Equilibrium Sorption of Structurally Diverse Organic Ions to Bovine

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Equilibrium Sorption of Structurally Diverse Organic Ions to Bovine Serum Albumin Luise Henneberger, Kai-Uwe Goss, and Satoshi Endo Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b06176 • Publication Date (Web): 20 Apr 2016 Downloaded from http://pubs.acs.org on April 26, 2016

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Environmental Science & Technology

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Equilibrium Sorption of Structurally Diverse

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Organic Ions to Bovine Serum Albumin

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Luise Henneberger,† Kai-Uwe Goss,†,‡ Satoshi Endo,†,§*

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† Helmholtz Centre for Environmental Research UFZ, Permoserstr. 15, D-04318 Leipzig,

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Germany

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‡ University of Halle-Wittenberg, Institute of Chemistry, Kurt-Mothes-Str. 2, D-06120 Halle,

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Germany

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§ Osaka City University, Urban Research Plaza & Graduate School of Engineering, Sugimoto 3-

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3-138, Sumiyoshi-ku, 558-8585 Osaka, Japan

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ABSTRACT

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Reliable partitioning data are essential for assessing the bioaccumulation potential and the

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toxicity of chemicals. In contrast to neutral organic chemicals, the partitioning behavior of

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ionogenic organic chemicals (IOCs) is still a black box for environmental scientists. Partitioning

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to serum albumin, the major protein in blood plasma, strongly influences the freely dissolved

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concentration of many chemicals (including IOCs), which affects their transport and distribution

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in the body. Because consistent datasets for partitioning of IOCs are rarely available, bovine

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serum albumin-water partition coefficients (KBSA/w) were measured in this study for 45 anionic

ACS Paragon Plus Environment

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and 4 cationic organic chemicals, including various substituted benzoic and naphthoic acids,

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sulfonates and several pesticides and pharmaceuticals. The results of this study suggest that

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binding to BSA is substantially influenced by the three dimensional structure of the chemicals

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and the position of substitutions on the sorbing molecules. For example, we found a difference of

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> 1.5 log units between isomeric chemicals such as 3,4-dichlorobenzoic acid and 2,6-

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dichlorobenzoic acid, 1-naphthoic acid and 2-naphthoic acid, and 2,4,6-trimethylbenzoic acid

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and 2,4,6-trimethylbenzenesulfonate. Conventional modeling approaches (e.g. based on octanol-

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water partition coefficients) poorly predict log KBSA/w of organic ions (R2 ≤ 0.5), partially

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because they do not capture the observed steric effects. Hence, alternative modeling strategies

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will be required for accurate prediction of serum albumin-water partition coefficients of organic

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ions.

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Introduction

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Partitioning of ionogenic organic chemicals (IOCs) into biological tissues and their

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constituents such as various lipids and proteins is of great importance, as it has direct

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implications for bioconcentration, bioaccumulation, and toxicity of IOCs. However, established

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models to predict relevant partition coefficients are almost exclusively focused on neutral

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organic chemicals including the neutral species of ionizable chemicals. For example, a simple

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regression with log Kow is widely used as a screening model, which sometimes gives a

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surprisingly

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Polyparameter linear free energy relationships (PP-LFERs) can give more accurate predictions

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for broader ranges of chemicals and partition phases.2, 3 These empirical models are convenient

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but applicable only for neutral chemicals. To our knowledge, there is only one model that was

good

approximation

(e.g.,

for

membrane-water

partition

coefficients).1


2

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developed specifically for the prediction of biopartitioning of IOCs in general.4 One reason for

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the absence of models may be the lack of experimental data that are measured under consistent

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conditions for partitioning of charged chemicals.

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Bioaccumulation models used for risk assessment often assume that the dominant sorption

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phase in organisms is the total lipid fraction. For IOCs storage lipids are expected to play a minor

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role while phospholipid membranes and the protein fraction of an organism are supposed to

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contribute significantly to the overall partitioning process.5 Serum albumin, the most abundant

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protein in blood plasma, is likely an important sorption phase for IOCs, because research on

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pharmaceuticals and endogenous chemicals has long shown that serum albumin binds a broad

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spectrum of chemicals, especially hydrophobic anions of medium size.6-8 Binding to serum

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albumin has major impact on transport and distribution of a target chemical in the body, because

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it increases the sorption capacity of the blood and decreases the free, unbound concentration of

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the chemical.9,

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serum albumin binding often determines the bound and the unbound fractions of the test

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chemical in the medium.11 Furthermore, serum albumin is often considered a generic protein to

