Biochimie 93 (2011) 1495e1501
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Biochimie
journal homepage: www.elsevier.com/locate/biochi
Research paper
A novel method for preparation of HAMLET-like protein complexes
Sergei E. Permyakov a, b, *, Ekaterina L. Knyazeva a, *, Marina V. Leonteva a, b, Roman S. Fadeev c,
Aleksei V. Chekanov c, Andrei P. Zhadan a, Anders P. Håkansson d, Vladimir S. Akatov c,
Eugene A. Permyakov a, b
a
Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia
Department of Biomedical Engineering, Pushchino State University, Pushchino, Moscow Region 142290, Russia
c
Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia
d
Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY 14214, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 March 2011
Accepted 4 May 2011
Available online 11 May 2011
Some natural proteins induce tumor-selective apoptosis. a-Lactalbumin (a-LA), a milk calcium-binding
protein, is converted into an antitumor form, called HAMLET/BAMLET, via partial unfolding and association with oleic acid (OA). Besides triggering multiple cell death mechanisms in tumor cells, HAMLET
exhibits bactericidal activity against Streptococcus pneumoniae. The existing methods for preparation of
active complexes of a-LA with OA employ neutral pH solutions, which greatly limit water solubility of OA.
Therefore these methods suffer from low scalability and/or heterogeneity of the resulting a-LA e OA
samples. In this study we present a novel method for preparation of a-LA e OA complexes using alkaline
conditions that favor aqueous solubility of OA. The unbound OA is removed by precipitation under acidic
conditions. The resulting sample, bLAeOA-45, bears 11 OA molecules and exhibits physico-chemical
properties similar to those of BAMLET. Cytotoxic activities of bLAeOA-45 against human epidermoid
larynx carcinoma and S. pneumoniae D39 cells are close to those of HAMLET. Treatment of S. pneumoniae
with bLAeOA-45 or HAMLET induces depolarization and rupture of the membrane. The cells are
markedly rescued from death upon pretreatment with an inhibitor of Ca2þ transport. Hence, the activation mechanisms of S. pneumoniae death are analogous for these two complexes. The developed
express method for preparation of active a-LA e OA complex is high-throughput and suited for development of other protein complexes with low-molecular-weight amphiphilic substances possessing
valuable cytotoxic properties.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
a-lactalbumin
Oleic acid
HAMLET
Cytotoxicity
Bactericidal activity
1. Introduction
Despite prominent achievements in development of novel
anticancer therapies, the need for new anticancer drug candidates
Abbreviations: OA, oleic acid (C18:1:9cis); CMC, critical micelle concentration;
a-LA, a-lactalbumin; intact a-LA, a-lactalbumin isolated from milk; hLA, human
a-lactalbumin; bLA, bovine a-lactalbumin; HAMLET(BAMLET), complex of
human(bovine) a-lactalbumin with oleic acid, prepared as described in [1,2];
bLAeOA-45, complex of bovine a-lactalbumin with oleic acid, prepared as
described in Materials and methods; reference bLA, sample of bovine a-lactalbumin
subjected to procedures described for preparation of bLAeOA-45, skipping addition
of oleic acid; MS, mass spectrometry; CD, circular dichroism; lmax, maximum
position of fluorescence emission spectrum; T1/2, mid-transition temperature; HEp2, human epidermoid larynx carcinoma cells; CFUs, colony-forming units.
* Corresponding authors. Institute for Biological Instrumentation of the Russian
Academy of Sciences, Institutskaya 7, Pushchino, Moscow Region 142290, Russia.
Tel.: þ7 4967 73 41 35; fax: þ7 4967 33 05 22.
E-mail addresses: [email protected] (S.E. Permyakov), elknyazeva@rambler.
ru (E.L. Knyazeva).
