Binding Affinity of Inorganic Mercury and Cadmium to Biomimetic

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Binding Affinity of Inorganic Mercury and Cadmium to Biomimetic Erythrocyte Membranes Mohamed Hassanin, Evan Michael Kerek, Michael H Chiu, Max Anikovskiy, and Elmar J. Prenner J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b10366 • Publication Date (Web): 28 Nov 2016 Downloaded from http://pubs.acs.org on November 29, 2016

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The Journal of Physical Chemistry

Binding Affinity of Inorganic Mercury and Cadmium to Biomimetic Erythrocyte Membranes Mohamed Hassanin§, a, b, Evan Kerek§, a, Michael Chiu§, Max Anikovskiy† and Elmar J Prenner§*

a

both authors contributed equally

b

current address: Alberta Biophotonics, Calgary, Alberta, T2L 2K8

§

Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada

†

Department of Chemistry, University of Calgary, Calgary, Alberta T2N 1N4, Canada

*

Corresponding Author

Email: [email protected]

List of Abbreviations Dynamic light scattering (DLS), Inorganic mercury (Hg), Inorganic cadmium (Cd), Isothermal titration calorimetry (ITC), ITC salt solution (ISS), large unilamellar vesicle (LUV), multilamellar vesicle (MLV), palmitoyl-oleoyl-phospho- (POP-), 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine

(POPC),

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine

(POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS).

1 ACS Paragon Plus Environment


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Abstract Inorganic mercury and cadmium are becoming increasingly prevalent due to industrial activity and have been linked to cardiovascular disease and diabetes. The binding affinity of Hg, Cd and their mixtures to biomimetic erythrocyte membranes was investigated by isothermal titration calorimetry in physiologically relevant media (100 mM NaCl, pH 7.4, 37 ºC). The thermodynamic parameters were not expressed per mole of lipid but as metals binding to liposomes. To our knowledge, this method is novel and provides a more intuitive approach to understand such interactions. The results demonstrated that Hg interacted with membranes in the following order: PC (phosphatidylcholine) > 85:15 PC:PE (phosphatidylethanolamine) > 85:15 PC:PS (phosphatidylserine) with the binding constants ranging from 109 – 233 M-1. In contrast, Cd interacted most readily with negatively charged PC:PS membranes but not with the remaining systems. Metal mixtures bind less to PC/PE membranes than the individual constituents. The large entropy contribution from these interactions suggests possible water release and/or reorganization upon Hg and Cd binding to membranes. Zeta potential data indicate that the process may be electrostatically driven. It is imperative to consider the chemical speciation of these metals in the presence of chloride to better understand metal-lipid interactions and their impact on biomembranes.


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1. Introduction Increased industrialization since the early 1800’s led to a greater mobilization of heavy metals such as inorganic mercury (Hg) and inorganic cadmium (Cd) from the earth’s crust into ecosystems1. As a consequence, chronic heavy metal toxicity is increasingly becoming a major health problem for developing and developed nations. Various studies show strong links between Hg and Cd and the etiology of various diseases. Cd is associated with an increased prevalence of breast cancer, while both heavy metals are linked with the proliferation of cardiovascular disease2, 3. In addition, inorganic cadmium is strongly linked with the occurrence and propagation of diabetes4, 5. Upon intake from contaminated food and absorption into the bloodstream, Hg and Cd interact with various plasma components such as glutathione6, human serum albumin7 and red blood cells (RBC’s)8. With regard to erythrocytes, these heavy metals target intracellular glutathione8, hemoglobin9, and various integral membrane proteins such as aquaporin 110 and the hexose transporter11. Despite the breadth of studies that examined Hg and Cd interactions with plasma proteins and peptides, fewer studies examined the interactions between these heavy metals and the lipid components of the red blood cell membrane, despite membrane lipids comprising ~ 75 % of the overall membrane surface area12, 13. Therefore, the goal of this study was to examine the interactions of Hg, Cd and mixtures thereof with biomimetic red blood cell membranes. Several studies examined the influence of Hg and Cd on human red blood cells. It has been reported that incubation of human erythrocytes with 1 µM Hg at 37 ºC for 24 hours in a Ringer solution supplemented with HEPES buffer increased phosphatidylserine (PS) exposure on the outer membrane leaflet 10 fold in comparison to controls as demonstrated by increased



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annexin-V binding to PS14. In a similar study, 5.5 µM Cd induced the same effect15. These results showed that Hg and Cd addition can disrupt erythrocyte phospholipid asymmetry eventually leading to eryptosis of RBC’s. Furthermore, vesicle formation was reported by Lim et al when human RBC’s were incubated with 5 µM Hg for 1 hour at 37 º C16. The group also observed a 50 % increase in PS presence on the outer RBC membrane leaflet and in vesicles that were released suggesting that this increased PS exposure to the blood plasma could stimulate thrombin activity and consequently clot formation. Glycerophospholipids (GPL) are the most abundant phospholipids in the RBC membrane making up ~ 38 % of the total molar lipid content. GPL’s are most often distinguished based on headgroup with the most common types being phosphocholine (PC), phosphoethanolamine (PE), and phosphoserine (PS) (Fig. S1). Based on headgroup structure, the overall ratio of PC:PE:PS in human RBC membranes is ~ 4:2:117. Furthermore, membrane lipids are asymmetrically distributed between the two leaflets where ~ 80-85 % of PC is found on the outer membrane leaflet, and 80-85 % of PE, and more than 96 % of PS are found on the inner leaflet17. Through fluorescence quenching of monomeric pyrene by Hg, it was observed that HgCl2 penetrates deep into the hydrophobic core of PS containing liposomes at pH 5.018. Experiments involving 199Hg and 31P NMR supported the hypothesis that Hg binds the amine group of PE and PS in a 1:3 metal: lipid ratio19, 20, 21. Furthermore, Hg was not observed to target the phosphate group of membrane lipids20, 21. Using X-ray diffraction spectroscopy, 10 µM Hg induced a 75 % decrease in intensity of X-ray signal of 5 mg/mL dimyristoyl – PC (DMPC) vesicles, indicating a membrane disordering effect upon Hg binding22. Finally, fluorescence spectroscopy studies


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concluded that 1 µM Hg, in the absence of NaCl, bound 100 µM liposomes in the order: 85/15 PC/PS > 85/15 PC/PE > PC (mol %)23. Nuclear magnetic resonance studies revealed that Cd bound PS >>> PC ≈ PE24. This implied that the interaction of Cd with lipids was largely electrostatic in nature and that the negatively charged serine headgroups are the most likely interaction sites. In a different study, 1 mM Cd expanded monolayers composed of animal Cephalin (0. 67 mg/mL), containing predominantly PE and PS, by 7.5 % at a surface pressure of 30 mN/m, relative to controls25. This pressure range, provides a good correlation between monolayer and bilayers in terms of lateral lipid packing26. X-ray diffraction spectroscopy has revealed that Cd weakly binds to PC and the strength of the interaction was similar to that of Ca2+

27

. Finally, it was shown by

fluorescence spectroscopy that in the absence of NaCl 1 µM Cd targets 100 µM 85/15 PC/PE > PC > 85/15 PC/PS >>> cholesterol containing vesicles23. In this study, the thermodynamic parameters of inorganic mercury and cadmium binding to different biomimetic large unilamellar vesicles (LUV’s) were measured. The enthalpy change (∆H), binding constants (Kb), free energy change (∆G), entropy change (-T∆S), and stoichiometry (n) of binding were determined from isothermal titration calorimetry (ITC) and Scatchard plots28. Since the first point of contact between heavy metals and the membrane occurs at the lipid headgroup of the outer membrane leaflet, model liposomes mimicking the lipid composition of these leaflets were used. Furthermore, since humans are exposed to different pollutants concurrently, the binding affinity of metal mixtures (1:1 Hg: Cd) to model membranes was also examined. Generally, ITC studies report thermodynamic information as per mole of lipid, which is less intuitive for the current experimental setting considering the binding of metal ions to liposomes. Thus, dynamic light scattering (DLS) was used to determine the size of the



