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\begin{document}

\title{The Effects of Magnesium, Zinc and Calcium Ions on Endotoxin-Plasmid DNA
Interaction at Various Cation Concentrations, pH Values and Incubation
Times}



\author[1]{Clarence Ongkudon}%
\author[2]{Elvina Clarie Dullah}%
\author[2]{nur Syahadatain Abdul Razak}%
\affil[1]{University Malaysia Sabah}%
\affil[2]{Universiti Malaysia Sabah}%


\vspace{-1em}



  
  \date{June 17, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
In plasmid DNA (pDNA) production from Gram-negative bacteria, endotoxin
has been known as the major contaminant. The separation becomes
difficult due to its ability to form a stable complex with pDNA apart
from sharing common properties like surface charge, molecular size,
temperature and pH stability. This study focused on the analysis of zeta
potential of endotoxin, theoretical number of cation bound per molecule
of endotoxin as well as binding tendency of cations towards endotoxin in
the presence of pDNA. These analyses were conducted under various
experimental conditions such as types of divalent metal cation, cation
concentration, pH and incubation time. The analysis of zeta potential at
different cation concentrations and pH values showed that
Mg\textsuperscript{2+} had the most significant effect on endotoxin
surface charge. The zeta potential of endotoxin reduced by a magnitude
of 43.55 mV, from -43.53 mV to 0.02 mV in the presence of 2.0 M
Mg\textsuperscript{2+}, and a magnitude of 44.12 mV , from -43.53 mV to
0.59 mV at the lowest pH level. However, in the analysis of theoretical
number of cation bound per molecule of endotoxin, Zn\textsuperscript{2+}
showed the highest number (0.6) compared to Ca\textsuperscript{2+}
(0.12) and Mg\textsuperscript{2+} (0.05). The tendency of
Zn\textsuperscript{2+} to preferentially bind with endotoxins forming
larger aggregated structure was also evident in the DNA gel
electrophoresis and transmission electron microscopic analysis.%
\end{abstract}%



\sloppy


\textbf{THE EFFECTS OF MAGNESIUM, ZINC AND CALCIUM IONS ON
ENDOTOXIN-PLASMID DNA INTERACTION AT VARIOUS CATION CONCENTRATIONS, pH
VALUES AND INCUBATION TIMES}

Elvina Clarie Dullah, Nur Syahadatain Abdul Razak, Clarence M. Ongkudon*

Biotechnology Research Institute, Universiti Malaysia Sabah, Kota
Kinabalu, Sabah, 88400, Malaysia

\textbf{*Corresponding author} ; Tel.: (+60)16-5091369; Fax:
(+60)88-320993

E-mail address: clarence@ums.edu.my (C.M. Ongkudon)

\textbf{ABSTRACT}

In plasmid DNA (pDNA) production from Gram-negative bacteria, endotoxin
has been known as the major contaminant. The separation becomes
difficult due to its ability to form a stable complex with pDNA apart
from sharing common properties like surface charge, molecular size,
temperature and pH stability. This study focused on the analysis of the
zeta potential values of endotoxin, the theoretical number of cations
bound per molecule of endotoxin as well as the binding tendency of
cations towards endotoxin in the presence of pDNA. These analyses were
conducted under various experimental conditions such as types of
divalent metal cation, cation concentration, pH and incubation time.

The analysis of zeta potential at different cation concentrations and pH
values showed that Mg\textsuperscript{2+} had the most significant
effect on endotoxin surface charge. The zeta potential of endotoxin was
reduced by a magnitude of 43.55 mV, from -43.53 mV to 0.02 mV in the
presence of 2.0 M Mg\textsuperscript{2+}, and a magnitude of 44.12 mV,
from -43.53 mV to 0.59 mV at the lowest pH level. However, in the
analysis of the theoretical number of cation bound per molecule of
endotoxin, Zn\textsuperscript{2+} showed the highest number (0.6)
compared to Ca\textsuperscript{2+} (0.12) and
Mg\textsuperscript{2+}(0.05). The tendency of Zn\textsuperscript{2+} to
preferentially bind with endotoxins forming a larger aggregated
structure was also evident in the DNA gel electrophoresis and
transmission electron microscopic analysis.