0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2011.05.002
is still urgent. One of the most intriguing classes of substances with
antitumor therapeutic potential is a set of natural proteins, selectively inducing apoptosis in tumor cells: apoptin, E4orf4, NS1, mda7, TRAIL, HAMLET and ELOA (for reviews, see [3,4]). Some of them
have shown positive results in pre-clinical and clinical studies
(mda-7, TRAIL and HAMLET). Aside from direct use of these
proteins for therapeutic purposes, they provide valuable information on molecular mechanisms used to defeat the biochemical
machinery of malignant cells. In this respect, HAMLET is of special
interest due to its ability to attack several critical organelles
(mitochondrion, proteasome and nucleosome), activating different
death pathways in tumor cells (for reviews, see [4,5]).
HAMLET represents a complex between a small calcium-binding
protein from human milk, a-lactalbumin (a-LA; for review, see [6]),
and oleic acid (OA) [1]. In the lactating mammary gland, a-LA serves
as a regulatory subunit of the lactose synthase enzyme complex [7].
The binding of OA to a-LA following a specific procedure converts it
into a form (HAMLET, Human Alpha-lactalbumin Made LEthal to
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S.E. Permyakov et al. / Biochimie 93 (2011) 1495e1501
Tumor cells), which induces death of tumor cells and undifferentiated cells, but spares healthy and mature cells in cell culture and
tissue [1,8e12]. Besides its tumoricidal activity, this complex also
exhibits bactericidal activity against antibiotic-sensitive and resistant
strains of Streptococcus pneumoniae [13,14]. Although a-LA is able to
bind other fatty acids [15], C18:1:9cis (OA) or C18:1:11cis (vaccenic acid) is
required to form an a-LA complex with pronounced antitumor
activity [16]. Meanwhile, human a-LA can be replaced by bovine,
equine, porcine or caprine a-LA without loss of the complex activity
[2]. Moreover, binding of OA to Ca2þ-binding equine lysozyme also
gives rise to tumoricidal activity, analogous to HAMLET [17]. This fact
and similar evidences discussed by Spolaore et al. [18] indicate that
the cytotoxic effect of HAMLET is mostly due to OA, which is cytotoxic
by itself [19e21], while the proteinaceous component of the complex
mainly serves as a carrier of the poorly water soluble OA [22e24].
Nevertheless, there are evidences that a-LA within HAMLET directly
affects some of key cellular processes [25], implying that both
components of the complex are important for its cytotoxicity.
The binding of OA to a-LA requires partial unfolding of the protein
[1], which can be achieved via Ca2þ depletion [26] or thermal
denaturation. The existing procedures for preparing tumoricidal aLA e OA complexes differ in the procedure of application of OA, in the
method of a-LA unfolding, and in the OA concentration used. The
original method employs chromatography of Ca2þ-free a-LA on
a DEAE-TrisacrylÒ M column preconditioned with OA followed by
elution of the complex by high salt concentration [1]. Other methods
are based upon direct binding of dissolved OA by Ca2þ-depleted
[21,27] or thermally denatured [28] a-LA. Only the method by
Knyazeva et al. takes into account low water solubility of OA at
neutral pH via use of OA concentrations below its effective critical
micelle concentration (CMC). The absence of such a control of CMC
values in other methods is fraught with formation of extended
phases of OA [22e24], making the a-LA e OA system highly
heterogeneous. The heterogeneity of this system is documented in
the work by Spolaore et al. [18]. Possibly this circumstance partly
rationalizes the fact that different oligomeric states of a-LA and
various OA to a-LA molar ratios were reported for active complexes
prepared using different approaches (for review, see [18]).
The CMC values of OA do not exceed 70 mM under optimized
solution conditions used in the method by Knyazeva et al. [21]. The
low water solubility of OA at neutral pH limits scalability of this
method. In the present work we present a novel method for
preparation of biologically active a-LA e OA complexes based on
the use of alkaline solvent conditions, greatly favoring aqueous
solubility of OA, thereby allowing us to overcome the scalability
limitation of the previous method. The developed low-cost express
method enables preparation under laboratory conditions of quantities of active sample suitable for animal testing and pre-clinical
studies. Furthermore, the use of extreme alkaline conditions
enables the extension of this method to other proteins, thus
opening up wide opportunities for preparation of protein
complexes with fatty acids or other low-molecular-weight amphiphilic substances with valuable cytotoxic properties. The proposed
method of dissolving of otherwise hardly water soluble cytotoxic
substances into the hydrophobic interior of natural proteins can be
exploited for cell delivery of these substances.
