Hemorrhagic activity of Bothrops venoms determined
by two different methods and relationship with proteolytic
activity on gelatin and lethality
Adolfo Rafael de Roodt*, Silvana Litwin, Juan Carlos Vidal1
Instituto Nacional de Produccion de Biologicos—A.N.L.I.S, Dr Carlos G. Malbran, Av. Velez Sarsfield 563, CP 1281 Buenos Aires, Argentina
Received 19 September 2002; accepted 9 December 2002
Abstract
The changes in hemorrhagic activity, proteolytic activity on gelatin and the lethal potency of four Bothrops venoms treated at
different pH values or with EDTA were studied. Venoms from B. alternatus, B. jararaca, B. moojeni and B. neuwiedii of
Argentina were preincubated at pH 5.8, 5.1 or 3.8 or with EDTA and the hemorrhagic activity expressed as size of the
hemorrhagic lesion or as the amount of hemoglobin extracted, the proteolytic activity on gelatin and the lethal potency were
determined. Although the MHDs recorded in rats were 19–56 fold higher than those recorded in mice, the A550 extracted per
gram of hemorrhagic haloes was very similar in rats or mice independent of the venom dose. Inhibition of proteolytic activity
after preincubation at pH 5.1 or 3.8, agrees with the decreased amount of hemoglobin extracted from the hemorrhagic haloes,
and with the increase in mean survival time after the i.p. injection to mice. Preincubation with EDTA resulted in 80% inhibition
of hemorrhagic activity of B. jararaca venom and complete inhibition with the other Bothrops venoms tested. Measurement of
the amount of hemoglobin extracted gives significant information in comparative studies, not available by measurement of the
size of hemorrhagic haloes.
q 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Venoms; Hemorrhage; Enzymatic activity; Bothrops; Snakes
1. Introduction
Envenomation by Bothrops bites is characterized by a
complex series of pathological alterations including hemor-
rhages, which perhaps represent one of the most conspic-
uous toxic activities in bothropic envenoming. After
Bothrops bite the hemorrhage may become systemic and
contribute significantly to the lethal potency of these
venoms. Hemorrhages are principally caused by metallo-
proteinases, enzymes that are responsible for degrading
proteins of extracellular matrix, they also have citotoxic
effect on endothelial cells and acts on components of
the haemostatic system (Kamiguti et al., 1996). Some
metalloproteinases induce hemorrhage by directly affecting
mostly capillary blood vessels cleaving in a highly selective
fashion key peptide bonds of basement membrane com-
ponents affecting the interaction between basement mem-
brane and endothelial cells (Gutierrez and Rucavado, 2000).
This hemorrhagic enzymes contains one single Zn2þ ion
per molecule (Bjarnasson and Fox, 1983, 1988, 1994;
Stocker et al., 1995) and have different molecular weights
due to the presence of additional protein domains and
structurally similar catalytic domain (Bjarnasson and Fox,
1994, 1995; Hite et al., 1992, 1994) which governs the
primary proteolytic specificity (Jia et al., 1996). The crystal
structure (Gomis-Ruth et al., 1993; Gutierrez and Rucavado,
2000; Zhang et al., 1994) show that they have a common
methionine turn below and C-terminal to a helical segment
containing two histidines (His 142 and His 146) of the three
histidines residues (His 152) involved in Zn2þ binding site
(Jia et al., 1996). Treatment at low pH values or with
0041-0101/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0041-0101(02)00392-6
Toxicon 41 (2003) 949–958
www.elsevier.com/locate/toxicon
1 In Memoriam.
* Corresponding author. Tel.: þ54-11-4303-1807–11x250; fax:
þ54-11-4303-2492.
E-mail address: [email protected] (A.R. de Roodt).
chelating agents produces structural alterations leading to
the irreversible loss of both, proteolytic and hemorrhagic
activities (Sanchez et al., 1995a).
Hemorrhagins are capable of degrading laminin, fibro-
nectin and Type IV collagen (Bjarnasson et al., 1988;
Baramova et al., 1990; Markland, 1998). In addition, these
enzymes hydrolyze fibrinogen, casein, dimethyl casein,
oxidized insulin B-chain (Mandelbaum et al., 1982;
Bjarnasson and Fox, 1994; Civello et al., 1983; Maruyama
et al., 1992; Sanchez et al., 1995 a,b) and gelatin (Bee et al.,
2001; Markland, 1998).