\textbf{INTRODUCTION}

Plasmid DNA has become a promising DNA-based vaccine and gene therapy
candidate for various acquired, infectious and genetic diseases {[}1{]}.
The production of recombinant pDNA using Gram-negative bacteria is less
expensive and time-efficient. However, the existence of toxic
lipopolysaccharides (LPS) in Gram-negative bacteria has become a major
issue in pDNA production. Endotoxins carry-over during pDNA isolation
decreases the purity of pDNA thus requiring additional purification
steps to ensure the safety of the final products {[}2{]}. Endotoxin
removal from pDNA is further complicated as endotoxins could form a
stable complex with pDNA apart from being heat and pH stable.

Endotoxins or lipopolysaccharides can be found on the outer layer of
Gram-negative bacteria. It is often released during cell growth or death
{[}3{]}. There are three major components of endotoxins which are the
o-specific antigen, core oligosaccharide, and lipid A {[}4{]}. Lipid A
is the most conserved part that causes the toxicity of endotoxins. A
minute amount of endotoxins in the human body could cause endotoxin
shock, inflammation, tissue injury and even lead to fatality {[}5{]}.
Thus, any drugs, particularly injectable drugs for humans have to be
screened for endotoxin contamination before the dosing protocol is
implemented {[}6{]}.

A previous study has found that divalent metal cations could affect and
act as selective precipitants for endotoxin including nickel ions,
magnesium ions, zinc ions, calcium ions and copper ions. These cations
were considered to have a high potential for removing endotoxins by
means of selective aggregation {[}7{]}. Thus, this manuscript intends to
report the effects of selected divalent metal cations which were calcium
ions (Ca\textsuperscript{2+}), magnesium ions (Mg\textsuperscript{2+})
and zinc ions (Zn\textsuperscript{2+}) on endotoxin and pDNA
interaction. The study was done based on several parameters such as
cation concentration, pH and incubation time. Theoretically, the ability
of cations to bind with endotoxins results in the preferential
aggregation hence precipitation of endotoxins thus leading to the high
recovery of pDNA.

\textbf{MATERIALS AND METHODS}

\textbf{List of materials}

Calcium chloride (CaCl\textsubscript{2}, Fisher Scientific), magnesium
chloride (MgCl\textsubscript{2}, Acros), zinc sulfate
(ZnSO\textsubscript{4}, Sigma-Aldrich), Tris base (Promega), Tris HCl
(Promega), acetic acid-glacial (Systerm), ethylene diamine triacetic
acid (EDTA, Amresco), ethidium bromide (EtBr, Sigma-Aldrich), agarose
(Amresco), 1kbp DNA ladder (Promega), blue/orange 6x loading dye
(Promega), ampicillin (Amresco), nutrient broth (Oxoid), PureYieldTM
Plasmid Miniprep System (Promega), arsenazo III (Fluka) and Endotoxin
E8029 (Sigma-Aldrich) were purchased from the respective local suppliers
in Malaysia.

\emph{Escherichia coli} \textbf{(\emph{E.coli}) cultivation for pDNA
isolation}

1 mL of \emph{E.coli} inoculum was transferred to 99 mL of nutrient
broth containing 50 \selectlanguage{greek}µ\selectlanguage{english}g/mL ampicillin and cultured overnight at
37\textsuperscript{o}C under 180 rpm. The growth was monitored by taking
an absorbance reading every 2 hours interval. Once the bacterial culture
reached the exponential phase, the culture was harvested and the plasmid
DNA was isolated using the PureYield\textsuperscript{TM} Plasmid
Miniprep System according to the manufacturer's instructions.

\textbf{Analysis of cation effects on endotoxin zeta potential at
different cation concentrations and pH values}

Zeta potential analysis was conducted to measure the effects of
magnesium (Mg\textsuperscript{2+}), calcium (Ca\textsuperscript{2+}) and
zinc (Zn\textsuperscript{2+}) ions on endotoxin surface charge. The pH
condition of each solution was fixed at pH 5. The endotoxin working
solution was made by dissolving the endotoxin stock solution in sterile
Milli-Q water to a final concentration of 8 \selectlanguage{greek}µ\selectlanguage{english}M. The working solution was
also used as the control solution. Cation concentrations of 0.1 M, 0.5
M, 1.0 M and 2.0 M were prepared using deionized water as the diluent.
The endotoxin working solution was added into separate microcentrifuge
tubes containing different concentrations of cation and incubated for 30
minutes at room temperature. The mixtures were then transferred into
separate zeta potential flow cells and analyzed using NanoPlus particle
analyzer (Particulate Systems) for 10 accumulation points and three
repetitions. The temperature was set at 25 \textsuperscript{o}C.