Human a-lactalbumin (hLA) was isolated from milk as described
[29,30]. Ca2þ-depleted bovine a-lactalbumin (bLA) and oleic acid
(C18:1:9cis) were from SigmaeAldrich Co. Chemistry grade 96% (v/v)
ethanol was treated with K2MnO4 and activated coal, followed by
double distillation; the absence of organic impurities was
confirmed spectrophotometrically. Stock solution of OA was
prepared by sonication of an ethanol suspension of OA for 3 min,
and stored at room temperature. HAMLET and BAMLET were
prepared using the original procedure [1,2]. Concentrations of a-LA
were evaluated using extinction coefficients A1%
1 cm;280 of 20.1 and
18.2 for bLA/BAMLET and hLA/HAMLET, respectively [31,32].
Ultra-grade H3BO3 was bought from Merck. Ultra-grade
ammonium bicarbonate, CaCl2 standard solution, DEAE-TrisacrylÒ
M, DMEM culture medium, crystal violet, Ruthenium Red and
propidium iodide were from SigmaeAldrich Co. Fetal bovine serum
was from HyClone. Todd-Hewitt medium and Bacto-Yeast extract
were from BD Biosciences. DiBAC4(3) was from Invitrogen. EDTA
standard solution and dialysis tubing were from Thermo Fisher
Scientific Inc. Water with conductivity of 18.2 MU cm was used.
HPLC grade acetonitrile was from Panreac.
2.2. Estimation of the effective critical micelle concentration of OA
Estimation of the effective critical micelle concentration of
OA was performed as previously described [21]. The appearance of
OA micelles was monitored photometrically at 335 nm. The lowest
estimate of OA binding capacity of a-LA, nmax, was found from the
dependence of effective CMC of OA upon protein concentration, [P]
(CMC0 is CMC value of free OA):
CMC ¼ CMC0 þ nmax $½P
(1)
2.3. Preparation of bLAeOA-45 state
6 ml of 0.6 mM Ca2þ-free bLA at pH 12.0 (5 mM KOH, 1 mM
EDTA) was titrated at 45 C by 12 ml aliquots of 1 M OA solution in
96% ethanol up to the effective CMC of OA, 22 mM. The unbound OA
was separated via acidification of the solution down to pH 2.0 and
centrifugation at 12,000g for 30 min at 40 C. The free from
unbound OA water phase was collected, and its pH was adjusted by
KOH to w5.5. The solution was triply dialyzed against 100-fold
volume of distilled water, freeze-dried and stored at 18 C. Bovine
a-lactalbumin, subjected to the described procedure, but skipping
addition of OA, will be referred to as “reference bLA”. The integrity
of bLAeOA-45 and reference bLA was confirmed by SDS-PAGE.
2.4. Quantitation of the content of OA in protein samples
2. Materials and methods
Quantitation of the content of OA in protein samples was performed using LC/MS system LCMS-2010EV (Shimadzu Co.), ESI probe
coupled to a single quadrupole detector. Sample of 0.1e1 mM a-LA in
50:50 (v/v) mixture of 20 mM ammonium bicarbonate pH 8.5 (buffer
A) and acetonitrile was applied to Shimadzu Silica 100 2.5 mm RP
4,4 50 mm column at 40 C. OA was eluted with buffer A:acetonitrile 20:80 (v/v) at 50 ml/min flow rate and infused into MS detector
(interface 4 kV, negative mode, SIM mode 281 0.5 u). The chromatogram was integrated and quantitated using a calibration curve.
The calibration was carried out using 0.01e100 mM OA in 96% ethanol.
