Comparative Biochemistry and Physiology Part C 124 (1999) 221 – 226
www.elsevier.com/locate/cbpc
Characterization of hemolytic activity of 3-alkylpyridinium
polymers from the marine sponge Reniera sarai
Petra Malovrh, Kristina Sepčić *, Tom Turk, Peter Maček
Department of Biology, Biotechnical Faculty, Uni6ersity of Ljubljana, Večna pot 111, 1111, Ljubljana, Slo6enia
Received 16 February 1999; received in revised form 28 July 1999; accepted 3 August 1999
Abstract
Polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai act as potent anticholinesterase
agents; in addition they show moderate hemolytic and cytotoxic activities. The hemolytic activity of poly-APS is due to their
detergent-like structure and behavior in aqueous solutions. In this work, the hemolytic activity of poly-APS is analyzed and
compared to that of structurally-related monomeric cationic surfactants. The influence of different divalent cations and lipids on
poly-APS induced hemolysis is discussed. The dimensions of lesions caused by poly-APS in erythrocyte membranes are determined
by the use of osmotic protectants. Finally, the possible role of poly-APS in their natural environment is proposed. © 1999 Elsevier
Science Inc. All rights reserved.
Keywords: 3-Alkylpyridinium polymers; Cationic surfactant; Detergent; Hemolytic activity; Marine sponge; Reniera sarai
1. Introduction
Marine sponges produce a variety of antiviral, antibacterial, cytotoxic, cytolytic, and hemolytic compounds. As hemolytic were proved proteins [8,9,17],
saponin-like peptides [23], and non-identified compounds [7,19]. Cytolytic agents, water soluble polymeric
3-octylpyridinium salts (poly-APS) (see Fig. 1), were
isolated from the marine sponge Reniera sarai [26,27].
Poly-APS are structurally and functionally related to
halitoxins, 0.5–25 kDa 3-alkylpyridinium polymers isolated from several sponges of the genus Haliclona
[5,24]. Poly-APS were determined to be a mixture of 29
(5520 Da) and 99 (18 900 Da) polymerized 3-octylpyridinium units [27]. In halitoxins, pyridinium rings are
connected by methyl-branched alkyl chains composed
Abbre6iations: CMC, critical micelle concentration; CPC, cetylpyridinium chloride; CTAB, cetyltrimethylammonium bromide; HC0.5,
concentration of surfactant causing 50% hemolysis in 2 min; IC0.5,
cation concentration producing 50% inhibition of hemolysis; PEG,
polyethyleneglycol; Poly-APS, polymeric alkylpyridinium salts.
* Corresponding author. Tel.: + 386-61-1233388; fax: +386-61273390.
E-mail address: [email protected] (K. Sepčić)
of 8–11 carbon atoms instead of the octyl chain in
poly-APS. To date, 3-alkylpyridinium oligomers and
polymers have been reported to be cytotoxic
[13,24,26,31], hemolytic [5,24,26], antibacterial [24], antifeeding [1], toxic to animals [5,24,31], antimitotic
[5,13], neurotoxic [5], and inhibitory for acetylcholinesterase [27–29], epidermal growth factors [10]
and muscarinic receptors [12].
In aqueous solution, poly-APS form larger structures
with an average hydrodynamic radius of 2393 nm
(mean9 SEM) and surfactant-like characteristics [27].
Our preliminary study has shown that besides a potent
and complex anticholinesterase activity [28], poly-APS
are hemolytic and cytotoxic for different cell lines including the transformed ones [26]. This study addresses
the characteristics of hemolytic activity of poly-APS as
compared to hemolysis induced by the chemically related pyridinium surfactants, cetylpyridinium chloride
(CPC), and cetyltrimethylammonium bromide (CTAB).
Cationic surfactants are known as hemolytic compounds [15,32,34] and are also used as antibacterial or
plaque-inhibiting agents [2]. We suggest that 3alkylpyridinium polymers, displaying a broad spectrum
of biological activities, may serve as unspecific defense
agents in marine sponges.
0742-8413/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved.
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P. Malo6rh et al. / Comparati6e Biochemistry and Physiology, Part C 124 (1999) 221–226
containing 3.0 ml of erythrocyte suspension in 0.13 M
NaCl, 0.02 M Tris/HCl, pH 7.4. The decrease of apparent absorbance was recorded at 700 nm using a Shimadzu 2100 UV/VIS spectrophotometer. All the
experiments were performed at 25°C. The suspension in
the cuvette was magnetically stirred. The surfactant
HC0.5 was defined as the concentration causing a 50%
decrease of absorbance in 2 min.
