
determined to be pGlu-Gly-Arg-Pro-Leu-Gly-Pro-Pro-Ile-Pro-Pro (pGlu denotes pyroglutamic
acid), which shared high homology with other BPPs. The molecular mass of TmF
was 1.1107 kD as determinated by electrospray ionization-mass spectrometry
(ESI-MS), which was in accordance with the calculated value of 1.1106 kD. The
potentiating “unit” of TmF to bradykinin-induced (BK-induced) contraction on
the guinea-pig ileum in vitro was (1.13±0.3) unit (mg/L), and TmF (5.0×10^－4 mg/kg) increased the
pressure-lowering-effect of bradykinin (5.0×10^－5 mg/kg) with
approximate descent value of (14±2) mmHg. In addition, TmF inhibited the
conversion of angiotensin I to angiotensin II, 2×10^－3 mg of TmF caused 50%
inhibition (IC50) of angiotensin- converting enzyme (ACE) hydrolyzing activity
to bradykinin.

Key words

angiotensin-converting enzyme; bradykinin; bradykinin-potentiating
peptide; TmF

Bradykinin first
discovered by Rocha e Silva et al.[1] is the hydrolyzed product of the
low-molecular-weight (LMW) kininogen by tissue kallikrein, or certain venom
kallikreins[2, 3]. It can induce the contraction of guinea-pig ileum in vitro,
and also caused the blood-pressure-lowering effect[4]. Furthermore, bradykinin
has been implicated in multiple physiological processes such as control of
blood pressure, contraction or relaxation of smooth muscle, inflammatory
responses, and induction of nociception and hyperalgesia[2, 5]. Interestingly,
it was found that there existed a factor in Bothrops jararaca venom which was
able to potentiate the biological actions of bradykinin[6, 7]. Moreover, this
factor could inhibit the enzymatic activity of angiotensin-converting enzyme
(ACE)[8], which was a cytoplasmatic membrane peptidase of endothelial cells
responsible for the conversion of angiotensin I to angiotensin II[6, 9, 10].
This factor, exhibiting both bradykinin-potentiating activity and inhibitory
activity to ACE, was designated as bradykinin-potentiating peptide (BPP) or ACE
inhibitor. Since then, many bradykinin-potentiating peptides have been
demonstrated and isolated from snake venoms[9, 11, 12], for example, five from
Agkistrodon blomhoffii[13], nine from Bothrops jararaca[14] and three from
Bothrops neuwiedi[7]. The analysis of the primary structures of these peptides
revealed that they belonged to 5－13 amino acid peptides with N-terminus pGlu and C-terminal tripeptide
Ile-Pro-Pro. However, up to date, the purified BPPs were only from the venom of
Agkistrodon or Bothrops genus, there was still no report on other genera. Is
there the existence of BPP in venom of other genera? It led us to investigated
the venom of T. mucrosquamatus, which distributes in most region of China,
especially Hunan province. Finally, a novel BPP termed TmF was purified and
characterized, which was an undecapeptide possessing dual activity.

1 Materials and Methods

1.1 Materials

The lyophilized
T. mucrosquamatus crude venom was from the stock of the Kunming Institute of
Zoology, the Chinese Academy of Sciences. Sephadex G-15 was from Pharmacia
(Uppsala, Sweden). RP-HPLC C18 column (Nava-Pak C18 column, 3.9 mm×300 mm) was
obtained from Waters. Bradykinin and pyroglutamate aminopeptidase (PAP) were
purchased from Sigma. Angiotensin-converting enzyme (ACE) was partially
purified from the rat plasma. Guinea pig and cat were from Kunming Medical
Institute. Other reagents used were of analytic grade from commercial sources.

1.2 Isolation process

The lyophilized
T. mucrosquamatus venom (2 g) was extracted with three times volumes of 70%
cold methanol followed by vacuum evaporation of the extracted fluid. The
remaining powder was dissolved in 3 mL of 50 mmol/L ammonium acetate (pH 4.7,
containing 0.1 mol/L NaCl) and chromatographed on a Sephadex G-15 column (2 cm×100
cm) previously equilibrated with the same buffer at a flow rate of 30 mL/h. The
fractions exhibiting the potentiating activity to bradykinin-induced
contraction on guinea-pig ileum in vitro were pooled, and then applied to a
RP-HPLC C18 column (Nava-Pak C18 column, 3.9 mm×300 mm) previously equilibrated
with 0.1% trifluoroacetic acid (TFA), the elution was performed with solution B
(acetonitrile, containing 0.1% TFA) with the gradient of 0%－20%, 20%－70%, 70%－100% at flow rate of 0.7 mL/min.
The peptide was monitored spectrophotometrically at 215 nm.

1.3 Mass spectrometry analysis

Electrospray
ionization-mass spectrometry (ESI-MS) was performed on a Finnigan LCQ DECA with
the spraying voltage 4 kV and the capillary temperature 150 ℃. The signal of
(M-H) was examined.