Hemorrhagic activity of snake venoms can be quantified
by the intradermal injection of a venom in an experimental
animal, which produces a readily observable hemorrhagic
halo on the dermic side of the skin, such as in the rabbit skin
test of Kondo et al. (1960), which have been adapted for rats
and mice (Theakston and Reid, 1983; Gutierrez and Chaves,
1980). This activity can be determined as minimal
hemorrhagic dose, that is represented as the amount of
venom which produces an hemorrhagic halo with an average
of the major perpendicular diameters ½ðd1 þ d2Þ=2� set to
1.0 cm (Theakston and Reid, 1983) or an hemorrhagic halo
of 1.0 cm2 (Gutierrez and Chaves, 1980). There were
performed several assays to study the hemorrhagic activity
like the measurement of hemoglobin in muscle inoculated
with venom (Ownby et al., 1984), the measurement of the
hemorrhagic halo in blood vessels that surround and supply
the chick embryo (Sells et al., 1997) or dog lung (Bjarnasson
and Fox, 1994) or the measurement of hemoglobin
contained in the hemorrhagic haloes in skin (Esmeraldino
et al., 1999; de Roodt et al., 2000). Approximate
quantification of the hemorrhagic activity by measurement
of the average diameter of the hemorrhagic haloes obtained
after the intradermical injection of venom (Kondo et al.,
1960; Oshaka et al., 1966; Theakston and Reid, 1983) is
specific, fast and reproducible. However, problems arise
when the hemorrhagic haloes obtained with several venoms
have similar sizes but differ in color intensity, reflecting
differences in hemoglobin content. In order to avoid this
inconvenient, the measurement of the amount of hemo-
globin in a sample of skeletal muscle injected with venom
(Ownby et al., 1984), scanning the color intensity of the
hemorrhagic haloes (Esmeraldino et al., 1999), or measure-
ment of the amount of hemoglobin extracted from the
excised hemorrhagic haloes (de Roodt et al., 2000) have
been proposed.
Data reported on Bothops venoms indicate that although
measurement of the size of the hemorrhagic haloes or the
amount of hemoglobin extracted from the excised hemor-
rhagic haloes are both functions of the intensity of the
response (i.e. hemorrhage), they give different results when
employed to quantify the same effect (de Roodt et al., 2000).
The fact that the amount of blood leaked as measured by
hemoglobin, appears not to be proportional to the size of the
hemorrhagic haloes rises severe questions on the validity of
measurements employing one or the other parameter. Since
this problem appeared frequently in our comparative studies
of the hemorrhagic activity in the venoms from different
Bothrops species from Argentina, we have compared the
results obtained by measuring the average diameters of the
haloes, the weights of the excised hemorrhagic haloes and
the amount of hemoglobin extracted from these excised
haloes produced after the i.d. injection to mice and rats of
either crude venoms or venom samples in which the
hemorrhagic activity was partially or completely inacti-
vated. Taking into account that treatment of hemorrhagins at
low pH values or with chelating agents like EDTA produces
structural alterations leading to the irreversible loss of both,
proteolytic and hemorrhagic activities (Sanchez et al.,
1995a), we evaluate the proteolytic and hemorrhagic
activities after total or partial inactivation.
These results were employed to determine the dose-
effect curves as well as to analyze the correlation between
hemorrhagic, proteolytic activity on gelatin and lethal
potency among those venoms and to compare the hemor-
rhagic activity by the measurement of the hemorrhagic halo
or the amount of hemoglobin extracted.
2. Materials and methods
2.1. Venoms
Whole venoms were obtained from healthy, adult
specimens of Bothrops (B.) alternatus; B. jararaca; B.
moojeni and B. neuwiedii, kept at the Serpentarium of the
Instituto Nacional de Produccion de Biologicos—A.N.L.I.S.
‘Dr Carlos G. Malbran’. The venoms were collected Petri
dishes, dried in vacuo and kept at 220 8C.
2.2. Animals
Mice (CF-1, 18–22 g) and rats (Whistar, 180–220 g)
were provided by the animal facility of the Instituto
Nacional de Produccion de Biologicos A.N.L.I.S. ‘Dr
Carlos G. Malbran’. They were kept under controlled
environmental conditions with dark/light cycles of 12 h and
received commercial rodent food and water ad libitum.
2.3. Reagents
All the reagents employed were analytical grade. Protein
determinations were performed according to Bradford
(1976) using the Bio-Rad Protein Assay Kit.
2.4. Determination of the activity and composition stability
of venom samples
The instability by autodegradation of Bothrops venoms
is well known (Vidal and Stoppani, 1970; Souza et al.,
2001). In order to establish the range of pH values of interest
A.R. de Roodt et al. / Toxicon 41 (2003) 949–958950
for studies of stability of proteolytic activity on gelatin, two
living specimens of B. alternatus were milked into a pH-Stat
(Radiometer, Copenhagen) titration vessel (0.5–5.0 ml) so
that the tips of the fangs passed through a layer of neutral
liquid paraffin, in order to prevent the exchange of CO2 with
the air. The pH, recorded continuously in the stirred sample
was 5.897 ^ 0.16 and varied by less than 0.01 pH units
during 12 h. Venom samples withdrawn after different
intervals of time were analyzed by SDS–PAGE. No
changes in the electrophoretic profile were observed during
12 h incubation. Once the pH conditions for optimum
stability were established, the dried venoms were dissolved
in 0.15 M NaCl, 20 mM sodium acetate buffer of the desired
pH into a (0.5–5.0 ml) titration vessel at 23 8C. The final pH
was controlled and, if necessary, adjusted in the pH-Stat
(kinetic mode) until a stable reading was obtained for 5 min.
The venom solutions were centrifuged for 10 min at 1200g
and the supernatant was placed in a clean titration vessel
kept at 23 8C. The pH was tested again and the sample was
kept under continuous stirring until use.