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represent various properties of the bulk protein fraction of organisms, including sorption

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properties,12 although this assumption may not always be valid and thus needs careful

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evaluation.13

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Moreover, fetal bovine serum is widely used for cell culture assays, where

In the literature, binding of a target chemical to protein, e.g., bovine serum albumin (BSA), is typically reported as association constant Ka [M-1]. For 1:1 binding Ka is defined as:

=

1


3


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where Cfree [mol/Lwater] is the freely dissolved molar concentration of the chemical, Cbound the

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molar concentration of the chemical bound to BSA, and [BSA] the free molar concentration of

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BSA in the solution. The extent of binding to BSA can also be expressed as BSA-water partition

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coefficient (KBSA/w [Lwater/kgBSA]), which is preferably used by environmental scientists and is

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defined as the ratio of the concentration of the target chemical in BSA (CBSA [mol/kgBSA]) to the

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freely dissolved concentration of the chemical: ⁄ = ⁄ 2

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In case the chemical concentration is so low that BSA binding is far below saturation, Ka and KBSA/w are convertible with a constant factor (i.e., Ka = 101.83 KBSA/w).14

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Large amounts of data are available in the literature for binding of pharmaceuticals (including

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also IOCs) to human serum albumin. However, reliability and comparability of these data are

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difficult to evaluate. As reviewed by Nilsson et al.,15 data for protein binding are often published

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without carefully controlling all the factors that can influence the partitioning process, e.g.,

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temperature; the pH value; concentrations of the test chemical, salt, and the protein; non-specific

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adsorption to the used equipment; equilibrium disturbances; and leaking of dialysis membrane.

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Moreover, pharmaceuticals are often multifunctional molecules with complex structure. A

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collection of data for such diverse chemicals is difficult to interpret in terms of structural

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influences on the binding constant. Systematically measured data for a series of chemicals with

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incremental changes in the substructure units (e.g., the number of Cl on the benzene ring) or data

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for a pair of chemicals that differ by only one structural feature may shed additional light on the

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binding mechanisms of IOCs to serum albumin.

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This study aims to elucidate how the molecular structure of organic ions (e.g., different basic

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structures, substitutions and charged functional groups) influences their partitioning to serum


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albumin and which mechanisms underlie the sorption process. To this end, we investigated the

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partitioning of systematically selected organic anions and cations to BSA, starting from simple

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compounds onwards to more complex structures. Particularly, many varyingly substituted

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benzenes and naphthalenes with an anionic functional group were included to study the

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influences of molecule structures on serum albumin binding. All measurement conditions were

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thoroughly controlled. Additionally, influence of pH value, dependence on the concentration of

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inorganic ions, and reversibility of BSA binding were determined experimentally. Finally, the

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data obtained were correlated with various descriptors to gain an insight into the requirements of

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successful modeling for serum albumin binding.

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Materials and methods

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Materials

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No. A3803) and used without further purification. BSA was chosen because of its good

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availability and comparability to the previous publication for neutral compounds.14 Water

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purified with a Milli-Q Gradient A10 system from Millipore was used. Methanol (Suprasolv)

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was obtained from Merck and acetonitrile (gradient grade) from Sigma Aldrich. Unless

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otherwise noted below, all sorption experiments were performed using Hanks’ balanced salt

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solution (HBSS, without phenol red and sodium bicarbonate, Sigma Aldrich) buffered with 10

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mM tris(hydroxymethyl)aminomethane (Tris) from Carl Roth. After addition of sodium

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bicarbonate and Tris, the pH value of HBSS was adjusted to 7.4 using 1 N HCl or NaOH

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solution from Merck. The exact salt concentrations of HBSS and a comparison with human

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plasma are shown in Table S3, SI. Ammonium acetate, formic acid, orthophosphoric acid, bis(2-

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hydroxyethyl)amino-tris(hydroxymethyl)methane

Bovine serum albumin (essentially fatty acid free) was purchased from Sigma Aldrich (Product

(Bis-Tris),

sodium

bicarbonate,

sodium


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chloride, sodium sulfate and sodium azide were purchased from Sigma Aldrich, Carl Roth or

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Merck.