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liposomes and based on the lipid concentration used and published values for the average area per molecule the number of liposomes in the solution was calculated. This allowed the assessment of how many metals were bound per liposome. Moreover, membrane surface potential studies were carried out to determine whether metal-lipid interactions were electrostatic in nature. In contrast to many previous papers on toxic metal membrane interactions, the impact of ionic strength on metal speciation was taken into account. This is important as it is known that Hg and Cd can form various chloride and hydroxide species depending on the Cl- concentration, pH and temperature29, 30. It is important to note that the use of Hg and Cd hereon are used as a generic term which does not imply that these metals are found as non-complexed ions at physiological conditions. The chemical speciation of Hg and Cd was determined by using the equilibrium speciation model program Visual Minteq 3.1 for given experimental conditions31. In 100 mM NaCl solution at pH 7.4 and 37 ºC, Hg is predicted to speciate into ~37% HgCl2, ~37% HgCl3-1, ~21% HgCl42- and ~5% HgClOH while Cd is predicted to form ~65% CdCl+, ~19% Cd2+ and ~16% CdCl2. The goal of this study was to quantify heavy metal interactions with RBC membrane lipids while taking metal speciation into account. This is crucial as different metal species likely have different binding affinities to lipid membranes compared to solvated Hg2+ and Cd2+ ions that are typically singled out in interaction studies. 2. Material and Methods 2.1 Materials 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

(POPC),

1-palmitoyl-2-oleoylsn-

glycero-3-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-Lserine (POPS) (Fig. S1) were obtained from Avanti Polar Lipids (Alabaster, AL) and used


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without further purification. HgCl2 and CdCl2 were purchased from Sigma-Aldrich (Oakville, ON). All solutions and lipid suspensions were prepared using deionized water supplied by a Millipore Synergy 185 water purification system (Billerica, MA). Previous experiments showed strong complexation of HEPES and TRIS buffers with Hg 19

. Furthermore, the formation of precipitate was observed upon adding Hg to MOPS and PIPES

and Cd to phosphate and hydrogen carbonate buffers. Therefore, no buffer was used in the ITC experiments. Instead, LUVs and metal solutions were prepared in water containing 100 mM of NaCl. The pH was adjusted to 7.4 with NaOH and did not change over the period of the experiments. This solution will be referred to as “ITC salt solution (ISS)” henceforth.

2.2 Lipid System Preparation Lyophilized lipids were weighed using a Sartorius Microbalance MC 5 (Göttingen, Germany) and dissolved in a 3:1 (v/v) mixture of chloroform: methanol. The solvent was evaporated under a constant flow of argon followed by an overnight incubation under vacuum to ensure complete removal of the solvent. The lipid films were hydrated using ISS to yield the final lipid concentration of 4 mM. This was done to ensure that the preparation always yielded 3 mM lipids which was the final concentration used for ITC. To remove the lipids from the container walls, the vials were vortexed, sonicated, and subjected to 5 cycles of freezing in liquid nitrogen and thawing in boiling water. To produce the LUVs the formed MLVs were extruded (Avanti Mini Extruder, Alabaster, AL) through a 100 nm polycarbonate filter. 2.3 Lipid Systems examined Three lipid systems were examined: 100% POPC, 85% POPC:15% POPE, and 85% POPC:15% POPS which will be referred to as PC, PC:PE, and PC:PS, respectively. Since 7 ACS Paragon Plus Environment


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phosphocholine is the most dominant headgroup on the outer membrane surface (~ 80-85 %), metal interactions to PC LUVs were examined first and was used as a reference to the remaining lipid systems. The addition of ~ 15 % of PE reflects the concentration found on the outer membrane surface

17

. Finally, in order to compare the affinity of Hg, Cd or mixtures to the PE

containing LUVs, PC:PS LUVs were made at the same molar ratio. Moreover, lipids with palmitoyl (16:0) and oleoyl (18:1) fatty acyl tails were used in this study since the majority of the lipids in human erythrocytes contain one saturated and one monounsaturated fatty acyl tail32.

2.4 Isothermal Titration Calorimetry Isothermal titration calorimetry experiments were conducted using a MicroCal ITC200 microcalorimeter (Malvern Instruments, Malvern, UK). The sample cell contained lipid suspensions at a total lipid concentration of 3 mM. In the case of PC:PS, 20 mM lipid suspension was used in order to obtain a measurable enthalpy. The metal solutions for injection contained 10 mM of Hg, Cd, or a 1:1 mixture (5 mM Hg + 5 mM Cd). Prior to measurements all samples were degassed for 4 minutes using the ThermoVac unit (Malvern Instruments, Malvern, UK). All ITC trials were conducted at 37 ºC and involved a total of 20 injections. To take into account the heats of dilution of metal and ISS injections into the sample cell, titrations of metal into ISS and ISS into LUV suspensions were also conducted. The enthalpy changes measured in the control experiments were subtracted from the data to obtain the net enthalpy of interaction. Furthermore, the net enthalpies determined from Hg only and Cd only injections into lipid suspensions were averaged to predict “Hg: Cd Calc” or the enthalpy changes that the metal mixture (1:1 Hg: Cd) would exhibit upon injection into a lipid suspension if the presence of one metal ion has no influence on the action of the other metal ion.


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2.5 Dynamic Light Scattering and Zeta (ζ) potential measurements The size of LUVs was measured using dynamic light scattering (DLS) DynaPro Titan (Wyatt Technology Corp, Santa Barbara, CA). Briefly, a 0.6 mM LUV suspension was made and degassed for 4 min. Experiments were performed in triplicate for each sample. Size distribution data were compiled and automatically plotted by the DynaPro software. The LUV size was used subsequently to determine the LUV concentration (see 2.6). In order to estimate the surface potential of liposomes, Zeta (ζ) potential experiments were conducted using a Malvern Zetasizer Nano ZS (Worcestershire, UK). Briefly, 1 ml of 0.5 mM lipid LUVs (suspended in ISS) was added to a zetasizer zeta potential folded capillary cell (DTS1060, Worcestershire, UK) and the lipid suspension was equilibrated for 10 minutes at 37 ºC. Conducting the experiments in the presence of up to 100 mM monovalent salt ensures that the measured ζ -potential values are a good approximation of membrane surface potential

33

. A total of 4 different trials, from two different sample

preparations, were explored for each of the lipid samples. Value averaging and t-test statistical analysis were both done using Microsoft Excel. 2.6 LUV Concentration Determination During extrusion lipids bind to the polycarbonate filter. As a result, the concentration of the extruded lipid suspension may vary from the original concentration (4 mM). To determine the total lipid concentration after extrusion a sensitive fluorometric method was used 34. Briefly, 50 µL lipid aliquots were collected from the pre-and post-extruded samples. The aqueous solvent was evaporated and the lipid pellet was redissolved in 100 µL chloroform. To help promote lipid dissolution, the samples were sonicated for 10 seconds and