2.1. Materials
2.5. Secondary structure content in a-LA
Human epidermoid larynx carcinoma (HEp-2) cells were from
the Russian Cell Culture Collection (Institute of Cytology, Russian
Academy of Sciences, St. Petersburg).
Secondary structure content in a-LA was determined from farUV circular dichroism (CD) spectra using the CDPro software
package [33] as previously described [21].
S.E. Permyakov et al. / Biochimie 93 (2011) 1495e1501
2.6. Thermal stability of a-LA
1497
S. pneumoniae D39 [34] was stored in glycerol stocks at 80 C.
Frozen stocks were used to seed Todd-Hewitt medium containing
0.5% Bacto-Yeast Extract. In late logarithmic growth phase the
bacteria were harvested by centrifugation at 1500g for 2 min and
suspended in PBS buffer (30 mM Na2HPO4, 10 mM KH2PO4, 120 mM
NaCl, pH 7.4) to an appropriate concentration for experiments. The
effect of a-LA samples on bacterial viability was assessed by plating
10-fold dilutions of treated and untreated bacteria on blood agar
and counting of viable colony-forming units (CFUs) after overnight
growth at 37 C. The effect of Ruthenium Red was assessed via
addition of protein complexes after 15 min pretreatment of the cells
with the inhibitor. The data were analysed using two-tailed
Student’s t-test.
strength) the respective CMC values reported for pH 8.3 under
similar solution conditions [21]. The maximal CMC value of
(2.1 0.6) mM is reached in the absence of metal cations at 20 C.
On the contrary, calcium ions (1 mM CaCl2) lower the CMC of OA
down to 4 mM, regardless of temperature. Similar values (3e4 mM)
were previously observed at pH 8.3 [21]. Since calcium ions drastically reduce water solubility of OA, they were avoided in further
optimization steps. It is further assumed that 1 mM EDTA is added.
Examination of the dependence of effective CMC of OA upon bLA
concentration at pH 12 and 45 C (Fig. 1) shows that OA binding
capacity of bLA, nmax (see Equation [1]), grows at bLA concentrations above 0.5 mM, which reflects loading of the low-affinity sites
of the protein (OA binding constants ca 103 M1). The use of bLA
concentrations above 0.6e1 mM would require EDTA concentrations above 1 mM for proper depletion of contaminating Ca2þ ions.
Since this high EDTA concentration may result in a binding of EDTA
to bLA [37], bLA concentration was restricted to 0.6 mM.
Optimization of the process of a-LA e OA interaction via selection of ionic strength and temperature conditions ensuring
maximal OA binding capacity of bLA (Table 1) shows that the most
favorable solution conditions are achieved in the absence of metal
cations at 45 C. The corresponding nmax value of 34 is about
twofold higher than the maximal nmax estimate for pH 8.3 [21].
The fact that negatively charged OA molecules efficiently associate with almost fully deprotonated protein at pH 12.0 (bLA
contains one Arg residue) suggests a crucial role of the hydrophobic
effect in stabilization of the bLA e OA complex. Thus, the process of
bLA association with OA can be regarded as dissolution of the
hydrophobic moiety of OA in the non-polar interior of the protein.
This mechanism seems to enable the binding of multiple OA
molecules to bLA (Table 1).
2.10. Measurement of bacterial outer membrane potential and
integrity of the membrane
3.2. Preparation of bLAeOA-45 sample
Thermal stability of a-LA was estimated spectrofluorimetrically
as previously described [21].
2.7. Calcium affinity of a-LA
Calcium affinity of a-LA was estimated from spectrofluorimetric
Ca2þ-titration of the Ca2þ-depleted protein using the cooperative
metal binding scheme as previously described [21].
2.8. Viability of human larynx carcinoma (HEp-2) cells
Viability of human larynx carcinoma (HEp-2) cells was estimated using crystal violet assay as previously described [21].