2.5. Proteolytic activity on gelatin
Gelatin was chosen as substrate to determine this activity
because in previous reports we found good correlation
between the liquefaction of gelatin and the MHD (cited in de
Roodt et al. (1999)). Gelatin (Sigma, from porcine skin, 300
Bloom) was suspended at 1% in 0.15 M NaCl, heated for 1 h
in a boiling water bath under continuous stirring, and
allowed to cool down up to 25 8C. Five ml were pipetted into
a 0.5–5.0 ml titration vessel of a pH-Stat (Radiometer) and
adjusted to 23 8C and pH 8.0 with 20 mN NaOH under a
current (15 ml/min) of humid argon. After stabilization, the
consumption of NaOH to maintain pH 8.0 was recorded for
10 min. hence, samples of different venom solutions were
added, and the consumption of alkali to maintain pH 8.0 was
recorded for additional 10 min period. The differences in
rates of alkali consumption in the presence and in the
absence of venom measure the rate of reaction. pH 8.0 was
routinely employed in order to improve titration efficiency.
One unit of proteolytic activity was defined as the
consumption of 1.0 nmol NaOH per min. Specific activities
were calculated in units per mg protein.
To determine the proteolytic activity of the venom
treated at different pH values, the proteolytic activity on
gelatin as a function of pH was studied by incubating the
venom solution for 1 h at pH values 5.8 (pH in which
the proteolytic activity is most stable), 5.1 (pH close with
the apparent pK value) and 3.8 (pH in which proteolytic
activity on gelatin is undetectable; see Results section).
Dried venoms (75–80 mg with B. jararaca; B. moojeni or B.
neuwiedii venoms; 63–65 mg with B. alternatus venom)
were dissolved in 1.8 ml of 0.15 M NaCl, 20 mM sodium
acetate buffer of the desired pH in a titration vessel at 23 8C.
The final pH was adjusted, if required in the pH-Stat.
After centrifugation, 1.4 ml of supernatant was transferred
to a clean titration vessel kept at 23 8C, the pH was tested
again and the sample was incubated under continuous
stirring. At different intervals of time 0.2 ml aliquots were
mixed with 0.28 ml of 0.15 M NaCl and the sample was
adjusted to pH 5.8 in the pHStat. The final volume was
adjusted to 0.5 ml, the time recorded and the enzymatic
activity on gelatin was measured using 25–50 ml samples as
described.
To determine the activity of the venom in the presence of
EDTA, Bothrops venoms (0.3 mg/ml) were incubated for
30 min at 23 8C in 0.15 M NaCl containing 3.0–1.0 mM
disodium EDTA and adjusted to pH 7.2. The stability of
proteolytic activity on gelatin was tested with venoms
treated at different pH levels and with venoms preincubated
with EDTA as described above.
2.6. Hemorrhagic activity
With all the Bothrops venoms employed, no significant
differences in hemorrhagic activity were observed with
samples used immediately after dissolution in 0.15 M NaCl
or after 1 h preincubation at pH 5.8.
To determine the MHD, different doses of each venom
(0.5–500 mg) adjusted to pH 5.8 were injected intradermi-
cally in mice, using 3–5 animals per dose level or in rats
using 3–5 points per dose level (Theakston and Reid, 1983).
To determine the inhibition of the hemorrhagic activity
of venom treated at different pH, venom samples were
preincubated at 23 8C for 1 h in 0.15 M NaCl, 20 mM
sodium acetate buffer at pH 5.8, 5.1 and 3.8. After
incubation, the samples were readjusted to pH 5.8 with the
pH-Stat, the final volume was adjusted and the hemorrhagic
activity was assayed by intradermal injection of 0.1 ml
samples in rats. Rats, under light anesthesia with ketamine
(50 mg/kg) were injected intradermically with three samples
(one preincubated at pH 5.8 in the back as control, one
incubated at pH 5.1 and another incubated at pH 3.8 in each
side) of up to 600 mg of each venom, in order to compare in
the same animal the effect of a control sample with that of
samples treated at different pH values.
To determine the inhibition of the hemorrhagic activity
of venom preincubated with EDTA, venom samples
(400 mg) were incubated for 30 min at 23 8C in 0.3 ml of
0.15 M NaCl containing 0.02 to 3.0 mM EDTA adjusted to
pH 7.4. These samples were employed to measure
hemorrhagic activity in rats (n ¼ 5 for each venom) using
a similar scheme. Venom without treatment was injected in
the back and EDTA-treated venom samples injected in the
sides. This methodology was also employed to decrease
individual variability of the response.
Three hours after the injection, the animals were
sacrificed, the skin was removed and the major perpendicu-
lar diameters of the hemorrhagic haloes were measured with
a caliper. The hemorrhagic haloes were immediately cut,
weighed and homogenized for 3.0 min in a Tenbroeker
tissue grinder with 5.0 ml of distilled water as described
A.R. de Roodt et al. / Toxicon 41 (2003) 949–958 951
previously (de Roodt et al., 2000). After centrifugation,
2.0 ml of the supernatants were transferred to glass tubes,
extracted with the same volume of chloroform and 100 ml of
clear aqueous phases (or dilutions) were pipetted in 96-well
plates in which the hemoglobin content was determined by
the absorbance at 550 nm directly measured (Al-Abdulla
et al., 1991) or by measurement of peroxidase activity as
described previously (de Roodt et al., 2000).
The hemorrhagic activity of each venom sample was
expressed (a) as the average diameter ð½d1 þ d2�=2Þ
measured in cm; (b) as the weight of the excised
hemorrhagic haloes (in g ^ SD) or (c) as the amount of
hemoglobin extracted from the excised hemorrhagic haloes
in A550 units per ml (^SD) of the aqueous phases.