1.4 Sequence analysis

TmF was digested
with PAP, and the resulted peptide was purified by RP-HPLC C18 (Nava-Pak C18 column,
3.9 mm×300 mm). The amino acid sequence of the PAP-treated peptide was
determined on Model 476A protein sequencer (Applied Biosystem, USA).

1.5 Biological assay

The
bradykinin-potentiating activity of TmF was determined on the isolated
guinea-pig ileum in vitro according to the method of Ferreiraet al.[7].
The increment of TmF to bradykinin-induced contraction was statistically
analyzed; the contraction effect of bradykinin alone was used as a control. One
potentiating “unit” was defined as the amount of the peptide per liter needed
to double the activity of bradykinin with a dose of 1 mg/L[15].

1.6 Arterial blood pressure

The adult male
cats (2－3 kg) in
normotensive state were anaesthetized with nembutal (30 mg/kg) according to the
method of Ferreira et al.[16], and then four groups of sample
combinations: bradykinin (5×10^－5 mg/kg)+TmF (5×10^－4
mg/kg), bradykinin (8×10^－5 mg/kg)+TmF (5×10^－4
mg/kg), bradykinin (5×10^－5 mg/kg)+TmF (1.0×10^－3
mg/kg) and bradykinin (8×10^－5 mg/kg)+TmF (1.0×10^－3
mg/kg) were respectively checked; the blood depression effect of bradykinin (5×10^－5
mg/kg) alone was used as a control. The data of arterial blood pressure were
recorded by LMS-2B physiological recorder.

1.7 ACE inhibition

Different doses
of TmF were incubated with ACE (5×10^－2 mg) in 1 mL Krebs solution at 37 ℃
for about 30 min, respectively, the residual activity of ACE was then assayed
with bradykinin. The hydrolyzing effect of ACE to BK in the absence of TmF was
used as a control.

2 Results

2.1 Purification and primary structure
determination of TmF

Three protein
peaks were observed in Sephadex G-15 gel filtration elution profile, and the
curve of bradykinin-potentiating effect of each tube was as below[Fig.1(A)].
The fractions in peak 1 exhibiting strong bradykinin-potentiating activity were
pooled, and then fractionated by a RP-HPLC C18 column, TmF appeared in the
first peak[Fig.1(B)].

Fig.1
Purification scheme of TmF from the venom of Trimersurus mucrosquamatus

(A) Gel filtration chromatography of the vacuum-evaporated powder on a
Sephadex G-15 column (2 cm×100 cm). The elution buffer used was 50 mmol/L
ammonium acetate (pH 4.7, containing 0.1 mol/L NaCl). Fraction volume of 3 mL
of each tube was pooled, and protein concentration was estimated by the
absorbance at 280 nm (―). The bradykinin-potentiating activity was assayed on
the guinea-pig ileum in vitro. The fractions in peak 1 (indicated by an arrow)
were pooled (—). (B) Collected fractions rechromatographed on RP-HPLC C18
column (Nava-Pak C18 column, 3.9 mm×300 mm) previously equilibrated with 0.1%
trifluoroacetic acid (TFA). The elution was performed with solution B
(acetonitrile, containing 0.1% TFA) with the gradient of 0%－20%, 20%－70%, 70%－100% at flow rate
of 0.7 mL/min. The eluted fractions were monitored spectrophotometerically at
215 nm. TmF was found in peak 1 (indicated by arrow).

The (M-H) signal
of ESI-MS showed the molecular mass of TmF to be 1.1097 kD (Fig.2).]

Fig.2 ESI-MS of TmF

The (M-H) of spectrum was
shown by electrospray ionization-mass spectrometry (ESI-MS) with the spraying
voltage 4 kV and the capillary temperature 150 ℃ (m/z=1109.7).

The PAP-treated peptide and the
released pyroglutamic acid were separated by a RP-HPLC C18 column. The
pyroglutamic acid was identified by comparison with the standard chromatography
profile of pGlu (data not shown). The amino acid sequence of the PAP-treated
peptide was determined to be Gly-Arg-Pro-Leu-Gly-Pro-Pro-Ile-Pro-Pro.

2.2 Bradykinin-potentiating effect of
TmF

Bradykinin caused guinea-pig ileum
contraction in vitro, whereas TmF can potentiate bradykinin-induced contraction
effect (Fig.3). The potentiating “unit” was statistically calculated to be
(1.13±0.3) mg/L; bradykinin (1 mg/L) was used as control.

Fig.3 Guinea-pig ileum contraction effect of bradykinin, bradykinin +TmF in
vitro

A 2-cm segment of guinea-pig ileum was suspended in 10 mL of Krebs
solution at 37 ℃. The contraction effect of bradykinin (1 mg/L) alone was first
assayed. The baseline was resumed by washing the strip and bath with Krebs
solution. Afterwards, TmF was added and preincubated for about 1 min following
the addition of same dose of bradykinin (1 g/L). The potentiating effect of TmF
to bradykinin-induced contraction was statistically analyzed. One potentiating “unit”
was defined as the amount of peptide per liter needed to double the
bradykinin-induced contraction with bradykinin dose of 1 mg/L.