2.9. Bactericidal assay
S. pneumoniae D39 were pelleted by centrifugation at 1500g and
washed twice by resuspension in PBS buffer (pH 7.4) after centrifugation. The bacterial pellet was resuspended in half of the initial
PBS volume and energized with 50 mM glucose for 15 min at 37 C.
Propidium iodide (40 mg/ml) and DiBAC4(3) (0.5 mM) were added to
the energized bacteria and 100 ml of bacterial suspension was
mixed with 100 ml of PBS alone or PBS containing Ruthenium Red in
each well of a 96-well microtiter plate (Falcon, BD Biosciences)
(final concentrations are 25 mM glucose, 20 mg/ml propidium
iodide and 0.25 mM DiBAC4(3)). The plate was placed into a Synergy
2 microplate reader (BioTek Instruments, Inc.) at 37 C and fluorescence was read for 40 min before addition of a-LA complexes to
allow the dyes to equilibrate. DiBAC4(3) fluorescence (485/20 nm
excitation, 520/25 nm emission) and propidium iodide fluorescence (485/20 nm excitation, 590/35 nm emission) were then read
for 1 h to monitor changes in membrane potential and integrity,
respectively.
The 0.6 mM solution of bLA was titrated by OA up to its effective
CMC value under optimized solution conditions: pH 12.0, 5 mM
KOH, 1 mM EDTA, 45 C. The intrinsically cytotoxic [19e21]
unbound OA was removed using the procedure employing low
solubility of fatty acids under acidic conditions. Acidification of the
solution down to pH 2.0 induces protonation of carboxylates of OA,
3. Results and discussion
3.1. Optimization of conditions for formation
of a-LA e OA complexes
The optimal conditions should ensure maximal OA binding
capacity of a-LA. This requirement entails the need for fulfillment of
two conditions: a-LA unfolding, previously shown to be critical for
association of OA [1], and high solubility of OA. Both conditions are
fully satisfied under extreme alkaline conditions of pH above 12
[35,36]. The CMCs of Ca2þ-free OA (1 mM EDTA) estimated at pH
12.0 exceed 2e105-fold (depending upon temperature and ionic
Fig. 1. The dependence of effective CMC of OA upon bLA concentration in the absence
of Ca2þ (1 mM EDTA) at pH 12.0 (5 mM KOH) and 45 C, in the absence/presence of
150 mM KCl (open/filled circles). The lines represent linear fits of the initial regions of
the experimental curves.
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S.E. Permyakov et al. / Biochimie 93 (2011) 1495e1501
Table 1
The dependence of OA binding capacity of bLA (nmax) at pH 12.0 (5 mM KOH) upon
calcium concentration, ionic strength and temperature. Protein concentration was
0.6 mM.
Temperature
20 C
45 C
1 mM EDTA
1 mM CaCl2
nmax, 150 mM KCl
nmax, no salt
nmax, 150 mM KCl
0
0
18 1
34 5
26 2
22 3
resulting in its precipitation. The precipitates were removed via
centrifugation. The resulting sample was adjusted to pH w5.5,
dialyzed against water and freeze-dried. The bLA sample subjected
to the same procedures, but without addition of OA, will be referred
to as “reference bLA”.
3.3. Physico-chemical characterization of bLAeOA-45 complex
The number of OA molecules per a-LA molecule in bLAeOA-45
sample estimated by LC/MS is 11, which is comparable to the
content of OA in an LAeOA-45 sample of human a-LA [21]. Thus,
about one third of the initially bound OA molecules are retained in
bLAeOA-45 sample. Notably, examination of the samples of other
proteins binding OA only non-specifically, which were subjected to
the same procedures used for preparation of bLAeOA-45 sample,
have shown for some of them at least an order of magnitude lower
OA content. This fact confirms efficacy of the acidic method of OA
removal and indicates that bLAeOA-45 sample retains OA molecules due to their specific binding.