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vortexed for a further 10 seconds. Finally, all tubes were centrifuged at 5000 rpm for 1 min using a Thermo IEC Micromax centrifuge (Thermo Scientific, Ottawa, ON) to pellet NaCl. 8 µg of Rhodamine 6G were dissolved in 2 mL of cyclohexane containing 1 µL of glacial acetic acid. The dye was excited at 535 nm and changes in its fluorescence upon lipid binding were monitored at 563 nm over the course of six 2 µL additions of the lipid solutions. The experiments were performed on a Cary Eclipse fluorescence spectrophotometer using 5 nm bandwidth for both slits (Agilent Technologies, Santa Clara, CA). The post-extrusion lipid concentration was calculated by comparing the slope of the known concentrations before extrusion to the slope after the LUV preparation. Once the LUV size and lipid concentration were determined, the molar concentration of LUVs (i.e. mol LUV/L) was estimated as outlined in the supplemental material section. Estimating the molar concentration of LUVs is important for determining the number of metals that bind per LUV once the was determined. To our knowledge, this was the first time that the ratio of bound metals/LUV was calculated to analyze ITC data. Since ITC can help in calculating the of metal-lipid interactions, it was also possible to calculate the fraction of total metal that bound to the various lipid systems. 2.7 Data Analysis 2.7.1 ∆Hmax Calculations and Scatchard Plot The metal-lipid interactions in the ITC experiments did not result in the typically observed sigmoidal binding curve necessary to determine the binding constant. Consequently, the ITC software could not be used to compute the and Scatchard plots28 were utilized. Therefore, the ratio of metal: lipid was used as shown in Equation 1: [] []

= × − [] ×

(1) 10

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[] represents the amount of metal bound to lipid

[]

[]

, where [] is the total

amount of lipid available for binding, [] is the concentration of free metal, is the association []

constant and n is the stoichiometry of the interaction. In this case a plot of

[]

vs. [] enables

the determination and . First, was determined. This value represents the binding of ~100% of metal to lipid. To this effect, a small volume of metal (2 µL of 10 mM) was injected into lipid solutions of increasing concentrations (0 to 20 mM) and the maximum enthalpy was obtained by fitting the data to Equation 2: ∆ = " exp&−' []( + ∆

(2)

where ", ', and ∆ are the fitting parameters. ∆ and [] represent the enthalpy change and lipid concentration, respectively. Increasing the concentration of lipid shifts the equilibrium to the complex formation thus promoting metal binding to the excess lipid. Upon calculating from the single injection experiments the fraction of bound metal with each injection was calculated as follows: *+

*+,-

= ./

&3(

Where ∆H is the ITC measured enthalpy for each injection step. The concentration of bound metal can be determined using Equation 4: ./ × []121 = []

(4)

where []121 is the total concentration of metal injected. Consequently, the concentration of unbound metal [M] can then be calculated by subtracting [ML] from [M]total. With the necessary [] and [] now known, the Scatchard plot was utilized to obtain the and n of metal-lipid binding.



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The binding constant(s) determined was then used to calculate the change in the Gibbs free energy (∆G using the gas constant R and the absolute temperature T. ∆3 was then used to calculate the entropy contribution, ∆4, using the Gibbs equation. Therefore, the measurement of ∆ was sufficient to provide a detailed thermodynamic characterization of metal-lipid interactions. Statistical analysis was conducted using the t-test in Microsoft excel to determine statistical significance in the binding constants of Hg to different lipids systems. 2.7.2 Number of bound Metals per LUV Analysis The number of Hg or Cd ions bound per a lipid molecule was calculated using the binding equation: []

= [][] = &[]

[]

5 6[](&[]5 6[](

(5)

where []7 and []7 are metal and lipid concentrations after the last injection in the ITC titration experiments. Solving for [] and dividing [] by [89] yields the number of metals bound per LUV. The fraction of metal bound to the lipids [ML] can be calculated as a percentage of the total amount of metals in the experiments. 2.7.3 Calculating Surface Concentration of Metal The investigations of the binding of Hg and Cd to LUVs composed of phospholipids POPC and POPS address both chemical binding as well as electrostatic interactions. Dissolving Hg and Cd in 100 mM NaCl is predicted to result in predominantly positive Cd and negative Hg charged complexes. Liposomes composed of zwitterionic POPC at pH 7.4 bear no net charge and will neither attract nor repel charged Hg or Cd species from the surface of the membrane. However, the addition of a negatively charged lipid POPS imparts a net negative charge to the 12 ACS Paragon Plus Environment

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surface of the membrane which repels ions of a like charge and attracts positively charged ions. This results in a higher concentration of charged species of Cd and a lower concentration of respective Hg species near the membrane surface compared to the bulk concentration. Additionally, the negatively charged membrane results in a decreased chlorine concentration near the membrane surface compared to the bulk solution thereby affecting the Hg and Cd speciation near the membrane surface. This concentration shifts due to electrostatic contribution is of importance as our previously discussed method of calculating the binding constants from the metal lipid interactions is directly dependent on the concentration of metal in solution. Thus, to account for the electrostatic contribution to the observed interaction between Hg and Cd and the PS containing system, this analysis was conducted to more accurately calculate the binding constant. To determine the overall charge of the metal near the surface, the concentration of chloride near the membrane surface was calculated using the Boltzmann Equation (Eq. 6). :;? = :/ 1:1 Hg: Cd > Cd. For comparative purposes, enthalpy changes that were calculated for the mixture of Hg and Cd are also shown. Interestingly, there was no apparent binding for Cd despite the presence of 20 mM lipid. However, experimentally obtained Hg: Cd enthalpy changes were on average 6-12 % lower compared to the values predicted by the calculations. This suggests that that Cd affects the interaction of Hg with POPC.



0.20

0.00 0.00 ∆H (Kcal/mol metal)

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5.00

10.00

15.00

20.00

-0.20

-0.40

-0.60

-0.80

-1.00

∆Hmax (Kcal/mol) Hg -2.214 Cd n/a Hg-Cd -1.456 -1.395 Hg-Cd Calc [lipid] (mM)

Figure 3. Net enthalpies obtained from single injections of metal solutions into various total concentrations of POPC. Each point represents an average of 3 trials from at least 2 different sample preparations of 2 µL 10 mM metal injections into 3 mM. Error bars = standard deviations of at least 3 trials from 2 different sample preparations.

3.2.3 Calculation of Kb using ∆Hmax and the Scatchard plot The concentration of metal-lipid complex at each injection was calculated using Eq. 5 and the Scatchard plot whose slope gives –Kb and the y-intercept n Kb. The Scatchard plots of Hg– PC and Cd-PC/PS interactions are presented in Fig. 4. For PC:PS, two regions of linearity were observed in the Scatchard plot consistent with a two binding site model. In this case, the slope of each binding site was calculated separately. The Kb and n of Hg binding to PC were calculated to be 233 ± 13 M-1 and 0.213 ± 0.012, respectively. Alternatively, the number of lipids per interaction site, 1/n, was determined to be 4.69 ± 0.27 PL/site.


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In the case of the Hg-PC interaction, ~ 19.7 % of the total metal in solution was bound to lipid at a pH 7.4, 100 mM NaCl and a temperature of 37 ºC corresponding to ~1.1x104 + 1.3x103 metals bound/LUV assuming only the outer leaflet (~55% of total lipids) are available targets for the metal. 55

160

50

140

45 YML/[M] (M-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


y = -232.57x + 49.59 R² = 0.9828

120

y = -1951.3x + 157.77 R² = 0.9857

100 40 80 35 60 30

40

25 20 0.00

y = -555.53x + 78.568 R² = 0.9943

20

0.02 Y 0.04 ML

0.06

0 0.00

0.05

0.10

0.15

YML

Figure 4. Scatchard plot of YML/[M] vs. YML for Hg-POPC (left) Cd-85/15 PC/PS Kb (right) and determination. YML = [ML]/[lipid].