The above-mentioned analyses were repeated using cations prepared at
different pH values (pH 1, pH 3 and pH 5). Cation concentration was
fixed at 0.5 M as it was the most suitable and stable concentration for
the Zeta potential analysis {[}8{]}.

\textbf{Analysis of the theoretical number of cations bound per
endotoxin molecule at different cation concentrations and incubation
times}

This experiment was done to analyze how much of the divalent metal
cations interacted with endotoxins based on the theoretical number of
cations bound per endotoxin molecule. The results were used to compare
the binding affinities of Ca\textsuperscript{2+},
Mg\textsuperscript{2+}and Zn\textsuperscript{2+} towards endotoxin.
Arsenazo III was used to bind specifically with free cations and form
complexes which showed significant spectrophotometric absorbance
reading. However, the binding of cations with Arsenazo III was highly
dependent on its pH whereby it was only distinguishable and strong at pH
7 and above, particularly at pH 9 {[}8{]}.

250 \selectlanguage{greek}µ\selectlanguage{english}M of Arsenazo stock solution was prepared by mixing 19.41 mg of
Arsenazo III in 100 mL of Milli-Q water. Arsenazo working solution was
prepared by mixing 20 mL of the stock solution with 80 mL of Milli-Q
water to obtain a concentration of 50 \selectlanguage{greek}µ\selectlanguage{english}M. The pH value was adjusted to
pH 9. The cation stock solutions were prepared at 500 mM using deionized
water as a diluent. These solutions were further diluted to 2.5, 5, 10,
20 and 40 \selectlanguage{greek}µ\selectlanguage{english}M and the pH was adjusted to pH 7. 20 \selectlanguage{greek}µ\selectlanguage{english}L of 50 \selectlanguage{greek}µ\selectlanguage{english}M endotoxin
was added into each microcentrifuge tube containing 20 \selectlanguage{greek}µ\selectlanguage{english}L of cations at
2.5, 5, 10, 20 and 40 \selectlanguage{greek}µ\selectlanguage{english}M respectively. The working endotoxin
concentration used in this experiment was an optimum concentration that
would give a distinct cation-endotoxin binding {[}8{]}. The solutions
were incubated for 30 minutes at 25 \textsuperscript{o}C before 50 \selectlanguage{greek}µ\selectlanguage{english}M
Arsenazo III was added and further incubated for 10 minutes at
25\textsuperscript{o}C. The solutions were transferred into a 96-well
microplate (Greiner Bio-One) and analyzed using a UV-Vis
spectrophotometer (SpectraMax M2) at 610 nm wavelength. Standard
Arsenazo III -- cation binding curves were generated using free cations
without the addition of endotoxin. The standard curves were used in
constructing the Scatchard plot of which the maximum theoretical number
of cations bound per endotoxin molecule could be found at the x-axis
intercept.

The above experiment was repeated at different incubation times (1, 1.5,
2 and 2.5 hours) and a fixed cation concentration of 40 \selectlanguage{greek}µ\selectlanguage{english}M.

\textbf{Analysis of cation effects on endotoxin--pDNA interaction at
different cation concentrations, pH values, and incubation times.}

The purpose of this study was to analyze the binding of cations with
endotoxins and/or pDNA at different cation concentrations, pH values and
incubation times. In cations-pDNA sample preparation, 5 \selectlanguage{greek}µ\selectlanguage{english}L of pDNA was
added into separate microcentrifuge tubes containing 0.5, 1.0, 1.5 and
2.0 M cations respectively. The solutions were gently mixed using a
pipette and incubated for 30 minutes at 25 \textsuperscript{o}C. Next,
cations-pDNA-endotoxin samples were prepared by adding 5 \selectlanguage{greek}µ\selectlanguage{english}L of pDNA and
5 \selectlanguage{greek}µ\selectlanguage{english}L of endotoxin into separate microcentrifuge tubes containing the
above-mentioned cations. The solutions were gently mixed before being
incubated. All samples were loaded in 1\% agarose gel containing TAE
buffer and electrophoresed at 65 V for 70 minutes {[}7{]}. The gel was
then soaked in EtBr for 10 minutes and viewed using Gel DocTM EZ Imager
(Bio-Rad).