The CD estimates of a-helical content for Ca2þ-depleted forms of
different bLA states at 5 C (pH 8.3) are shown in Table 2. Reference
bLA exhibits a-helical content lowered by (4.6 0.7)%, evidencing
some structural perturbations caused by the procedures used for
preparation of bLAeOA-45 sample. At the same time, the bLAeOA45 state has the same a-helical content as intact protein. Hence,
modification of bLA with OA improves its a-helicity. An analogous
effect is observed for BAMLET (Table 2) and hLA e OA complexes
[21]. This effect is also observed at 45 C.
Spectrofluorimetric measurement of thermal unfolding of the
Ca2þ-free bLAeOA-45 shows that its thermal stability is equivalent
to that of the BAMLET state, being lowered by about 8 C with
respect to both intact and reference bLA samples (Table 2). This
effect is in agreement with the previous data, evidencing a decrease
in stability of apo-a-LA upon association with OA [21,38]. The
lowered stabilities of the Ca2þ-free bLA complexes with OA are in
line with red shifts (9e12 nm) of their fluorescence emission
spectra (Table 2), which indicate higher accessibility of emitting
tryptophans to the solvent. A similar, but less pronounced, effect
(7 nm red shift) is observed for reference bLA. Calcium-binding
(1 mM CaCl2) lowers this effect to 5 nm. The structural perturbations in reference bLA affect protein’s secondary structure and the
environment of emitting tryptophans, but do not change thermal
stability of bLA.
Calcium binding (1 mM CaCl2) renders all bLA states studied
equally stable (mid-transition temperatures of 61e62 C). Despite
that, fluorescence spectrum of bLAeOA-45 is red-shifted by 11 nm
in comparison with intact bLA and BAMLET. Hence, in the case of
bLAeOA-45, the association with OA increases solvent accessibility
of emitting tryptophans of the Ca2þ-bound protein, without
affecting its thermal stability.
Spectrofluorimetric measurements of calcium affinity of various
bLA states at 45 C show that association of OA does not affect
protein calcium affinity, but decreases cooperativity of Ca2þbinding by bLAeOA-45 (Table 2). Since calcium affinity data for
bLAeOA-45 and reference bLA are indistinguishable, the modification of the protein by OA does not affect calcium affinity of bLA.
Overall, our data show that conformational and physicochemical properties of bLAeOA-45 state are similar to those of
BAMLET.
3.4. Cytotoxic effects of bLAeOA-45 upon HEp-2
and S. pneumoniae D39 cells
The concentration dependencies of the cytotoxicites of bLAeOA45, HAMLET and respective control samples on human larynx
carcinoma (HEp-2) cells in vitro are shown in Fig. 2. Intact and
reference bLA at concentrations up to 1 mM were shown to be nontoxic. Oleic acid exerts toxic effect on HEp-2 cells only at concentrations above 300 mM. The IC50 values for bLAeOA-45 (about
20 mM) and HAMLET (40 mM) differ insignificantly.
The comparison of bactericidal activities of bLAeOA-45 and
HAMLET against S. pneumoniae D39 is shown in Table 3. In the
range of protein concentrations of 50e150 mg/ml (3.5e10.6 mM)
bLAeOA-45 reduces viability of the bacteria by 2.4e6.2 orders of
magnitude, which is close to the bactericidal activity of HAMLET.
Intact a-LA at a concentration of 150 mg/ml does not exert any
bactericidal activity. To evaluate the specific contribution of OA to
the bactericidal activity, we exposed bacteria to a concentration of
OA, equivalent to the content of OA in 10.6 mM of bLAeOA-45
sample. An OA concentration of 106 mM exhibits a bactericidal
activity of 6.3 0.2 orders of magnitude, which is similar to activity
for 10.6 mM of bLAeOA-45. Hence, the cytotoxic effect of bLAeOA45 sample could be ascribed primarily to the toxic action of OA. The
same conclusion follows from the cytotoxicity data presented in
Fig. 2 and the previous data on the hLA e OA complexes [21]. These
facts favor the view that the proteinaceous component of the LA e
OA complexes mainly serves as a carrier of the otherwise poorly
soluble cytotoxic component, oleic acid.