3.2.4 Thermodynamic parameters of metal interactions with PC, PC:PE, and PC:PS model systems Table 1 provides a summary of the ∆Hmax determined for each lipid system whereby the highest enthalpies were -11.5 and -9.4 kJ/mol for Hg into PC and the PC/PE systems respectively while enthalpies for the Hg/Cd mixtures were -4.9 and -7.0 kJ/mol for the PC and PE systems. The lowest enthalpy found in this study was -1.75 for Cd and the PC/PS system. Upon



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determination of ∆Hmax for each of the lipid systems and calculation of Kb, the free energy (∆G) and entropy contribution (-T∆S) of metal-lipid interactions were calculated. Overall, Hg- PC binding was enthalpy driven (∆Hmax > -T∆S) while no interaction was observed in this case for Cd (Table 1). The measured binding constant of the metal mixture to PC was 23% lower than the predicted value as depicted by Hg: Cd Calc. Finally, the number of lipids per interaction site was between 4 and 7 for this metal system. Following the last injection of metal, between 17.5-20.7% of the metal was bound to the LUVs. Similarly to the results for PC, Hg interactions with PC:PE were enthalpy driven while Hg: Cd binding to PC:PE had a three times higher entropy contribution of 5.99 kJ/mol compared to 1.95 with Hg alone. The Kb determined for the binding of Hg to PC:PE was 20 % lower than the calculated affinity for pure PC LUVs and these results were significant (p < 0.05). Moreover, the binding constant obtained from the binding of the metal mixtures was 2.7 times lower than expected from calculations (Hg: Cd Calc). Additionally, only 7.9% of metal was bound to the LUVs making this the weakest binding observed in this study. The number of lipids /interaction site for Hg was ~6 for the PC:PE system and ~2.5 for the Hg/Cd mixture which shows that fewer binding sites are occupied by metal when both Hg and Cd are present. The binding of Hg to PC:PS LUVs was exothermic but the data could not be fit to provide the ∆Hmax as the enthalpies obtained did not level off with increased lipid concentrations. However, it was observed that the Kb reached a plateau when ∆Hmax was varied as shown in Fig. S2. Since the number of lipids/interaction site varied in the range of ~4-10 in previous systems, we estimated that the Kb of Hg-PC/PS would correspond to a ∆Hmax for which the Scatchard plot shows a 1/n value between 4-10. Taking this into account, the Kb was estimated to be ~ 109-113 M-1. While this did not allow for an accurate determination of the remaining thermodynamic


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parameters, it provided an estimate to allow for a comparison of the binding affinities of Hg for PC, PC/PE and PC/PS. On the other hand, the Scatchard plot for Cd and PC/PS displayed a nonlinear upward trending pattern in which the first 3 data points had a much higher slope compared to the rest of the data (Fig. 4 Right). This curve is consistent with Scatchard plots for systems with two binding sites as previously described in detail 36. The slope of the first 3 data points yielded a Kb of 1953 + 177 M-1 which will be referred to as Site 1 while the Kb for Site 2 was 556 + 72 M-1 using the surface concentrations of Cd (See 2.7.3). The determination of ∆Hmax utilized a single injection of Cd by varying the concentration of lipid until saturation occurred. As the concentration of Cd after this single injection falls within the range of metal for which binding Site 1 was calculated, the enthalpy for binding Site 2 cannot be determined. ∆G’s of -19.5 and -16.3 KJ/mol were determined for Sites 1 and 2 respectively while the -T∆S component was unable to be calculated for Site 2 without the ∆Hmax. In contrast to results from Hg and previous lipid systems, the interaction between Cd and PC/PS was mostly entropy driven with a -T∆S of -17.8 KJ/mol which was the largest determined in this study. As two binding constants were determined, the percentage of metal bound was calculated at the end of the 3rd injection (corresponds to binding to Site 1) as well as the last injection (corresponds to binding to Site 2). While only 11.6% Hg was bound to the PC/PS LUVs after the last injection, 94.8% of Cd was bound after the 3rd injection while ~56% was bound at the end of the titration. This was the highest percentage of metal bound to the LUVs in this study compared to the next highest which was only 19.7% Hg bound to POPC after the last injection. Overall, the binding constants of Hg and Cd for each lipid system in this study are shown in Fig. S3.



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Table 1. Summary of Thermodynamic parameters obtained for the binding of Hg, Cd and 1:1 Hg:Cd to different RBC biomimetic LUVs. 1/n is the lipids/interaction site. ∆Hmax

POPC

(KJ/mol)

Hg

2

Kb (x10 -1

M )

-9.43 ± 0.27 2.3 ± 0.1

Cd

NI

∆G (KJ/mol)

-T∆S (KJ/mol)

1/n

% metal bound

-14.1 ± 0.1 -4.62 ± 0.31 4.69 ± 0.27 19.7 ± 0.7

NI

NI

NI

NI

NI

# metals 4

bound (x10 ) /LUV 1.07 ± 0.13 NI

Hg-Cd -6.37 ± 0.31 1.9 ± 0.2

-13.6 ± 0.3 -7.20 ± 0.30 6.29 ± 0.67 17.5 ± 1.3

0.95 ± 0.13

Hg-Cd -5.80 ± 0.42 2.5 ± 0.3 Calc

-14.3 ± 0.3 -8.50 ± 0.54 7.10 ± 0.94 20.7 ± 1.7

1.13 ± 0.15

∆Hmax POPC/ POPE (KJ/mol)

2

Kb (x10 -1

M )

Hg

-11.52 ± 0.57

1.8 ± 0.2

Cd

NI

NI

∆G (KJ/mol)

-T∆S (KJ/mol)

1/n

% metal bound

-13.5 ± 0.3 -1.95 ± 0.66 6.34 ± 0.25 16.8 ± 1.6 NI

NI

NI

-10.9 ± 0.3 -5.99 ± 0.56 2.47 ± 0.35 7.9 ± 0.9

Hg-Cd -6.99 ± 0.28 1.9 ± 0.1 Calc

-13.5 ± 0.2 -6.48 ± 0.33

10.32 ± 0.733

16.9 ± 0.8

2

Kb (x10

4

bound (x10 ) /LUV 1.06 ± 0.11

NI

Hg-Cd -4.93 ± 0.43 0.7 ± 0.1

∆Hmax POPC/ POPS (KJ/mol)

# metals

0.50 ± 0.06 1.07 ± 0.07 # metals

-T∆S (KJ/mol)

1/n

M )

∆G (KJ/mol)

% metal bound

~1.1

-

-

-

11.6 + 0.2

0.62 + 0.04

Cd Site -1.75 ± 0.21 19.5 ± 1.7 -19.5 ± 0.2 -17.8 ± 0.2 12.4 ± 1.0 1

94.8 ± 0.4

0.72 ± 0.05

Cd Site 2

55.9 + 1.4 3.41 + 2.39

Hg

-

-

-1

5.6 + 0.7 -16.3 + 0.3

-

6.8 + 0.2

4

bound (x10 ) /LUV

3.3 ζ- potential measurement results To correlate the binding behaviour of Hg and Cd to the lipid systems examined, zeta potential measurements were conducted. Zeta potential was highest for PC:PS >> PC > PC:PE


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(Fig. 5). The zeta potential of PC:PS LUVs was 9 times more negative in comparison to the PC and PC:PE liposomes and these results were statistically significant (p < 0.01).