The above experiment was repeated at different pH values and a fixed
cation concentration of 1.0 M. The samples were electrophoresed in 1 \%
agarose gel and viewed using Gel DocTM EZ Imager (Bio-Rad).

The same experiment was repeated at different incubation times (0, 10,
30, 50 and 60 minutes), 1.0 M of cation concentration and pH 5. A
summary of the effects of cations on endotoxin--pDNA interaction was
drawn based on the profiles of the DNA electrophoresis.

\textbf{Transmission electron microscopy (TEM)}

The effects of Zn\textsuperscript{2+} on endotoxin and pDNA morphology
were further visualized under TEM. The optimal concentrations of
endotoxin and cation used for the TEM analysis were 3.125 \selectlanguage{greek}µ\selectlanguage{english}M and 0.05 M,
respectively.

The TEM images were taken for endotoxin, Zn\textsuperscript{2+} and the
complexes of endotoxin-cation and endotoxin-cation-pDNA. The endotoxin
and cation solutions were homogenized by sonication for 10 minutes. 2 \%
phosphotungstic acid was added into the solutions and mixed well prior
to transferring 2.5 \selectlanguage{greek}µ\selectlanguage{english}L of the solutions onto a carbon grid. The grid was
air-dried and micrographs were taken using the Tecnai, G2 Spirit Biotwin
electron microscope at magnifications of 2.9k and 13k.

\textbf{RESULTS AND DISCUSSION}

\textbf{Effect of divalent metal cations on endotoxin zeta potential}

Zeta potential is a measure of electrostatic repulsion or attraction
among particles. It can be used to indicate the stability of particles
in a solution based on their zeta potential values. A magnitude of \selectlanguage{ngerman}± 40
mV and larger indicates that the particles are well dispersed, while a
lower magnitude indicates that the particles are easily aggregated. The
colloidal stability based on zeta potential is presented in Table 1.

\textbf{{[}Table 1{]} {[}Table 2{]}}

The addition of 0.5 M Mg\textsuperscript{2+}, Ca\textsuperscript{2+} and
Zn\textsuperscript{2+} led to reduction of endotoxin zeta potential from
-43.53 mV to -0.52, -11.85, and -14.3 mV respectively (Table 2). The
downward trend of the magnitude of endotoxin zeta potential was observed
along with increasing cation concentration. Apparently at 2.0 M
Mg\textsuperscript{2+}, Ca\textsuperscript{2+} and
Zn\textsuperscript{2+}, the endotoxin particles were effectively
destabilized as indicated by the near-zero zeta potential values.

Endotoxins treated with pH 5 Mg\textsuperscript{2+},
Ca\textsuperscript{2+} and Zn\textsuperscript{2+} showed a significant
decrease of zeta potential values from -43.53 mV to -0.52, -11.85 and
-14.30 mV respectively. As expected, the zeta potential values of
endotoxin reached the destabilization phase at pH 3 (Table 3).

\textbf{{[}Table 3{]}}

Based on these results and Table 1, it was evident that the colloidal
stability of endotoxins was highly affected by pH and cation
concentration. The magnitude of the effect exerted by
Mg\textsuperscript{2+}, Ca\textsuperscript{2+} and
Zn\textsuperscript{2+} ions on endotoxins was in the order of
Mg\textsuperscript{2+}\textgreater{}Ca\textsuperscript{2+}\textgreater{}Zn\textsuperscript{2+}with
Mg\textsuperscript{2+} having the most profound effect on endotoxins
zeta potential.

Dudev and Lim {[}9{]} highlighted that Mg\textsuperscript{2+} and
Ca\textsuperscript{2+} promoted biological membrane stability through
charge neutralization when binding occurred to the carboxylated and
phosphorylated lipid headgroups. This experiment suggested that treating
endotoxins with high concentration and low pH of both
Mg\textsuperscript{2+} and Ca\textsuperscript{2+} led to the
destabilization of endotoxins by means of charge neutralization. The
interaction of cations with endotoxins was probably via cations binding
with lipids. In addition, the alteration of endotoxin charge by
Mg\textsuperscript{2+} and Ca\textsuperscript{2+} can be further
explained based on the study done by Lam \emph{et al.} {[}11{]}.
Divalent cations specifically Mg\textsuperscript{2+} and
Ca\textsuperscript{2+} play an important role in maintaining the
integrity of bacterial outer membrane by electrostatically altering its
permeability {[}11{]}. These lead to charge inhomogeneity on the
endotoxin surface where the inversion of endotoxin charge occurs
{[}11{]}.