2.3 Hypotensive effect

The normal
diastolic pressure of cat was (65±5) mmHg, bradykinin alone caused (5±3) mmHg
descent to the normal blood pressure, while TmF could potentiate
bradykinin-induced pressure-lowering effect with (14±2) mmHg; it was about
three-fold to bradykinin alone (Table 1).

Table
1   Hypotensive effect of TmF to
bradykinin-induced blood-pressure-lowering effect in cat

The blood-pressure-lowering
effect of two doses of bradykinin was respectively recorded. And then, the
different combinations of TmF + bradykinin were checked. The decreasing values
of blood pressure were analyzed. Results are shown as x±s (n=3).

2.4 ACE inhibition effect

The ACE activity
curve descended with the increasing of TmF concentration, 2 μg of TmF
approximately caused IC₅₀ of angiotensin-converting enzyme
hydrolyzing activity to bradykinin.

3 Discussion

The TmF was
purified from the venom of T. mucrosquamatus. The molecular mass of TmF was
1.1107 kD, which was in accordance with the calculated value of 1.1106 kD. The
complete amino acid sequence of TmF was determined to be pGlu-Gly-Arg-Pro-Leu-Gly-Pro-Pro-Ile-Pro-Pro,
which showed a high homology with venom BPPs from other genera. Except BPP5a,
their sequences were conserved with N-terminal pGlu and C-terminal tripeptide
Ile-Pro-Pro. The variable amino acids usually exist at the middle position with
Gly, Pro, Arg or Trp (Table 2). The sequences of both BPPA and TmF were
identical except one amino acid difference, although two undecapeptides were
isolated from the venom of Agkistrodon andTrimeresurus genera, respectively.
That indicated that venom BPPs probably came from a same ancestor.

Table
2 Amino acid sequences of TmF and alignment with other bradykinin-potentiating
peptides

*Data of this work. The
conserved amino acids at N- and C-termini are bolded; one amino acid difference
between TmF and BPPA was red.

cDNA cloning of
BPPs revealed that the nucleotide sequences encoding different types of BPPs
were tandemly aligned in precursor, one or more mature BPPs or its analogues
were postulated to arise from the same ancestral peptide-coding region[17,18].

Like other venom
BPPs, TmF also exhibited dual biological or pharmacological activity. The
potentiating “unit” of TmF was (1.13±0.3) unit (mg/L), it was lower than those
of captopril and bradykinin-potentiator B, whereas higher than those of BPF5a
and peptide P (Table 3).

Table
3 Comparison of the potentiating “unit” of TmF, captopril,
bradykinin-potentiator B, BPf5a and peptide P

Pu*,
potentiating unit: milligrams of peptide per liter to double the magnitude of
contraction of a dose of bradykinin (1 g/L) on guinea-pig ileum in vitro.
Results are shown as x±s (n=3).

Angiotensin-converting
enzyme (ACE), a zinc-metallopeptidase releasing a C-terminal dipeptide[19], can
catalyze the breakdown of bradykinin into inactive products[20]. TmF, an ACE
inhibitor, can block the ACE, thus the ACE activity decreased with the
increasing dose of TmF (Fig.4).

Fig.4 Inhibition of the angiotensin-converting enzyme by TmF

Different doses of TmF were incubated with angiotensin-converting
enzyme (50 μg) in 1 mL of Krebs solution at 37 ℃ for about 30 min,
respectively, the residual activity of ACE was then assayed with bradykinin.
The hydrolyzing effect of ACE to BK in the absence of TmF was used as a
control. Results are shown as x±s (n=3).

The mechanism of
dual biological activity exhibition of venom BPPs was complicated. Cushman et
al.[21, 22] assumed that BPP was a competitive inhibitor to kininogenase
II, which was capable of degrading bradykinin, thus bradykinin-potentiating
effect was increased indirectly. However, He et al.[23] pointed out that
the bradykinin-potentiating effect of these peptides on the bradykinin-induced
contraction of guinea-pig ileum in vitro was not disturbed when kininogenase II
was inhibited, that means there exists no interaction between BPPs and
kininogenase II. Whereas this viewpoint didn t exclude the inhibition mechanism
of BPP to kininogenase II in vivo[24－26]. In further research on venom BPPs, a new type of peptide POL
236[27] isolated from the venom of Crotalus atrox exerted only
bradykinin-potentiating activity without inhibitory activity to ACE, although
the amino acid sequence of POL 236 was identical to peptide P, which exhibited
dual biological activities, except only one amino acid difference. So the real
mechanism of BPP dual biological activity remains to be clarified. Nevertheless,
some recent studies indicate that the biological effects of bradykinin are
exerted through the activation of one transmembrane G-protein-coupled receptor
denoted as B2 receptor[2], venom BPPs block B2 receptor desensitization,
thereby potentiating bradykinin effect beyond blocking its hydrolysis[28].

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__________________________________________

Received: January 13, 2003   Accepted: April 18, 2003

This work was supported by a grant from the
Yunnan Youth Science Foundation of China (No. 1999C0019Q)

*Corresponding author: Tel, 86-871-5192476;
Fax, 86-871-5191823; e-mail, [email protected]
or [email protected]