Lipid System PC

PC:PE

PC:PS

0 -5 ζ-Potential (mV)

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-10 -15 -20 -25 -30 **

-35

Figure 5. ζ-potential studies of various lipid systems. Results are averages of 4 trials. [lipid] = 0.5 mM. Applied voltage = 5 V. Error bars = SD. ** = p < 0.01 compared to pure POPC. 4. Discussion In order to gain a better understanding of the mechanism of chronic toxicity of Hg and Cd on cellular and organ function, it is very important to investigate the interactions of the metals with biomolecules. In this study, the thermodynamic parameters of heavy metals binding to model membranes were determined by ITC using the fact that heat exchanges due to binding can be directly quantified without any labelling. When metals enter the blood stream, there are a number of different targets they may interact with while the most prevalent cell they encounter are red blood cells, which make up 23 ACS Paragon Plus Environment


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45% of the volume37. Consequently, LUVs mimicking the outer leaflet of these cells were formulated using the lipids shown in Fig. S1 for the current investigation as the metals will interact with this leaflet first. As a result of the differences in these metals affinity for chlorides, under our experimental conditions, Hg and Cd form different complexes which leads to a pronounced difference in their affinities for different phospholipids. Due to the formation of predominantly positively charged complexes, it was predicted that Cd would have a higher affinity to the net negatively charged PS lipid and a lower affinity for zwitterionic PC and PE lipids. In contrast, Hg forms complexes with predominantly 2-4 chlorides leading to neutral and negatively charged species that likely change the way Hg interacts with these same lipids. The first lipid used in this screen was the main component of the outer leaflet of RBCs, the partly unsaturated POPC. The interactions of Hg with these membranes were exothermic (Figs. 1A and 2). The Scatchard plot for this system yielded a Kb of 233 + 13 (Table 1) while the enthalpy recorded for the interaction of Cd with PC was nearly zero (Fig. 1B and 2). In comparison to the limited number of contributions in the literature, another ITC study on the interaction of La3+ to PC determined a Kb of ~4500 M-1 in 100 mM NaCl, at pH 5.6 and 27 ºC which is ~19x stronger than for Hg38. As La is a trivalent metal that is predicted to be ~93% La3+ under these experimental conditions by using Visual Minteq, a much stronger binding to the phosphate in PC membranes is expected. In contrast, the formation of neutral and negative (i.e. HgCl2, HgCl3-, HgCl42-) species under the same conditions very likely results in much weaker binding. However, Hg bound PC LUVs ~223x stronger than monovalent alkali metals39. The interaction of the monovalent alkali metals and La3+ with PC membranes were endothermic38,39 whereby the driving force was presumed to be the release of water molecules from the metal 24 ACS Paragon Plus Environment

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hydration shells. The fact that the Hg-POPC interaction had a net negative enthalpy (See Fig.2) could indicate a more complex binding mechanism. One possible explanation is a reorganization of the Hg species by a release of Cl- upon binding. Indeed, there is evidence for an increase of free Cl- upon Hg binding to PE and PS lipids19. Whereas the likely target of the positively charged monovalent and La3+ ions is the negatively charged phosphate of PC, the permanent positive charge of +1 in the choline headgroup is presumed to attract the negatively charged HgCl3- and HgCl42-. On the other hand, Cd did not interact with POPC in the presence of 100 mM NaCl (Figs. 2 and 3) presumably due to the formation of CdCl+ species in the presence of salt. In contrast, the divalent Cd present in the absence of chloride was shown to interact with PC membranes by fluorescence studies23. In order to investigate the discrepancy between the experimental (Hg: Cd) and predicted (Hg: Cd Calc) metal mix binding affinity to PC membranes (Table 1), ITC trials of Cd injections into Hg, and vice versa, were conducted (Fig. S4). Interestingly, Hg injections into Cd were slightly exothermic while those of Cd into Hg were endothermic. It is possible that both metals could indeed be interacting with one another or that the presence of one metal may affect the chloride speciation of the other, resulting in a rearrangement of metal species and consequently to a change in the measured enthalpy. Since lower binding affinities were observed in the presence of the metal mixture (See Table 1), the proportion of Hg-chloride species responsible for binding may decrease when Cd is available. Alternatively, Hg may increase the fraction of particular Cd specie(s) thereby enhancing the binding affinity of the latter metal to PC membranes resulting in antagonistic competition for the available binding sites. Two such species could be CdCl2 and CdCl3- which increase with higher [Cl-]. As the [Cl-] used in the ITC



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studies was ~ 100 times greater than the concentrations of both heavy metals combined, the influence of one metal on the speciation of another would be predicted to be negligible. It is more probable that the effect of one metal on the speciation of the other occurs on a more localized level, specifically near the membrane surface where metal-lipid interactions take place. In that regard, the presence of Hg-chloride complexes at the lipid-water interface may release chloride19 and may thus increase the overall available Cl- near the membrane surface and thereby increase the local concentration of CdCl3- and CdCl2 promoting the binding of these complexes. Considering that humans are exposed to multiple chemicals concurrently, the results show that the presence of one contaminant may impact and in some cases enhance the binding, and possibly overall toxicity, of another. In contrast to pure PC vesicles, the binding affinity of Hg to PC:PE LUVs was statistically different (p < 0.05) and 20% lower. Interestingly, the binding of Hg to PC:PE membranes was even more enthalpy driven compared to pure PC membranes (Table 1). These differences in Hg binding can be correlated with the structural differences between PC and PE. The primary amine of PE lipids form rigid networks via hydrogen bonding of one NH3+ group to a neighbouring PO4- group which is thought to considerably reduce the hydration of PE membranes compared to PC40. It was previously reported that the hydration of PC lipids is more than twice that of PE, although this number varies depending on lipid chain length and the presence of double bonds41. Assuming that Hg binds to PE, a disruption and/or breaking of these hydrogen bonds could occur and would lead to a net increase in the number of bonds between water and PE lipids. The effect of different metal cations on membrane surface potential and curvature has been shown to depend heavily on metal and lipid hydration states as well as the tendency for these metals to disrupt the lipid hydrogen bonding networks42.


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As the intrinsic pKa of the primary amine group of PE is ~ 9.643, this group carries a net positive charge at a pH of 7.4 and may therefore play a role in the binding of negatively charged Hg-chloride complexes. Moreover, the binding of Hg to PC:PE monolayers deposited using a physiologically relevant subphase (1 mM phosphate, 100 mM NaCl, pH 7.4) depended greatly on the proportions of PC and PE present44 suggesting that lipid packing may also influence the accessibility of the lipids and thus the targeting of Hg to the membrane. Nonetheless, the obtained results agree with fluorescence spectroscopy and X-ray diffraction data which show a greater preference of Hg for PC over PE membranes22,23. However, these data contrast with others showing a stronger Hg binding to the primary amine of PEs 19,20,45. These differences can be attributed to the metal/lipid ratio as well as different salt and temperature conditions. Both studies in which Hg was found to bind PE stronger used 74 mM acetate buffer between pH 4619,20 while studies that showed Hg’s preference for PC used distilled water22 and 1 mM phosphate 100 mM NaCl buffers pH 7.4 23. Studies that utilize 100 mM NaCl in the buffers have a large difference in chloride compared to studies where the chloride comes from HgCl2 dissolved in solution. This lends support to the hypothesis that different Hg complexes likely have different affinities for PC and PE phospholipids. As seen for the PC membranes, Cd exposure of PC/PE yielded enthalpies that were close to zero which suggests that no interaction took place. This observation contrasts previous results that showed Cd binding of PE over PC lipids at micromolar concentrations23,44. Again, the difference in experimental conditions must be considered as the first study23 was conducted in a phosphate buffer at pH 7.4 with no chloride while the second study44 used a 1 mM phosphate, 100 mM NaCl at pH 7.4. As Cd readily forms a precipitate with phosphate46, it is likely that the