In addition, the carboxylated group of keto-deoxyoctulosonate (KDO) in
endotoxins is responsible to increase the lateral interactions among
endotoxin molecules via divalent cation bridging. However, they become
protonated when the physiological pH is more acidic, hence the reason
for the charge conversion {[}12{]}.

\textbf{Theoretical number of divalent metal cations bound per endotoxin
molecule}

A Scatchard plot was used to determine how much cations were bound per
molecule of endotoxin {[}8{]}. The graph was constructed by plotting the
ratio of bound cation over free cation on the y-axis, versus the ratio
of bound cation over endotoxin molecule on the x-axis. The constructed
graph showed either a positive or negative cooperative binding profile
between cations and endotoxins. When the binding of a ligand increases
the apparent affinity of a receptor and the chance of another ligand
binding, it indicates positive cooperativity. While negative
cooperativity decreases the binding affinity of a ligand and makes
binding of another ligand less likely {[}13{]}.

Based on the constructed Scatchard plot, incubation of endotoxins at
different concentrations of Mg\textsuperscript{2+} showed negative
cooperativity with a maximum theoretical number of 0.05 cations bound
per molecule of endotoxin (Figure 1A). In contrast, exposing endotoxins
with Ca\textsuperscript{2+} and Zn\textsuperscript{2+} showed positive
cooperativity whereby Zn\textsuperscript{2+} had the highest theoretical
number of 0.6 cations bound per molecule of endotoxin (Figure 1C)
followed by Ca\textsuperscript{2+} with 0.12 cations bound per molecule
of endotoxin (Figure 1B). This finding further supports the previous
study done by Ongkudon et. al., {[}8{]} where a similar Scatchard
profile for Mg\textsuperscript{2+} was obtained. The negative binding
cooperativity of Mg\textsuperscript{2+} implies that the binding
affinity of Mg\textsuperscript{2+} towards endotoxins decreases with
increasing cation concentration. This finding also suggests that
endotoxins have the highest binding capacity for
Zn\textsuperscript{2+}followed by Ca\textsuperscript{2+} and
Mg\textsuperscript{2+} as evident from their respective maximum
theoretical number of cations bound per endotoxin molecule.

\textbf{{[}Figure 1{]}}

The effect of incubation time on the theoretical number of cations bound
per endotoxin molecule was shown in Figure 2A, Figure 2B and Figure 2C.
Apparently, increasing the incubation time of cation-endotoxin
interaction led to an increased theoretical number of cation bound per
endotoxin molecule. However, the magnitude of increment was relatively
low indicating that the interaction was instantaneous at the start of
the incubation period and might have equilibrated before the first hour
of incubation was completed. At 1 hour of incubation, the theoretical
number of cation bound per endotoxin molecule for
Mg\textsuperscript{2+}, Ca\textsuperscript{2+} and
Zn\textsuperscript{2+} were 0.05, 0.15 and 0.79 respectively.
Interestingly, Mg\textsuperscript{2+} and Ca\textsuperscript{2+} could
further bind with endotoxins after 1 hour of incubation while
Zn\textsuperscript{2+} almost reached its saturated condition. At 2.5
hours of incubation, these values increased to 0.5, 0.22 and 0.8
respectively. This result also indicated that Zn\textsuperscript{2+} had
the highest tendency to bind with endotoxins in the shortest time
compared to Ca\textsuperscript{2+} and Mg\textsuperscript{2+}.

\textbf{{[}Figure 2{]}}

The positive cooperativity of Ca\textsuperscript{2+} and
Zn\textsuperscript{2+} was already expected as these cations were
speculated to have specific binding affinities towards endotoxins which
led to the discovery of selective endotoxin precipitant {[}7{]}. In a
biological system (transporting ions to the cell via the outer
membrane), Ca\textsuperscript{2+} seems to mediate most of its effects
through a specific adaptor protein, whereas Zn\textsuperscript{2+} is
able to interact directly with a range of different target proteins
{[}14{]}. The ability of Zn\textsuperscript{2+} to interact directly
with biomolecules might also influence its binding affinity towards
endotoxin molecules as seen from the Scatchard plot. The molecular
details of the binding affinity of these cations towards endotoxins can
be further explored using advanced techniques including nuclear magnetic
resonance, molecular dynamic simulation and fluorescence lifetime
imaging.