2.7. Lethal potency
The lethal potency of samples of the different Bothrops
venoms preincubated at different pH values after readjust-
ment to pH 5.8 or after treatment with EDTA was
determined.
After 1 h preincubation at pH 5.1 or 3.8 as described
above, the Bothrops venom solutions were adjusted to pH
5.8, and their lethal potency were studied on mice by
comparing the mean survival time after the i.p. injection of a
(nominal) dose of 3.0 LD50 (i.p.) of the treated venom
samples and control venoms.
To determine the lethal potency of venoms previously
treated with EDTA, 5.0 mg/ml of venom of B. alternatus
and B. jararaca were incubated for 30 min at 23 8C in
0.15 M NaCl, 0.1 M EDTA, 20 mM Sodium acetate buffer
pH 6.5. The sample was chromatographed in a Sephadex G-
25 column pre-equilibrated with 0.15 M NaCl–20 mM
sodium acetate buffer pH 5.8, in order to eliminate the
excess of EDTA. As controls there were used samples
treated in the same way but in the absence of EDTA. After
measurement of protein content of the samples, mice were
injected i.p. with nominal 3.0 LD50 of treated venom or
venom control.
2.8. Statistics
All data are presented as mean ^ SD. Linear and non-
linear regression analysis as well as tests for statistical
significance were performed by using the combined
Prisma—StatMate software (GraphPad Software, San
Diego, CA).
3. Results
3.1. Proteolytic activity
The specific proteolytic activities (units per mg
protein) of the Bothrops venoms employed were 508 ^ 33
(B. alternatus), 232 ^ 35 (B. neuwiedii); 260 ^ 40
(B. moojeni) and 156 ^ 35 (B. jararaca).
Except for the sample preincubated at pH 5.8, which
changed by less than 10% with time without significant
changes in the SDS–PAGE profiles, the proteolytic activity
on gelatin ða1; a2;…:; anÞ obtained from a venom sample
preincubated at pH values lower than 5.8 for t1; t2;…; tn min
decreased with the time of incubation as an exponential
decay of the form a2=a1 ¼ exp½2k0ðt2 2 t1Þ� and the
apparent first-order constant (k0) for each pH value was
calculated by non-linear regression analysis of the activity
vs. time curves.
The proteolytic activity on gelatin of the Bothrops
venoms studied was significantly inhibited by preincubation
at pH values lower than 5.0 (Table 1, Fig. 1B). For each
fixed pH value, the rate of enzyme inactivation could be
described as an exponential decay, the value of the apparent
first-order constant k 21 (inactivation) increased as the pH
decreased. The decrease in proteolytic activity on gelatin of
B. alternatus, B. neuwiedii and B. moojeni venoms after
incubation at different pH values exhibited a similar profile.
The enzymatic activity decreased smoothly with time at pH
5.8 (k0 , 0.12 h21 with B. alternatus to k0 , 0.135 h21 with
B. moojeni venom). The rates of inactivation increased at pH
5.5 (k0 , 0.18 h21 with B. neuwiedii venom; k0 , 0.32 h21
Table 1
Effect of preincubation at different pH values on hemorrhagic activity of Bothrops venoms
Venoms pH 5.8 pH 5.1 pH 3.8
Average diameter
(cm)
Hemoglobin
(A550)
Average diameter
(cm)
Hemoglobin
(A550)
Average diameter
(cm)
Hemoglobin
(A550)
B. alternatus 0.94 ^ 0.16 0.43 ^ 0.04 0.66 ^ 0.06 0.25 ^ 0.10 0.18 ^ 0.09 0.06 ^ 0.02
B. neuwiedii 1.07 ^ 0.18 0.12 ^ 0.06 0.86 ^ 0.10 0.08 ^ 0.02 0.24 ^ 0.16 0.02 ^ 0.01
B. moojeni 1.13 ^ 0.07 0.23 ^ 0.05 0.99 ^ 0.11 0.14 ^ 0.02 0.54 ^ 0.10 0.04 ^ 0.02
B. jararaca 0.94 ^ 0.43 0.38 ^ 0.02 0.88 ^ 0.08 0.30 ^ 0.03 0.22 ^ 0.02 0.05 ^ 0.03
The hemorrhagic activity of the different venoms treated at different pH values was determined by measuring the average diameters or by
the hemoglobin extracted from the hemorrhagic halo. The diameters are expressed in cm as the mean ^ SD. The hemoglobin extracted is
expressed in A550/ml as the mean ^ SD.
A.R. de Roodt et al. / Toxicon 41 (2003) 949–958952
with B. alternatus and B. moojeni venoms) and at pH 5.0
(k0 , 0.38 h21 with B. neuwiedii venom and k0 , 0.53 with
B. alternatus and B. moojeni venoms). Higher rates of
inactivation were observed at pH values lower than 5.0. The
plots of residual activity after 1 h incubation as a function of
pH (Fig. 1A) were fitted to simple sigmoid curves with pK
values about 5.3. The plots of the logarithm of the residual
activity after 1 h incubation as a function of the pH (Fig. 1B)
exhibited an inflection at pH 5.3 in which the slope changes
from 0 to 1.0 and a second one at pH 4.5, in which the slope
changes from 1.0 to 3.0. The inflection at pH 5.3 may
represent the molecular dissociation constant of the first
ionic species to be protonated as the pH is moved down from
that of maximum stability. Compared to the magnitude of
the known group constants, it is close to that reported for the
imidazolium group of histidine. Further changes in slope in
the logarithmic plot suggest that the ionization of more than
one group may be involved in enzyme stability. The pK
values may be shifted if a conformational change facilitates
the loss of protons or permits the binding of a polyvalent
cation (Tripton and Dixon, 1979).