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net charge of Cd in these studies was considerably different than in this study under phosphate free conditions which likely has a large impact on Cd-lipid interactions. Interestingly, the metal mixture bound PC:PE membranes much less strongly than calculated based on the addition of the single metal contributions; the presence of Cd decreased the overall binding constant to 70 M-1 compared to 180 M-1 for Hg alone (Table 1). These results imply that Cd may indeed bind PE-containing membranes although the net enthalpy is close to zero. It is important to note that the absence of signal in ITC does not disprove binding since the interactions of Cd with PE may involve endothermic and exothermic components that are similar in magnitude and would cancel out resulting in an overall net zero enthalpy. As such, the binding of Hg to the membrane is reduced due to greater competition with Cd for the same lipid interaction sites. The last system was a binary mixture of PC and the negatively charged which exhibited weak binding with an estimated Kb of 109-113 M-1 which is less than half of what was determined for PC alone. This is consistent with the formation of negatively charged Hg-chloride complexes that are electrostatically repulsed by PS. The presence of chloride and the pH are the determining factors regulating the interactions of Hg with anionic lipids since Hg abolished the membrane phase transition of DMPS MLVs in 74 mM acetate buffer pH 5.845. On the other hand, the negative charge of PS will attract the positive Cd-chloride complexes in which two binding sites were observed in the Scatchard plot (Fig. 4 Right) with Kb values of 19.5 x102 and 5.6 x102 M-1, respectively (Table 1). Indeed, our results show that after the third injection 94.8% of the Cd was bound to the LUVs while only 55.9% of metal was bound at the end of the titration which is consistent with high affinity binding to site 1 (Table 1). A lower percentage of Cd bound at the end of the titration can be explained by a saturation of the


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PS lipid sites with Cd after which additional injections of metal have no effect. It is then likely that the much stronger initial binding site corresponds to the more exposed carboxyl group while the weaker binding site is the buried phosphate group. Fluorescence studies with the molecule Laurdan show that Cd induces larger changes in membrane rigidity in POPS compared to POPE 47

. As the only difference in these lipid structures is the serine carboxyl group in POPS, the effect

of Cd is a result of the accessibility and closer proximity of this negative charge to aqueous phase compared to the less accessible phosphate group. In order to quantify the differences in electrostatic attraction, the Zeta potential of the model membranes was measured as shown in Fig 5. PC/PS showed a negative membrane potential of -27 mV while PC and PC:PE had potentials of -2.6 and -0.6 mV respectively. As stated earlier, at physiological [Cl-] and pH about 19 % of Cd are divalent while 65% are comprised of CdCl+ and 19 % are CdCl2. As such, ~ 84% of the total Cd species in solution carry a net positive charge allowing for favourable charge-charge attractions to the negatively charged PS. Indeed, Cd interacted most strongly with the various model systems examined in the order PS >> PE = PC (Fig. S3). The binding of Cd to PC:PS vesicles was observed to have a negative enthalpy while the overall interaction was largely entropy driven (Table 1) which was similar to the interaction of Ca2+ with 70:30 PC:PG membranes48. Another study also found that the binding of Ca2+, Mg2+ and Sr2+ to DMPG was also largely entropy driven with only a 5-10% negative enthalpy contribution in the ∆G49 which was very similar to a 9% enthalpy contribution from the binding of Cd to PC:PS in this study (Table 1). Thus, water reorganization or release of water from both divalent metal and anionic lipid hydration shells plays a substantial role in these binding mechanisms. On the other hand, the binding constant of Ca2+ to PC:PG liposomes was 1.4 x 103



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M-1 which was 28% smaller than the Kb for Site 1 despite the higher concentration of the negatively charged lipid (i.e. 85:15 PC:PS vs. 70:30 PC:PG. A difference in results are expected as PG only has one negatively charged group at the phosphate in the backbone while PS has two, the phosphate in the backbone and the carboxyl in the headgroup. Nonetheless, results indicate that Cd has a higher affinity to negatively charged membranes than Ca2+. It was also observed that Ca2+ in the presence of salt did not bind to pure PC vesicles

38

but bound 4:1 PC:PS

membranes in the absence of salt but the interaction was completely diminishing at 100 mM NaCl 50. These studies support the conclusion that Cd behaves in a similar manner as Ca2+ when targeting PC and PC:PS membranes while the binding of Cd is likely stronger as we could still observe an interaction in 100 mM NaCl. However, it is important to note that Cd species and Ca2+ targeting of negatively charged bilayers will also depend on the overall charge localization and distribution in the lipid headgroups. PS has a partially buried negative phosphate moiety and an exposed carboxyl group within the zwitterionic serine group in contrast to one negative charge on the PG located at the phosphate group. Indeed, the finding that Cd has a 2x higher lipid/water partition coefficient for DMPS than into DMPG vesicles by using NMR supports the claim that the localization and environment of the charged group(s) may have an influence on Cd interactions with lipid membranes

24

. Therefore, the overall net negative charge of PS could be responsible for the

attraction of Cd and/or CdCl+ to the membrane surface after which the phosphate moiety is targeted by the metal. Furthermore, packing defects in the PC/PS lipid mixture have to be considered that may facilitate the binding of Cd to phosphate groups inPC or PS. Given that the lipids/interaction site were ~12 for Cd-PC/PS, the binding of Cd may not be entirely specific for PS. On the other hand, it is unlikely that Cd would interact with more than 2 PS lipids at any


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given moment since the area of a Cd ion is about 3 Å2 area/lipid

52

51

compared to between 55-65 Å2

. Ca2+ was indeed proposed to bridge two PG or two PS phospholipids48,53.

Nonetheless, it is possible that the binding of the Cd complex under our experimental conditions to PC:PS membranes may induce membrane lipid clustering. While this binding was strong, it was observed that higher NaCl concentrations (0.8 mM and 1.8 mM NaCl) abolished the effect of Cd on the membrane phase transition of pure DMPG and DMPA MLVs while reducing the effect by 50% on DMPS lipid membranes 24. This implies that the interaction of Cd with anionic lipids strongly depends on both the shielding of lipid charges by Na+ ions as well as the speciation of Cd into CdCl2 and CdCl3-1 formed under very high salt conditions. It is important to note that the binding of Cd to the membrane might also depend on lipid packing and the fraction of available PS. The expansion of PC:PS monolayers was observed to depend heavily on the PC/PS ratio albeit no direct relation between metal binding and the ratio of PS: total lipid could be deduced44. The results obtained herein and previous work in the literature suggest that the interactions of Hg and Cd interactions with lipid membranes not only depend on the salt, pH and temperature at but also upon the lipid type and packing, that will define metal speciation and the access to specific binding sites (e.g. primary amine, phosphate etc.) on the membrane. 5. Conclusions A quantitative thermodynamic characterization of Hg, Cd, and metal mixture binding to RBC biomimetic membranes was provided to add to the overall understanding of metal-lipid interactions. Most importantly it was found that the binding affinity of the metal mixtures to pure



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PC membranes is higher compared to the values found for the single metals indicating that the presence of one metal may synergistically enhance the binding of another. Hg was observed to bind RBC model membranes in the order PC > PC:PE > PC:PS in a largely entropy driven reaction suggesting liberation of water molecules upon metal binding. Binding was based on electrostatic interactions of the mostly negatively charged chloride species of Hg while Cd solely interacted electrostatically with PS in an overall entropy driven reaction. The above results enhance our knowledge of metal-lipid interactions, and may also contribute to a greater understanding of heavy metal induced RBC toxicity.