Summarizing, bLAeOA-45 and HAMLET samples are equally
effective with respect to both HEp-2 and S. pneumoniae D39 cells.
3.5. The initial stages of bLAeOA-45 action
upon S. pneumoniae D39 cells
Pretreatment of S. pneumoniae D39 cells with 30 mM Ruthenium
Red, an inhibitor of Ca2þ transport, significantly prevents the bLAe
OA-45einduced death of the cells, indicated by a reduction of logdeath from 3.1 0.5 to 1.48 0.17 (protein concentration is 75 mg/
ml), which suggests that Ca2þ transport is directly involved in the
cytotoxic action of this complex. This is analogous to the 51% log-
Table 2
Physico-chemical properties of various states of bLA: a-helical content, maximum position of fluorescence emission spectrum (lmax) and mid-transition temperature (T1/2) for
Ca2þ-depleted protein (1 mM EDTA), equilibrium Ca2þ association constant (K) and Hill coefficient (n) at 45 C. Buffer conditions: pH 8.3, 20 mM H3BO3eKOH, 150 mM NaCl.
Protein state
a (5 C), %
Intact bLA
Reference bLA
bLAeOA-45
BAMLET
31.0
26.4
31.0
38.4
0.5
0.5
0.5
0.5
lmax (10 C), nm
324.6
331.4
336.2
333.6
0.1
0.1
0.1
0.1
K, M1
T1/2, C
37.4
38.1
30.0
29.5
0.8
1.0
0.7
1.4
(1.1
(2.0
(2.0
(1.2
n
0.2)
0.3)
0.3)
0.2)
106
106
106
106
0.95
0.65
0.68
0.98
0.02
0.08
0.04
0.01
S.E. Permyakov et al. / Biochimie 93 (2011) 1495e1501
1499
Fig. 2. Concentration dependences of cytotoxicites against human larynx carcinoma
cells evaluated using the crystal violet assay for bLAeOA-45 (open circles), HAMLET
(open triangles), OA (filled triangles), reference bLA (filled circles) and intact bLA (open
squares).
reduction recently shown for HAMLET [14]. Similar results are
observed for bLAeOA-45einduced death of S. pneumoniae D39 cells
upon their pretreatment with other inhibitors of Ca2þ transport:
100 mM of tetrandrine or dichlorobenzamil (reduction of log-death
to 1.3 0.6 and 1.2 1.2, respectively). The involvement of Ca2þ
transport in the initial stages of cytotoxic action of hLA e OA
complexes was previously shown on plasma membrane of Chara
coralline [39].
To assess the effects of bLAeOA-45 and HAMLET on bacterial
plasma membrane the depolarization and integrity of the
membranes were monitored using the fluorescence dyes DiBAC4(3)
and propidium iodide, respectively. DiBAC4(3) is known to enter
depolarized cells where it binds to intracellular proteins or
membranes, which causes an enhancement in its fluorescence [40].
Propidium iodide is unable to penetrate into intact cells, but binds
to cellular DNA in damaged cells, resulting in appearance of red
fluorescence [41]. Addition of bLAeOA-45 or HAMLET to pneumococci results in a depolarization of the bacterial membrane within
2 min after the addition, as monitored by DiBAC4(3) fluorescence
(Fig. 3A). The kinetics of disturbance of integrity of the membranes
detected by fluorescence of propidium iodide (Fig. 3B) suggests that
the membrane depolarization leads to membrane damage. Both
these effects were greatly inhibited by Ruthenium Red, suggesting
a role of calcium in the activation of depolarization and cell death.
Intact a-LA does not cause depolarization or rupture of bacterial
membrane. These effects are in accord with the previous data on
plasmalemma of C. coralline [39].
Taken together, the experimental data demonstrate a high
degree of similarity in the initiation of bacterial death induced by
bLAeOA-45 and HAMLET.
Table 3
The bactericidal activities of bLAeOA-45 and HAMLET against S. pneumoniae D39.