\textbf{Effect of divalent metal cations on pDNA-endotoxin complexes}

The interaction between endotoxins and pDNA was further studied based on
the tendency of cations to bind either with endotoxins or pDNA. This was
done by observing the pDNA band migration and intensity in 1\% agarose
gel electrophoresis. Three parameters were studied including cation
concentration, pH value, and incubation time of pDNA-cation and
pDNA-endotoxin-cation complexes.

The effect of cation concentration on pDNA and pDNA-endotoxin complexes
are shown in Figure 3. Apparently, the pDNA band intensity and migration
towards the positive terminal of the electrophoresis unit decreased with
increasing cation concentration. This was due to the charge
neutralization effect exerted by the cations on the negatively charged
pDNA. It was also observed that Ca\textsuperscript{2+} and
Mg\textsuperscript{2+} had an indistinguishable effect on the
pDNA-endotoxin complex when compared to pDNA alone. However, the pDNA
band intensity was rather strong and less smeared in the
Zn\textsuperscript{2+}-pDNA-endotoxin complex compared to
Zn\textsuperscript{2+}-pDNA. This observation indicated that
Zn\textsuperscript{2+} had preferentially bound to endotoxins in the
pDNA-endotoxin complex leaving pDNA to migrate freely forming a more
intense band compared to Zn\textsuperscript{2+}-pDNA.

\textbf{{[}Figure 3{]}}

The effect of pH on cation-pDNA and cation-pDNA-endotoxin complexes were
rather interesting. Regardless of the presence or absence of endotoxins,
the trends of pDNA band intensity and migration were similar among all
cations Figure 4. This result indicated that acidic pH could interfere
with the interaction between endotoxins and cations as observed from the
Zn\textsuperscript{2+}-pDNA and Zn\textsuperscript{2+}-pDNA-endotoxin
complexes whereby no profound changes were observed.

\textbf{{[}Figure 4{]}}

Theoretically, the longer the incubation time, the less intense the pDNA
band as a result of the increased number of cation-pDNA binding. In this
experiment, cation concentration and pH values were fixed at 0.5 M and
pH 5 respectively, while incubation time was varied from 0 to 60
minutes. Based on Figure 5, no significant changes in the pDNA band
intensities were observed from 0 to 60 minutes for
Mg\textsuperscript{2+} and Ca\textsuperscript{2+} in both cation-pDNA
and cation-pDNA-endotoxin complexes. It indicated that the binding of
Mg\textsuperscript{2+} and Ca\textsuperscript{2+} with pDNA was more
favorable and rapid as compared to binding with endotoxins. This
observation was in agreement with the results portrayed in the Scatchard
plot analysis whereby it took a longer time (more than 1 hour) for
Mg\textsuperscript{2+} and Ca\textsuperscript{2+} to increase their
binding with endotoxins. However, Zn\textsuperscript{2+} seemed to
affect pDNA and pDNA-endotoxin complexes in a distinct way. Apparently,
the binding of Zn\textsuperscript{2+} with pDNA was also rapid but less
homogenous compared to Mg\textsuperscript{2+} and
Ca\textsuperscript{2+}thus resulting in smeared pDNA bands. When
endotoxin was present, the pDNA band intensity increased as a result of
preferential binding of Zn\textsuperscript{2+} with endotoxins which
occurred in a short time as observed in both the pDNA band and Scatchard
plot.

\textbf{{[}Figure 5{]}}

Based on the above observations, Mg\textsuperscript{2+} and
Ca\textsuperscript{2+} did not interfere with the pDNA-endotoxin
complexes. These results could be due to the antagonistic effect of
these cations on the pDNA molecule folding transition {[}17{]}. In
addition, the binding of pDNA with \emph{E. coli} -endotoxin in the
presence of Ca\textsuperscript{2+} has also been reported
previously{[}18{]}. Their work further reported the occurrence of
Ca\textsuperscript{2+}-mediated cell-membrane disintegration during the
movement of endotoxin-absorbed DNA into the cell cytosol {[}18{]}. This
phenomenon could be related to the results obtained in this research for
Ca\textsuperscript{2+}, whereby pDNA might be bound to endotoxins
instead of Ca\textsuperscript{2+}, leading to insignificant changes in
pDNA band intensity. The insignificant changes in pDNA band intensity in
the presence of Ca\textsuperscript{2+} and Mg\textsuperscript{2+}could
also imply that these cations probably act as mediators in forming
complexes of pDNA and endotoxins {[}15{]}.