Supporting Information •

We describe how the [LUVs] was calculated using the radii of LUVs determined with DLS as well as the area occupied per lipid headgroup from the literature.

•

Table S1 contains the size of the LUVs of each system determined DLS as well as the [LUVs] based on these sizes

•

Figure S1 shows the structures and net charges of the lipids used in this study

•

Figure S2 shows the binding constant of Hg to the 85/15 POPC/POPS system as a function of the enthalpy of the interaction

•

Figure S3 is a summary of the binding constants of Hg and Cd determined for the 3 lipid systems in this study

•

Figure S4 shows ITC data for injections of Hg into Cd and Cd into Hg

Acknowledgements This work was supported by a NSERC Discovery grant to EJP. 32 ACS Paragon Plus Environment

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References (1)

Nriagu, J. O. A History of Global Metal Pollution. Science. 1996, 272 (5259), 223–224.

(2)

Peters, J. L.; Perlstein, T. S.; Perry, M. J.; McNeely, E.; Weuve, J. Cadmium Exposure in Association with History of Stroke and Heart Failure. Environ. Res. 2010, 110 (2), 199– 206.

(3)

Virtanen, J. K.; Voutilainen, S.; Rissanen, T. H.; Mursu, J.; Tuomainen, T.-P.; Korhonen, M. J.; Valkonen, V.-P.; Seppänen, K.; Laukkanen, J. A.; Salonen, J. T. Mercury, Fish Oils, and Risk of Acute Coronary Events and Cardiovascular Disease, Coronary Heart Disease, and All-Cause Mortality in Men in Eastern Finland. Arterioscler. Thromb. Vasc. Biol. 2005, 25 (1), 228–233.

(4)

Edwards, J. R.; Prozialeck, W. C. Cadmium, Diabetes and Chronic Kidney Disease. Toxicol. Appl. Pharmacol. 2009, 238 (3), 289–293.

(5)

Satarug, S.; Garrett, S. H.; Sens, M. A.; Sens, D. A. Cadmium, Environmental Exposure, and Health Outcomes. Cien. Saude Colet. 2011, 16 (5), 2587–2602.

(6)

Fuhr, B. J.; Rabenstein, D. L. Nuclear Magnetic Resonance Studies of the Solution Chemistry of Metal Complexes. IX. Binding of Cadmium, Zinc, Lead, and Mercury by Glutathione. J. Am. Chem. Soc. 1973, 95 (21), 6944–6950.

(7)

Lau, S.; Sarkar, B. Inorganic mercury(II) Binding Components in Normal Human Blood



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Serum. J. Toxicol. Environ. Health 1979, 5 (5), 907–916. (8)

Rabenstein, D. L.; Isab, A. A. A Proton Nuclear Magnetic Resonance Study of the Interaction of Mercury with Intact Human Erythrocytes. Biochim. Biophys. Acta - Mol. Cell Res. 1982, 721 (4), 374–384.

(9)

Weed, R. Interaction of Mercury with Human Erythrocytes. J. Gen. Physiol. 1962, 45 (3), 395–410.

(10)

Yukutake, Y.; Tsuji, S.; Hirano, Y.; Adachi, T.; Takahashi, T.; Fujihara, K.; Agre, P.; Yasui, M.; Suematsu, M. Mercury Chloride Decreases the Water Permeability of Aquaporin-4-Reconstituted Proteoliposomes. Biol. Cell 2008, 100 (6), 355–363.

(11)

Vansteveninck, J. Localization of Erythrocyte Membrane Sulfhydryl Groups Essential for Glucose Transport. J. Gen. Physiol. 1965, 48 (4), 617–632.

(12)

Grasberger, B.; Minton, A. P.; DeLisi, C.; Metzger, H. Interaction between Proteins Localized in Membranes. Proc. Natl. Acad. Sci. U. S. A. 1986, 83 (17), 6258–6262.

(13)

Zwaal, R. F. A.; Demel, R. A.; Roelofsen, B.; van Deenen, L. L. M. The Lipid Bilayer Concept of Cell Membranes. Trends Biochem. Sci. 1976, 1 (2), 112–114.

(14)

Eisele, K.; Lang, P. A.; Kempe, D. S.; Klarl, B. A.; Niemöller, O.; Wieder, T.; Huber, S. M.; Duranton, C.; Lang, F. Stimulation of Erythrocyte Phosphatidylserine Exposure by Mercury Ions. Toxicol. Appl. Pharmacol. 2006, 210 (1–2), 116–122.

(15)

Sopjani, M.; Föller, M.; Dreischer, P.; Lang, F. Stimulation of Eryptosis by Cadmium Ions. Cell. Physiol. Biochem. 2008, 22 (1–4), 245–252.

(16)

Lim, K. M.; Kim, S.; Noh, J. Y.; Kim, K.; Jang, W. H.; Bae, O. N.; Chung, S. M.; Chung, J. H. Low-Level Mercury Can Enhance Procoagulant Activity of Erythrocytes: A New Contributing Factor for Mercury-Related Thrombotic Disease. Environ. Health Perspect.


Page 34 of 40

Page 35 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2010, 118 (7), 928–935. (17)

Zachowski, A. Phospholipids in Animal Eukaryotic Membranes: Transverse Asymmetry and Movement. Biochem. J. 1993, 294 ( Pt 1, 1–14.

(18)

Boudou, A.; Desmazes, J. P.; Georgescauld, D. Fluorescence Quenching Study of Mercury Compounds and Liposome Interactions: Effect of Charged Lipid and pH. Ecotoxicol. Environ. Saf. 1982, 6 (4), 379–387.

(19)

Delnomdedieu, M.; Boudou, A.; Georgescauld, D.; Dufourc, E. J. Specific Interactions of Mercury Chloride with Membranes and Other Ligands as Revealed by Mercury-NMR. Chem. Biol. Interact. 1992, 81 (3), 243–269.

(20)

Girault, L.; Lemaire, P.; Boudou, A.; Dufourc, E. J. Inorganic Mercury Interactions with Lipid Components of Biological membranes:31P-NMR Study of Hg(II) Binding to Headgroups of Micellar Phospholipids. Water, Air, Soil Pollut. 1995, 80 (1–4), 95–98.

(21)

Girault, L.; Lemaire, P.; Boudou, A.; Debouzy, J. C.; Dufourc, E. J. Interactions of Inorganic Mercury with Phospholipid Micelles and Model Membranes. A 31P-NMR Study. Eur. Biophys. J. 1996, 24 (6), 413–421.

(22)

Suwalsky, M.; Ungerer, B.; Villena, F.; Cuevas, F.; Sotomayor, C. P. HgCl2 Disrupts the Structure of the Human Erythrocyte Membrane and Model Phospholipid Bilayers. J. Inorg. Biochem. 2000, 81 (4), 267–273.

(23)

Le, M. T.; Gailer, J.; Prenner, E. J. Hg2+ and Cd2+ Interact Differently with Biomimetic Erythrocyte Membranes. Biometals 2009, 22 (2), 261–274.