Protein concentration,
mg/ml
50
75
100
150
Log (control CFUs)/CFUs
HAMLET
3.6
4.8
6.0
6.5
0.9
0.8
1.1
1.3
bLAeOA-45
2.4
3.1
6.0
6.2
0.3
0.5
1.1
0.7
Fig. 3. The kinetics of the response of S. pneumoniae D39 cells to bLAeOA-45
(50e150 mg/ml, solid curves) or HAMLET (100 mg/ml, dotted curves) fluorimetrically
monitored using 0.25 mM DiBAC4(3) (A) or 20 mg/ml propidium iodide (B). The results
represent a ratio of fluorescence intensities for cells treated and untreated by a-LA
sample. bLAeOA-45/HAMLET was added 40 min after the dyes.
4. Conclusions
The use of extreme alkaline solution for preparation of the
biologically active bLA e OA complex enables us to achieve both aLA unfolding and higher water solubility of OA. The latter factor
facilitates saturation of bLA with OA. The bLA e OA complex was
formed under optimized conditions ensuring maximal OA binding
capacity of bLA. Notably, the OA concentration was kept below its
effective CMC value, which ensures the absence of extended phases
of OA and the absence of sample heterogeneity related to them.
Furthermore, the sample was subjected to removal of the unbound
OA via solution acidification, centrifugation and dialysis. The
resulting complex, bLAeOA-45, contains about 11 molecules of OA
per bLA molecule, and possesses secondary structure, thermal
stability and calcium affinity closely resembling those of the
BAMLET state. The cytotoxic activities of bLAeOA-45 with respect
to HEp-2 and S. pneumoniae D39 cells were shown to be equivalent
to those for HAMLET. Importantly, the initial stages of the cytotoxic
effect of bLAeOA-45 against S. pneumoniae cells are very similar to
those for HAMLET: depolarization of the bacterial membrane
followed by its rupture. Both effects are dependent upon Ca2þ
1500
S.E. Permyakov et al. / Biochimie 93 (2011) 1495e1501
transport. Overall, the bLAeOA-45 sample is in many ways equivalent to HAMLET/BAMLET. The developed low-cost express method
for preparation of the HAMLET-like complex lacks the scalability
limitation of the previous method and is well suited for preparation
under regular laboratory conditions of quantities of the active
complex necessary to conduct animal testing and pre-clinical
studies.
The shown crucial role of hydrophobic effect in stabilization of
the a-LA e OA complex opens up the possibility to prepare similar
complexes of alkaline-denatured proteins with fatty acids and
other low-molecular-weight amphiphilic substances, possessing
cytotoxic properties with respect to malignant cells. The possibility
of dissolving cytotoxic hydrophobic substances in the non-polar
interior of natural proteins opens up a new avenue for cell
delivery of these substances. Further efforts aimed at development
of the complexes of this kind are required.
Conflict of interests
We confirm that there is no conflict of interest in this
manuscript.
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
Author contributions
[19]
Sergei E. Permyakov was involved in experiment design, data
treatment and analysis, and manuscript preparation. Ekaterina L.
Knyazeva was involved in data collection, data treatment and
analysis, manuscript preparation. Marina V. Leonteva, Roman S.
Fadeev, Aleksei V. Chekanov and Andrei P. Zhadan were involved in
data collection, data treatment and analysis. Anders P. Håkansson
was involved in experiment design, data collection, data treatment
and analysis, and manuscript preparation. Vladimir S. Akatov was
involved in experiment design. Eugene A. Permyakov was involved
in experiment design, and manuscript preparation.
[20]
[21]
[22]
[23]
[24]
Acknowledgements
This work was supported by grant to P.E.A. from the Program of
the Russian Academy of Sciences «Molecular and Cellular Biology»,
grant to K.E.L. from the Dynasty Foundation, and grants to A.P.H.
from the Bill and Melinda Gates Foundation (Grant 53085), the JR
Oishei Foundation, and The American Lung Association (Grant RG123721-N).
[25]
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