The rate of inactivation of proteolytic activity on gelatin
with B. jararaca venom changed only poorly in the range
from pH 5.8 (k0 , 0.09 h21) to pH 5.0 (k0 , 0.12 h21). The
rate increased significantly (k0 , 0.42 h21) at pH 4.5 an
increased further at lower pH values. The plot of residual
activity after 1 h preincubation as a function of pH (Fig. 1A)
fitted a simple sigmoidal curve with an apparent pK value
about 4.2. The plot of logarithm of the residual activity after
1 h preincubation as a function of pH (Fig. 1B) showed
again several linear portions with slopes of about 3.0 (from
pH 3.5 to 4.0); about 1.0 (pH 4.0–4.5) and became almost
horizontal (slope zero) from pH 4.5 to 5.8. The inflection at
pH 3.8 may reflect the ionization of an acididic group, like
the active site Glu 143.
No proteolytic activity on gelatin could be detected with
any of the venoms employed in this study after 1 h
incubation at pH 3.8.
On this basis, proteolytic activity on gelatin and
hemorrhagic activity of each venom were compared after
preincubation of the venom samples for 1 h at three pH
values, namely (a) pH 5.8, at which proteolytic activity is
most stable, (b) at pH 5.1, close to the apparent pK values
and (c) at pH 3.8, at which proteolytic activity on gelatin is
undetectable.
Incubation of B. alternatus, B. neuwiedii and B. moojeni
venoms with 1.0 mM EDTA resulted in complete inacti-
vation of proteolytic activity on gelatin. On the other hand,
after incubation of B. jararaca venom with 1.0 mM EDTA
proteolytic activity on gelatin was incompletely inactivated,
and a fraction (17–30%) of the initial proteolytic activity
remained still measurable.
3.2. Hemorrhagic activity
The results, expressed as average diameters of the
hemorrhagic haloes ð½d1 þ d2�=2Þ and as amount of
hemoglobin extracted (A550) are presented in Tables 1 and
3. With all the Bothrops venoms employed, no significant
differences in hemorrhagic activity were observed with
samples used immediately after dissolution in 0.15 M NaCl
or after 1 h preincubation at pH 5.8. The porcentual
inhibitions by preincubation at different pH levels are
presented in Table 2. The MHD found in rats was about
170 ^ 20 mg for B. alternatus, 140 ^ 15 mg for B.
jararaca, 210 ^ 30 mg for B. neuwiedii and 150 ^ 50 mg
for B. moojeni. The MHD in mice of these venoms was
9.0 ^ 0.3 mg for B. alternatus, 2.5 ^ 0.5 mg for B.
jararaca, 17.1 ^ 0.4 mg for B. neuwiedii and 30 ^ 4 for
B. moojeni venom (Table 3).
The values of hemoglobin extracted from hemorrhagic
haloes were very similar for each venom, whereas the
MHDs showed a high variation, ranging the relation MHD
rats / mice from five-fold with B. moojeni venom to 56 with
B. jararaca venom. As shown in Fig. 2 and Table 3, plots of
the ratio [average amount of hemoglobin extracted]/[weight
in grams of the excised hemorrhagic halo] is a constant k,
Fig. 1. (A) Plots of residual proteolytic activity on gelatin of
Bothrops venoms after 1 h preincubation at 23 8C as function of pH.
The data are presented as mean ^ SD (n ¼ 3). (X) B. alternatus,
(P) B. jararaca, (A) B. neuwiedii; (B) plots of the logarithm of
residual activity (as percentage) of Bothrops venoms after 1 h
preincubation at 23 8C as a function of pH. (B) B. alternatus, (P) B.
jararaca, (A) B. neuwiedii. (V) B. moojeni.
A.R. de Roodt et al. / Toxicon 41 (2003) 949–958 953
characteristic of each Bothrops venom and independent of
the venom dose. The numerical values of k (average A550/
ml extracted per gram of excised hemorrhagic haloes) were
3.54 [95% i.c. 3.13–3.95] for B. alternatus, 2.33 [2.14 to
2.52] for B. jararaca, 1.67 [1.24–1.89] for B. moojeni and
1.16 [0.83–1.49] for B. neuwiedii. (i.e. close to those
already published for these venoms in experiments on mice,
see Table 3).
In all the cases the incubation of the venoms at pH 5.1
reduced the hemorrhagic activity. The hemorrhage was
inhibited in over 80% when the residual activity was
determined by measurement of the hemoglobin from the
hemorrhagic halo. When the residual activity was estimated
by measurement of the average diameters of the hemor-
rhagic halo, the inhibition was estimated in the order of 50–
80%. See Table 2.