2.3. Estimation of pore size
Fig. 1. Chemical structure of poly-APS (a); cetylpyridinium chloride
(b); cetyltrimethylammonium bromide, (c).
2. Materials and methods
2.1. Materials
Poly-APS were purified from the marine sponge R.
sarai as described previously [27]. A series of poly-APS
solutions were prepared by diluting the 1.3 mg ml − 1
stock solution in water.
Stock solutions of CPC and CTAB (both Sigma,
USA) were prepared in erythrocyte buffer (0.13 M
NaCl, 0.02 M Tris/HCl, pH 7.4) to achieve a final
concentration of 300 mg ml − 1. The chemical structure
of the surfactants is presented in Fig. 1. For osmotic
protection, we used glucose, sucrose (both Kemika,
Croatia) and a series of polyethyleneglycols (all Sigma,
USA) with molecular weights of 1, 1.5, 2 and 4 kDa,
respectively. A stock solution of 600 mM saccharides
was prepared in water. Sphingomyelin (Serva, Germany), and L-a-phosphatidylcholine, gangliosides,
cholesterol and phosphatidic acid (all Sigma, USA)
were assayed for inhibition of the hemolysis induced by
poly-APS. Lipids were prepared at 100 mg ml − 1 in
ethanol or water (gangliosides). To study the effect of
divalent cations on surfactant-induced hemolysis, water
solutions of CaCl2, CoCl2, HgCl2, MnCl2, NiCl2 and
ZnCl2 (all Kemika, Croatia) were used. Their stock
solutions were prepared at a concentration of 600 mM
in water.
2.2. Hemolytic acti6ity of poly-APS
Hemolytic activity was measured on bovine erythrocytes as described before [4,16]. Typically, 10 – 50 ml of
water-dissolved surfactant was added into a cuvette
The size of lesions produced by poly-APS in erythrocyte membranes was estimated by using osmotic protectants as described by Belmonte et al. [4]. Osmotic
protectants of different sizes were added to erythrocyte
suspension at a 30 mM final concentration. After mixing, poly-APS at their HC0.5 were added and the time
course of hemolysis was followed for 10 min. To assure
that the observed protection of hemolysis was not the
consequence of vesiculation, we repeated the experiment by adding the protectant at the stage of cell lysis
[25]. As a parameter of the time necessary for the sugar
molecules to enter the cells through the poly-APS induced pores, we used t1/2 − t 01/2, where t 01/2 was the time
to 50% lysis without, and t1/2 was the time to lysis with
osmotic protectants [4]. Accordingly, the rate of solute
diffusion through the pore was proportional to 1/(t1/
2− t 01/2). The obtained data were fitted using the
Renkin equation [22] to estimate the functional radius
of the pore.
2.4. Inhibition of hemolysis with lipids
The effect of different lipids on poly-APS induced
hemolysis was studied using a competition method [33].
Four microlitres of the stock solution of different lipids
were added to 3.0 ml of erythrocyte suspension to
obtain a final lipid concentration of about 10 − 7 M. The
mixture was incubated for 1 min, then poly-APS at
their HC0.5 were added, and the time course of hemolysis was monitored for 10 min.
2.5. Effect of di6alent cations
To study the effect of divalent cations on hemolysis
induced by poly-APS or CPC, to 3 ml of erythrocyte
suspension supplemented with 0–10 mM CaCl2,
MnCl2, CoCl2, NiCl2, ZnCl2, and HgCl2, surfactants
were added at their HC0.5, and the hemolytic activity
was measured as described. We determined an IC0.5,
cation concentration increasing the half time of hemolysis t1/2 by two-fold, i.e. producing 50% inhibition. In
controls without hemolytic agents no hemolysis occurred within 30 min.
The effect of EDTA on divalent cation-induced inhibition of hemolysis was studied as follows. To an
P. Malo6rh et al. / Comparati6e Biochemistry and Physiology, Part C 124 (1999) 221–226
Fig. 2. Time course of hemolysis caused by poly-APS. Concentrations
of poly-APS from the right to the left: 0.12; 0.22; 0.31; 0.61; 0.92; 1.2;
3.1; 6.1 and 9.2 mg ml − 1. See Section 2 for details.
erythrocyte suspension with an apparent absorbance of
0.5 at 700 nm poly-APS or CPC were added at their
HC0.5. When absorbance dropped to 0.25, ZnCl2 was
added at a 1 mM final concentration, and after 70 s, a
final 5 mM EDTA was added.