In contrast, adding Zn\textsuperscript{2+} to pDNA-endotoxin complexes
resulted in a preferential binding of endotoxins with
Zn\textsuperscript{2+}. Besides the charge neutralization effect, the
slower mobility of pDNA-cations and pDNA-endotoxin-cations in the
agarose gel was also due to the larger size of pDNA aggregates formed
compared to free pDNA molecules {[}15{]}. The presence of two DNA bands
on the agarose gel indicates superhelical and circular pDNA {[}16{]}.
Superhelical pDNA is a highly mobile and conserved form of pDNA compared
to circular pDNA hence the difference in their DNA band intensity as
well as mobility. Although Zn\textsuperscript{2+} could significantly
interrupt the stable pDNA and endotoxin complexes, however, the pDNA
molecules were not fully dissociated from Zn\textsuperscript{2+} since
the band intensity was less than the control (pDNA without
Zn\textsuperscript{2+} and endotoxin). This observation correlates to
reduced pDNA recovery from endotoxin-pDNA complexes treated with
Zn\textsuperscript{2+} as reported previously by Ongkudon and Danquah
{[}7{]}\textbf{.}

Transmission electron microscopic (TEM) observation of
Zn\textsuperscript{2+}, endotoxins and pDNA interaction

It has been found that Zn\textsuperscript{2+} could bind with endotoxins
more preferably compared to Ca\textsuperscript{2+} and
Mg\textsuperscript{2+} even when pDNA was present. The binding of
Zn\textsuperscript{2+} with endotoxins in the presence of pDNA was
further observed at the nanometer scale using TEM. Figure 6 shows the
endotoxin E8079 particles without Zn\textsuperscript{2+} and pDNA. The
micrograph reveals that endotoxins were not aggregated. By adding 0.05 M
Zn\textsuperscript{2+} (in the form of ZnSO\textsubscript{4}) into the
endotoxin solution, a highly aggregated form of endotoxin was observed
in Figure 7. Notably, the addition of pDNA into the
endotoxin-Zn\textsuperscript{2+} solution showed no obvious changes in
the morphology (Figure 8).

\textbf{{[}Figure 6{]}}

\textbf{{[}Figure 7{]}}

\textbf{{[}Figure 8{]}}

Based on the electron micrographs, it could be said that endotoxins have
a unique binding preference toward Zn\textsuperscript{2+}. Although pDNA
and endotoxins were both negatively charged, in the presence of
Zn\textsuperscript{2+}, the aggregated form of endotoxins was not
disrupted which was in agreement with the previous findings {[}7{]}.
This observation indicated that the presence of pDNA did not effectively
interfere with the binding of endotoxins and cations. It was also
suggested that introducing Zn\textsuperscript{2+} into the endotoxin
suspension could lead to the compaction and self-rigidification of
endotoxin monomers {[}19{]}.

\textbf{CONCLUSION}

This study intended to provide further evidence on the effects of three
divalent metal cations, i.e; Mg\textsuperscript{2+},
Ca\textsuperscript{2+} and Zn\textsuperscript{2+} on endotoxins and pDNA
binding. It was hypothesized that Zn\textsuperscript{2+} could bind with
endotoxins more preferably as opposed to Mg\textsuperscript{2+}and
Ca\textsuperscript{2+} thus resulting in the separation of endotoxins
and pDNA. It could be seen that the addition of Zn\textsuperscript{2+}in
endotoxin-pDNA complex resulted in preferential binding of
Zn\textsuperscript{2+} with endotoxins as indicated by the highest
theoretical number of cation bound per endotoxin molecule and high
intensity of pDNA band in the DNA gel electrophoresis.

This study also showed that parameters such as cation concentration, pH
and incubation time greatly affected the endotoxin surface charge as
well as binding with cation hence separation from pDNA. In summary, the
effects of divalent metal cations on endotoxin increased in the order of
Mg\textsuperscript{2+}\textless{} Ca\textsuperscript{2+} \textless{}
Zn\textsuperscript{2+}.

\textbf{ACKNOWLEDGEMENT}

This project was funded and supported by the FRGS research grant scheme
of the Ministry of Higher Education, Malaysia and Universiti Malaysia
Sabah, Malaysia.

\textbf{Data availability statement:} Data openly available in a public
repository that issues datasets with DOIs

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