(24)

Girault, L.; Boudou, A.; Dufourc, E. J. 113Cd-, 31P-NMR and Fluorescence Polarization Studies of cadmium(II) Interactions with Phospholipids in Model Membranes. Biochim. Biophys. Acta - Biomembr. 1998, 1414 (1–2), 140–154.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(25)

Suzuki, Y.; Matsushita, H. Interaction of Metal Ions and Phospholipid Monolayer as a Biological Membrane Model. Ind. Health 1968, 6 (3), 128–133.

(26)

Marsh, D. Lateral Pressure in Membranes. Biochim. Biophys. Acta 1996, 1286 (3), 183– 223.

(27)

Lis, L. J.; Lis, W. T.; Parsegian, V. A.; Rand, R. P. Adsorption of Divalent Cations to a Variety of Phosphatidylcholine Bilayers. Biochemistry 1981, 20 (7), 1771–1777.

(28)

Scatchard, G. The Attractions of Proteins for Small Molecules and Ions. Ann. N. Y. Acad. Sci. 1949, 51 (4), 660–672.

(29)

Jurinak, J. J.; Baes, C. F.; Mesmer, R. . The Hydrolysis of Cations; Wiley Interscience: Toronto, 1976; Vol. 40.

(30)

Hahne, H. C. H.; Kroontje, W. Significance of pH and Chloride Concentration on Behavior of Heavy Metal Pollutants: Mercury(II), Cadmium(II), Zinc(II), and Lead(II)1. J. Environ. Qual. 1973, 2 (4), 444.

(31)

Gustafsson, J. P. Visual MINTEQ 3.1. KTH Department of Land and Water Resources Engineering: Stockholm, Sweden 2011.

(32)

Myher, J. J.; Kuksis, A.; Pind, S. Molecular Species of Glycerophospholipids and Sphingomyelins of Human Erythrocytes: Improved Method of Analysis. Lipids 1989, 24 (5), 396–407.

(33)

Aveyard, R.; Haydon, D. . An Introduction to the Principles of Surface Chemistry; Cambridge University Press: Cambridge, Great Britain, 1973; pp 87–88.

(34)

Schiefer, H.-G. G.; Neuhoff, V. Fluorometric Microdetermination of Phospholipids on the Cellular Level. Hoppe-Seyler´s Zeitschrift für Physiol. Chemie 1971, 352 (7), 913–926.

(35)

Winiski, A. P.; Mclaughlin, A. C.; Mcdaniel, R. V; Eisenberg, M.; Mclaughlin, S. An


Page 36 of 40

Page 37 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Experimental Test of the Discreteness-of-Charge Effect in Positive and Negative Lipid Bilayers. Biochemistry 1986, 25, 8206–8214. (36)

Bordbar, A. K.; Saboury, A. A.; Moosavi-Movahedi, A. A. The Shapes of Scatchard Plots for Systems with Two Sets of Binding Sites. Biochem. Educ. 1996, 24 (3), 172–175.

(37)

Rodkiewicz, C. M.; Sinha, P.; Kennedy, J. S. On the Application of a Constitutive Equation for Whole Human Blood. J. Biomech. Eng. 1990, 112 (2), 198–206.

(38)

Lehrmann, R.; Seelig, J. Adsorption of Ca2+ and La3+ to Bilayer Membranes: Measurement of the Adsorption Enthalpy and Binding Constant with Titration Calorimetry. Biochim. Biophys. Acta 1994, 1189 (1), 89–95.

(39)

Klasczyk, B.; Knecht, V.; Lipowsky, R.; Dimova, R. Interactions of Alkali Metal Chlorides with Phosphatidylcholine Vesicles. Langmuir 2010, 26 (24), 18951–18958.

(40)

McIntosh, T. J. Hydration Properties of Lamellar and Non-Lamellar Phases of Phosphatidylcholine and Phosphatidylethanolamine. Chem. Phys. Lipids 1996, 81 (2), 117–131.

(41)

Nagle, J. F.; Wiener, M. C. Structure of Fully Hydrated Bilayer Dispersions. Biochim. Biophys. Acta 1988, 942 (1), 1–10.

(42)

Kraayenhof, R.; Sterk, G.; Wong Fang Sang, H.; Krab, K.; Epand, R. Monovalent Cations Differentially Affect Membrane Surface Properties and Membrane Curvature, as Revealed by Fluorescent Probes and Dynamic Light Scattering. Biochim. Biophys. Acta 1996, 1282, 293–302.

(43)

Tsui, F. C. C.; Ojcius, D. M. M.; Hubbell, W. L. L. The Intrinsic pKa Values for Phosphatidylserine and Phosphatidylethanolamine in Phosphatidylcholine Host Bilayers. Biophys. J. 1986, 49 (2), 459–468.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(44)

Le, M. T.; Hassanin, M.; Mahadeo, M.; Gailer, J.; Prenner, E. J. Hg- and Cd-Induced Modulation of Lipid Packing and Monolayer Fluidity in Biomimetic Erythrocyte Model Systems. Chem. Phys. Lipids 2013, 170–171, 46–54.

(45)

Delnomdedieu, M.; Boudou, A.; Desmazès, J.-P.; Georgescauld, D. Interaction of Mercury Chloride with the Primary Amine Group of Model Membranes Containing Phosphatidylserine and Phosphatidylethanolamine. Biochim. Biophys. Acta - Biomembr. 1989, 986 (2), 191–199.

(46)

Ayati, M.; Madsen, H. E. L. Solubility Product of the Cadmium Phosphate Cd5H2(PO4)4·4H2O at 37 °C. J. Chem. Eng. Data 2001, 46 (1), 113–116.

(47)

Kerek, E. M.; Prenner, E. J. Inorganic Cadmium Affects the Fluidity and Size of Phospholipid Based Liposomes. Biochim. Biophys. Acta - Biomembr. 2016, 1858 (12), 3169–3181.

(48)

Arseneault, M.; Lafleur, M. Isothermal Titration Calorimetric Study of Calcium Association to Lipid Bilayers: Influence of the Vesicle Preparation and Composition. Chem. Phys. Lipids 2006, 142 (1–2), 84–93.

(49)

Garidel, P.; Blume, A. Interaction of Alkaline Earth Cations with the Negatively Charged Phospholipid 1,2-Dimyristoyl- Sn -Glycero-3-Phosphoglycerol: A Differential Scanning and Isothermal Titration Calorimetric Study. Langmuir 1999, 15 (17), 5526–5534.

(50)

Sinn, C. G.; Antonietti, M.; Dimova, R. Binding of Calcium to Phosphatidylcholine– phosphatidylserine Membranes. Colloids Surfaces A Physicochem. Eng. Asp. 2006, 282– 283, 410–419.

(51)

Island, R. Differences in Cadmium and Mercury Uptakes by Hepatocytes : Role of Calcium Channels 1 The Liver Plays an Important Role in the Dis- Position of Cadmium (


Page 38 of 40

Page 39 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cd ) and Mercury ( Hg ). The Accumulation of Cd in Hepatocytes Is Rapid , Biphasic , Non Energy Depe. 1991, 3. (52)

Kučerka, N.; Tristram-Nagle, S.; Nagle, J. F. Structure of Fully Hydrated Fluid Phase Lipid Bilayers with Monounsaturated Chains. J. Membr. Biol. 2006, 208 (3), 193–202.

(53)

Ohki, S.; Ohshima, H. Divalent Cation-Induced Surface Tension Increase in Acidic Phospholipid Membranes. Biochim. Biophys. Acta - Biomembr. 1984, 776 (1), 177–182.



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Binding Affinity of Inorganic Mercury and Cadmium to Biomimetic

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