Treatment of B. alternatus and B. neuwiedii venoms with
0.15 mM EDTA and of B. moojeni venom with 0.3 mM
EDTA resulted in the complete loss of hemorrhagic activity
(measured by both, the average diameter of the hemorrhagic
haloes or the amount of hemoglobin extracted) after the i.d.
injection to rats. On the other hand, incubation of B.
jararaca venom with EDTA up to 0.3 mM inhibited its
hemorrhagic activity after intradermal injection to rats up to
20–26% (by the average diameter of the hemorrhagic
haloes) and up to 70–83% (by measurement of the amount
of hemoglobin extracted from the excised hemorrhagic
haloes).
3.3. Lethal potency
With all the Bothrops venoms employed, no significant
differences in lethal potency were observed with samples
used immediately after dissolution in 0.15 M NaCl or after
1 h preincubation at pH 5.8. The mean survival times were
30–40 min., and all the animals died within 1 h post
injection. Post mortem gross pathological examination
showed generalized hemorrhages in all cases.
Preincubation of Bothrops venoms at pH 5.1 increased
the mean survival time in all cases (Fig. 3). With B.
neuwiedii venom, the mean survival time increased to 1.3 h,
however, all the animals died 2 h after injection. With B.
jararaca venom the mean survival time was about 1.2 h and
15% of the animals survived 24 h after injection. With B.
alternatus and B. moojeni venoms, the mean survival times
were 2.3 and 2.8 h, respectively, and 14–20% of the animals
survived 24 h after the injection. Post-mortem gross
pathological examination again showed generalized
hemorrhages.
Preincubation of Bothrops venoms at pH 3.8 strongly
reduced the lethal potency of all the venoms tested (Fig. 3).
However, except for B. moojeni venom, 16–20% of
Table 2
Inhibition of proteolytic activity on gelatin and hemorrhagic activities using venoms treated at different pH values
Venom Percentage of inhibition of venoms treated at pH 5.1 Percentage of inhibition of venoms treated at pH 3.8
Inhibition of
proteolysis
Inhibition of
hemorrhage
(A550/ml)
Inhibition of
hemorrhage
(diameter)
Inhibition of
proteolysis
Inhibition of
hemorrhage
(A550/ml)
Inhibition of
hemorrhage
(diameter)
B. alternatus 42.0 ^ 2.1 42.0 ^ 16.8 30.0 ^ 2.7 100.0 86.0 ^ 28.7 80.9 ^ 40.1
B. neuwiedii 34.3 ^ 3.2 33.3 ^ 8.3 20.0 ^ 5.0 100.0 83.3 ^ 41.7 77.6 ^ 51.7
B. moojeni 45.0 ^ 5.0 39.0 ^ 5.6 12.0 ^ 1.3 100.0 82.6 ^ 41.3 52.2 ^ 9.7
B. jararaca 18.2 ^ 1.1 21.0 ^ 2.1 6.0 ^ 5.4 100.0 86.8 ^ 52.1 76.6 ^ 7.0
The table indicates the proteolytic activity on gelatin and the hemorrhagic activity of venoms treated at different pH levels. The values in
table represent the percentage of inhibition of the proteolytic activity on gelatin and the hemorrhagic activity determined by measurement of
diameters of the hemorrhagic halo or by the hemoglobin extracted from the hemorrhagic halo. The values are expressed as the mean ^ SD.
Table 3
Hemorrhagic activity in mice and rats determined by both methods
Venom MHD mice MHD rats MHD rats/mice k Rats (A550/ml g21) k Mice (A550/ml g21) k Rats/mice
B. alternatus 9.0 ^ 3.0 170 ^ 20 19 3.54 (3.13–3.95) 3.61 (2.91–4.31) 0.98
B. jararaca 2.5 ^ 0.5 140 ^ 15 56 2.33 (2.14–2.52) 3.19 (2.86–3.53) 0.73
B. moojeni 30.0 ^ 4.0 150 ^ 50 5 1.67 (1.24–1.89) 2.17 (1.97–2.51) 0.78
B. neuwiedii 17.1 ^ 0.4 210 ^ 30 12 1.16 (0.83–1.49) 1.02 (0.95–1.08) 1.13
The table indicates the values of MHD (expressed as mg ^ S.D. of venom) or the hemoglobin extracted from 1 g of hemorrhagic halo
expressed as k (A550/ml g21). The 95% c.i. are indicated into brackets. MHD rats/mice indicates the relation between MHD of rats and mice in
each venom. k rats/mice indicates the relation in hemoglobin extracted per gram of hemorrhagic halo between rats and mice.
A.R. de Roodt et al. / Toxicon 41 (2003) 949–958954
the animals died about 2 h after the injection of B. neuwiedii
venom and a similar percentage of animals died 24–48 h
after the injection of B. alternatusand B. jararaca venoms,
without exhibiting generalized hemorrhages upon post
mortem gross pathological examination.
When mice were inoculated with venom previously
treated with EDTA as described, the survival time was over
12 h to mice injected with B. jararaca venom and over 24 h
to those injected with B. alternatus venom. The controls
died 30–45 min. post injection.
4. Discussion
The inhibition of the proteolytic and hemorrhagic
activities by preincubation of venoms at different pH values
or with EDTA was observed in all the cases, however some
discrepancies were found in the expected values of
inhibition depending of the method used to determine the
hemorrhagic activity (Tables 1 and 2).