To test the effect of divalent cations on hemolysis
induced by a hyposmotic solution, erythrocyte suspensions supplemented with divalent cations were rapidly
mixed with an equal volume of deionized water and the
time course of hemolysis was followed as described
above.
3. Results
Poly-APS induced hemolysis in a dose-dependent
manner. Its time course was sigmoidal (Fig. 2) as were
those produced by CPC or CTAB (not shown). In Fig.
3, the t1/2 values of hemolysis induced by surfactants
are plotted against their concentration, and the estimated values for HC0.5 are tabulated as compared to
critical micelle concentration of surfactants. To compare the hemolytic activity of poly-APS with that of
CPC and CTAB on a molar basis, we used the molecular weight of the 3-octylpiridinium moiety (190.16 g
mol − 1) to calculate the concentration of 3-octylpyridinium monomers in erythrocyte suspension. It is obvious that poly-APS are less active than CPC and CTAB.
The hemolytic activity of poly-APS was abolished in
the presence of phosphatidic acid (Fig. 4) but it was not
affected by the other assayed lipids.
Poly-APS mediated hemolysis was apparently attenuated by osmotic protectants with a m.w. higher than 1.5
kDa (Fig. 5a). The high viscosity of solutions precluded
the use of osmotic protectants with a m.w. exceeding
4.0 kDa. As well, the protection was preserved if the
osmotic protectant was added at the stage of cell lysis,
223
Fig. 3. Comparison of hemolytic activities of cationic surfactants.
Time necessary to achieve 50% lysis of bovine erythrocytes, t1/2, is
plotted vs. surfactant concentration: poly-APS (); CPC () and
CTAB (
). Each result is the mean value 9SEM of three experiments. Inset: a comparison of critical micelle concentration (CMC)
and hemolytic activity (HC0.5) for CTAB, CPC, and poly-APS. CMC
values are taken from [30] for CPC, [14] for CTAB, and [27] for
poly-APS. For poly-APS, we used the molecular weight of the
3-octylpyridinium moiety (190.16 g mol − 1) to calculate the concentration of 3-octylpyridinium monomers in erythrocyte suspension.
excluding the possibility that a vesiculation process
competed with hemolysis [25]. An analysis of hemolysis
data using the Renkin plot, i.e. the relative diffusion
rate of the osmoticant molecule vs. its size, is shown in
Fig. 5b. The best fit to the Renkin equation gave an
estimate of a pore-effective radius of about 2.9 nm. In
the presence of 1.0 kDa polyethyleneglycol, the
hemolytic activity of CPC was decreased by 80% in
contrast to poly-APS which remained fully active (Fig.
5c). This suggests that poly-APS make larger pores
than CPC in erythrocyte membranes.
Zn2 + and Hg2 + exhibited the strongest inhibition of
the surfactant-induced hemolysis, while Ni2 + , Mn2 + ,
Fig. 4. Inhibition of poly-APS induced hemolysis by different lipids.
Surfactant was at HC0.5, the lipid concentration was about 10 − 7 M.
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P. Malo6rh et al. / Comparati6e Biochemistry and Physiology, Part C 124 (1999) 221–226
Fig. 5. Poly-APS-induced lysis of bovine erythrocytes in the presence of osmotic protectants (indicated by arrows), a; Renkin plot reporting the
relative diffusion rate of the osmotic protectant molecule vs. its size. Hydrated radii of the osmotic protectants are: 0.42 (glucose); 0.54 nm
(sucrose); 0.9 nm (1.0 kDa PEG); 1.2 nm (1.5 kDa PEG); 1.4 nm (2.0 kDa PEG) and 2.0 nm (4.0 kDa PEG), b; effect of 1.0 kDa
polyethyleneglycol on hemolysis caused by poly-APS and CPC, c. Each result is the mean value 9 SEM of three experiments.
Ca2 + and Co2 + were considerably less effective (Table
1). At a 1 mM final concentration, Zn2 + was inhibitory
even when added after poly-APS or CPC, while the
addition of 5 mM EDTA completely restored hemolytic
activity as represented for poly-APS in Fig. 6. None of
the used cations were able to inhibit hyposmotically
induced hemolysis. In fact, Hg2 + induced a complete
inhibition of hemolysis but only when preincubated
with an erythrocyte suspension for more than 5 min
(not shown).