The most remarkable observation was that, with all the
Bothrops venoms employed, the rates of inactivation of
proteolytic activity on gelatin at pH 5.1 describes quanti-
tatively the decrease in the amount of hemoglobin extracted
from the excised hemorrhagic haloes (Table 2). In fact, the
pseudo-first order rate constants (k0) for the decay of
proteolytic activity on gelatin at pH 5.1 were about 0.58 h21
(B. alternatus), 0.42 h21 (B. neuwiedii), 0.55 h21 (B.
moojeni) and 0.20 h21 (B. jararaca). Thus, after 1 h
preincubation at pH 5.1, the initial activity will be reduced
Fig. 2. Plot of A550 per gram of excised hemorrhagic haloes (k) as
function of the venom dose in rats. The value of k for each venom is
obtained from the intercept and is expressed as mean [95% c.i.
limits] (n ¼ 5). (B) B. alternatus (k: 3.54 [3.13–3.95]), (P) B.
jararaca (k: 2.33 [2.14–2.52]), (V) B. moojeni (k: 1.57 [1.14–
2.09]), (A) B. neuwiedii (k: 1.16 [0.83–1.49]).
Fig. 3. Effect of preincubation for 1 h at different pH values of Bothrops venoms on the mean survival time of mice after intraperitoneal
injection. Mice (groups of, at least 6 animals per curve) were injected intraperitoneally with samples of B. jararaca (A) B. neuwiedii (B) B.
alternatus (C) and B. moojeni (D) venoms preincubated for 1 h at 23 8C at pH 3.8 (a), 5.1 (b) and 5.8 (c) at a (nominal) dose of 3.0 DL50. Each
curve was obtained by triplicate and the percentage of surviving animals is presented as a function of the time in hours after injection.
A.R. de Roodt et al. / Toxicon 41 (2003) 949–958 955
by 42% (B. alternatus), 34.3% (B. neuwiedii), 45% (B.
moojeni) and 18.2% (B. jararaca), in close agreement with
the degrees of inhibition found experimentally when the
hemoglobin content of the hemorrhagic area were measured
(Table 2). After preincubation at pH 3.8, in agreement with
the complete inhibition of proteolytic activity on gelatin, the
decrease in the amount of hemoglobin extracted from
the hemorrhagic haloes ranged between 80 and 90% with all
the Bothrops venoms studied (Tables 1 and 2).
The effect of preincubation at different pH values can be
interpreted as the result of the distribution of the enzyme(s)
protein(s) into different ionic species. While the distribution
of the enzyme(s) into different ionic species at pH 5.8 is
surely different that that prevailing at pH 8.0 (i.e. the value
at which the enzyme activity is measured), most of the ionic
species are recovered as catalytically active enzyme. At
lower pH values, a significant fraction of the enzymes(s)
undergoes irreversible inactivation. Readjustment to pH 5.8
after incubation will favor the distribution towards poten-
tially catalytically active species, however, the fraction lost
due to irreversible inactivation will not be recovered.
Preincubation of Bothrops venoms at pH 5.1 or 3.8
increased the mean survival time after the i.p. injection to
mice (Fig. 3), and post-mortem examination suggests that
this effect is related to the inhibition in hemorrhagic activity.
The magnitude of these increases ranged from about two-
fold (B. jararaca), 2.5 to 3-fold (B. neuwiedii) up to 4 to 5-
fold (B. alternatus and B. moojeni). This sequence is
consistent with the decrease in the amount of hemoglobin
extracted from the hemorrhagic haloes, rather than with the
decrease in size of the hemorrhagic haloes.
When the venom was treated by EDTA, no hemor-
rhages were detected and the mean survival time of mice
inoculated with B. alternatus or B. jararaca venoms was
over 24 or 12 h, respectively, while the controls died in
30–45 min. Treatment of B. alternatus, B. moojeni and
B. neuwiedii venoms with 1.0 mM EDTA resulted in
complete inactivation of proteolytic activity on gelatin, as
well as in hemorrhagic activity after the intradermal
injection to rats. In contrast, preincubation of B. jararaca
venom with 1.0 mM EDTA inhibited proteolytic activity
on gelatin by about 85%. The remaining hemorrhagic
activity after i.d. injection to rats was about 20% by the
measurement of diameters and about 73% by the amount of
hemoglobin extracted from the hemorrhagic haloes near the
value of inhibition observed in the proteolytic activity of
this venom after EDTA treatment. The results with
B. jararaca venom suggest that the hemorrhagins of this
venom differ in some characteristics with those from the
other venoms studied and/or that other venom components
are involved in the hemorrhagic process on the site of
injection of this venom.
The concordance between the hemorrhage (specially
when it was determined by the measurement of
hemoglobin), the proteolytic activity and the lethal
potency, suggests a relevant participation of hemorrhagic
metalloproteinases (hemorrhagins) in the lethality caused
by Bothrops venoms. Bothropic venoms produce gener-
alized hemorrhages due the procoagulants and thrombin-
like enzymes, which lead to the fibrinogen consumption
(Kamiguti et al., 1996; Mandelbaum et al., 1982).