4. Discussion
Cytotoxic and cytolytic activities of 3-alkylpyridinium polymers observed so far [5,24,26] at least in
part may be explained by their surfactant-like characteristics. Studies of the surfactant/membrane interactions have shown that the hydrophile/lipophile balance
of the surfactant plays the major role in membrane
solubilization, electrostatic factors being less important
[11,34]. The hemolytic activity of detergents has been
reported to be (i) inversely proportional to their critical
micelle concentration (CMC) [20]; (ii) proportional to
the length of the alkyl chain [15,34], and (iii) affected by
the character of their ionic head [32]. However, the
polymeric structure of 3-alkylpyridinium salts do not
resemble the classic design of the typical surfactants
such as CPC and CTAB with both hydrophilic heads
and hydrophobic tails free. Moreover, octyl chains
flanked by two pyridinium rings in the case of polyAPS appear considerably shorter than the cetyl group
of CPC and CTAB. Despite these differences, poly-APS
surprisingly share many functional characteristics with
the monomeric surfactants. First, both the characteristics of t1/2 vs. concentration plot and the HC0.5 value
for poly-APS are comparable to those of CPC and
CTAB (see Fig. 3). Second, experiments with osmotic
protectants suggest that poly-APS produce discrete lesions in erythrocyte membranes. The resulting pores of
about 5.8 nm in diameter appear slightly larger than
those provoked by CPC. In comparison, an estimate
for sodium dodecylsulfate and Triton X-100 pores was
4 nm [25]. Furthermore, the divalent cations Zn2 + and
Hg2 + are common inhibitors of poly-APS and CPC
(Table 1). These values are in accordance with reports
Table 1
Effect of different divalent cations on surfactant-induced hemolysisa
Cation
Poly-APS IC0.5 (mM)
CPC IC0.5 (mM)
Zn2+
Hg2+
Ni2+
Ca2+
Mn2+
Co2+
0.3 90.05
0.04 90.009
\3
\10
\3
\1
0.2 9 0.01
0.2 9 0.04
2 90.24
\10
\3
\1
a
Each result is the mean value 9SEM of three experiments. PolyAPS and CPC were used at their HC0.5. IC0.5 is the cation concentration increasing the half time of hemolysis t1/2 two-fold, i.e. producing
50% inhibition of hemolysis.
P. Malo6rh et al. / Comparati6e Biochemistry and Physiology, Part C 124 (1999) 221–226
225
References
Fig. 6. Effect of ZnCl2 and EDTA on time course of hemolysis
induced by poly-APS at HC0.5. Additions of ZnCl2 and EDTA are
indicated by the arrows. Numbers in parenthesis are the final concentrations.
of a Zn2 + mediated inhibition of hemolysis by both
cationic [3,21], anionic, and non-ionic hemolytic agents
[25]. A higher inhibitory efficiency of Hg2 + with polyAPS may reflect the interaction of different negatively
charged forms of mercury chloride (II) in water
[HgCl24 − , HgCl2(OH)22 − ] [18] with a polycationic structure of poly-APS bound into the cell membrane. The
polycationic structure of poly-APS could be a reasonable explanation for their preference for the negatively
charged lipid as demonstrated for phosphatidic acid.
One reason for Zn2 + and other divalent cations
mediated inhibition of hemolysis could be their ability
to stabilize the erythrocyte membrane [6]. However, in
our study Zn2 + did not inhibit hyposmotically evoked
hemolysis. This fact and the ability of Zn2 + to inhibit
already progressing lysis suggest that Zn2 + neither
stabilizes nor prevents the binding of poly-APS to the
membrane. Rather, we suggest that divalent cations
close the resulted pores.
In conclusion, our results demonstrate that hemolytic
activity of poly-APS is comparable to that of cationic
surfactants CPC and CTAB despite the polymeric nature of poly-APS and their considerably shorter alkyl
chain. As polycationic surfactants poly-APS show
strong preference for negatively charged lipids. They
induce a coloid–osmotic type of hemolysis by producing discrete lesions, 5.8 nm in diameter, in the cell
membrane. Although occurence of poly-APS in marine
sponges is not completely understood, there is emerging
evidence that these natural surfactants, possessing a
large variety of biological activities, may serve as unspecific antibiotics and repelling agents.
Acknowledgements
This work was supported by the Ministry of Science
and Technology of the Republic of Slovenia.
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