Metalloproteinases contribute to the haemostatic disturb-
ances acting on some points of the haemostatic system
(Kamiguti et al., 1996; Markland, 1998), by the loss of
the vascular integrity (Bjarnasson and Fox, 1994;
Gutierrez and Rucavado, 2000) and facilitating the
generalization of the venom components destroying the
extracellular matrix and blood vessels at the site of bite
(Anai et al., 2002). In this work we could observe that
when the proteolytic activity of the venoms was inhibited,
there were not observed generalized hemorrhages and the
lethal potency of the venoms diminished, indicating the
importance of this enzymes in the systemic envenoming
by Bothrops snakes.
However, although the hemorrhagic activity seems to be
very important in the lethality caused by bothropic venoms,
the contribution of other components to the lethal potency
resulted evident when no macroscopic hemorrhages were
detected in rats or in mice killed by venom in which the
hemorrhagic activity was inhibited. In fact, except for B.
moojeni venom, 15–20% of the mice injected i.p. with
Bothrops venoms preincubated at pH 3.8 died 2 h after
injection (B. neuwiedii) or about 24 h after injection (B.
alternatus and B. jararaca), without evidences of general-
ized hemorrhages upon post-mortem gross pathological
examination.
The differences between the inhibition of proteolytic
activity and hemorrhagic activity using both methods can be
related with the parameters that we are measuring. The
hemoglobin content in the hemorrhagic halo reflects the
amount of blood leakage from blood vessels after its rupture.
Although the area of the hemorrhagic spot reflects the
hemorrhagic activity of the venom, not necessarily indicates
the amount of red blood cells in the tissue but may reflect the
dispersion of the leakage or extravasation of red blood cells
in this area. This interpretation of the hemorrhagic activity
may reflect changes in vascular permeability that not
necessarily indicate the rupture of blood vessels or
destruction of the extracellular matrix, the principal factors
in the production of hemorrhages. These facts make difficult
the interpretation of the real hemorrhagic potency, specially
in comparative studies and may explain the lack of
concordance between the proteolytic activity and hemor-
rhage determined by both methods.
Having the appropriate control, the simple and repro-
ducible measurement of the size of the hemorrhagic haloes
will be the method of choice. However, some problems can
be found when the comparison between different venoms
has to be done.
Leakage of blood is the inherent effect of hemorrhagic
activity induced by Bothrops venoms, however differences
in potency of the hemorrhagic activity in different venoms
A.R. de Roodt et al. / Toxicon 41 (2003) 949–958956
are likely to occur, given the microheterogeneity among
the hemorrhagic metalloproteinases, and that usually,
multiple forms having significant different potencies (Jia
et al., 1996) are observed even in a single venom
(Bjarnasson and Fox, 1994; Mandelbaum et al., 1982).
This is consistent with the observation of differences in
maximum effect (YMax) among Bothrops venoms in mice
when the amount of hemoglobin extracted was plotted as a
function of the venom dose, that showed big differences in
the hemoglobin content from hemorrhagic areas of a similar
size (de Roodt et al., 2000).
The close agreement between the degree of inhibition of
proteolytic activity on gelatin by preincubation at different
pH values or after treatment with EDTA with the decrease in
hemorrhagic activity expressed as the amount of hemo-
globin extracted, strongly suggest that this method reflects
real differences in potency of hemorrhagic activity (Tables 2
and 3) since some differences are not detected when the
hemorrhagic activity is studied by the size of the hemor-
rhagic haloes. In fact, having the MHD in rats of
B. alternatus (about 170 mg), B. jararaca (about 140 mg),
B. moojeni (about 150 mg) or B. neuwiedii venoms (about
210 mg) there is no way to predict that the amount of
hemoglobin extracted (A550/ml) from a similar hemorrhagic
halo will be about three-fold higher with B. alternatus and
B. jararaca venoms or two-fold higher with B. moojeni
venom than the extracted with B. neuwiedii venom, unless
the A550 be measured (Table 3).
When the differences between the MHD of a snake
venom were compared in different species, we found very
big differences, ranging since 5–56 fold. However, when
the k (A550/ml g21) of the different venoms in rats or mice
were compared, the values of A550/ml g21 found for each
venom were quite similar with differences ranging from 2 to
27% (Table 3). The value of k for each venom was defined
previously (de Roodt et al., 2000) as the slope of the plots of
amount of hemoglobin (A550/ml) extracted as a function of
the weight (in g) of the excised hemorrhagic haloes, so that,
if H is the A550/ml extracted from a hemorrhagic halo
weighing Wgrams, is H ¼ k £ W : Therefore kð¼ H=WÞ is a
constant and seems to be an intrinsic property characteristic
of each venom related with the hemorrhagic potency and
independent on the venom dose (Fig. 2).
Combined with those data on inactivation by EDTA, the
results of pH-stability of the hemorrhagic activity of
Bothrops venoms indicate that the measurement of hemo-
globin well correlates with the activity of proteases that acts
on gelatin and with the lethal potency of the venoms. The
measurement of the hemorrhagic haloes may not be a
reliable parameter to determine the remaining hemorrhagic
activity of a venom after such treatments. Consequently, it
can be assumed that the amount of hemoglobin extracted
from the hemorrhagic haloes produced by Bothrops venoms
rather than their size, seems to reflect more accurately the
potency of their hemorrhagic activity.
Acknowledgements
The authors are very grateful to the anonymous
reviewers by their helpful comments to improve the quality
of the paper.
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