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The chemicals used for the binding experiments were different organic acids and bases, or salts

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of them, all of which have only one ionizable functional group and are more than 99 % ionized at

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pH 7.4. To investigate the influence of different substitutions on the binding constant, a series of

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benzoic acids, naphthoic acids and sulfonates were chosen for the dataset. More complex

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compounds (i.e., pesticides and pharmaceuticals) were included, because of their environmental

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relevance. All test chemicals are listed in Table 1 and had purity of at least 98%. More details on

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the test chemicals (e.g., CAS number, provider, analytical method used for quantification,

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chemical structure, pKa values and recovery from control experiments) can be found in the

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Supporting Information Table S1. In this work, weak acids and bases are always denoted with

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the chemical names of their neutral species (e.g., 4-chlorobenzoic acid) because they are more

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common than the names for the ionic species (e.g., 4-chlorobenzoate), despite the fact that weak

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acids and bases in our test solutions are always predominantly in their ionic form and that KBSA/w

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reported in this work is thus for the ionic species.

121 122

Dialysis experiments

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detail.16 In short: a custom-made dialysis cell that consists of two half-cells (total volume approx.

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10 mL) and dialysis membranes from Spectrum Laboratories (type Spectra/Por 4 RC with a

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molecular weight cutoff of 12-14 kD) was used. One half-cell of the dialysis unit received 5 mL

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HBSS buffer and the other half-cell 4.9 mL BSA solution (1-50 g/L). All samples were spiked

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with 100 µL of a dilution of the test chemical in HBSS, which was prepared from a concentrated

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stock solution in methanol (final concentration of methanol in the system ≤0.5 vol%), and the

Our experimental procedure for the dialysis experiments has been described previously in


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dialysis cells were equilibrated at 37°C. Three to four replicates were prepared. Aliquots of 100

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µL were taken from the buffer side of the BSA samples after two and three days (with no

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significant difference observed between the two time points). The concentration of the test

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chemicals (Cfree) was quantified in all samples as described in the instrumental analysis section,

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CBSA was obtained from the mass balance calculation, and log KBSA/w was calculated using eq 2.

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Test chemicals for which the fraction bound to BSA was less than 20 % were excluded from the

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dataset. At high concentrations BSA possibly causes a colloid osmotic pressure that leads to a

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volume shift in the dialysis cell. However, this was not observed in our experiments. In

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preliminary experiments we also determined the amount of proteins that passes the dialysis

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membrane using the Bradford assay. Only 0.01 % of the total protein were found to diffuse

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through the membrane, which is not expected to influence the determination of KBSA/w in our

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experiments. Control samples without BSA were also prepared and measured in parallel. If the

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recovery for a test chemical from the control was consistently below 95 % or above 105 %, the

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concentration in the BSA samples was corrected according to the recovery. This correction is

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justified, because a consistent deviation from 100 % recovery was found to result from the first

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dilution step of the methanolic stock solution in HBSS, which should cause exactly the same

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error in the dose amounts for BSA and control samples. Test chemicals with recoveries below 90

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and above 120 % were excluded from the dataset. The average recovery from the control

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samples for the remaining test chemicals was 91-117 %. All sorption experiments were

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performed for individual chemicals and not with mixtures. Additionally, in all experiments the

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amount of the bound test chemicals was kept well below the amount of BSA (i.e., ≤ 0.1 mol/mol

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at equilibrium, see also Table S2, SI), to avoid saturation of the binding sites of BSA. More

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details on the dialysis experiments are listed in Table S2, SI (e.g., concentration of stock


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solutions in methanol, initial water phase and measured equilibrium water phase concentrations

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of all test chemicals, concentration of BSA solution used for the dialysis experiments).

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Reversibility of BSA binding

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performed for a subset of the test chemicals. Dialysis cells with BSA were prepared as described

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above, but additionally with 300 mg/L sodium azide to prevent microbial activity. This was

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necessary to extend the experimental time without causing precipitation of BSA. Additional

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experiments showed no significant influence of sodium azide on the partitioning of benzoic acid,

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2-phenoxyacetic acid and 2-methoxy-1-naphthoic acid to BSA (data not shown). After

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equilibrium was established (three days) the buffer-containing half-cell was emptied completely

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and 5 mL of fresh buffer were added. Additional three days were given for re-equilibration and

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the buffer was sampled again. With both equilibrium buffer concentrations after three and six

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days, log KBSA/w was calculated, assuming the mass conservation. For samples taken after six

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days, the removal of test chemical due to clearance of buffer after three days was considered in

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the mass balance calculation. If binding to BSA was fully reversible and no mass loss occurred,

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the determined KBSA/w after three and six days should be the same. If either (or both) of the

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conditions is not fulfilled, KBSA/w calculated for the six days sample should become larger than

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that for the three days sample.

170 171

Dependence of BSA binding on pH value and salt concentration

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this study sorption of 2,6-dichlorobenzoic acid to BSA was measured at pH 6, 7 and 8 at a

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constant concentration of Cl- (150 mM). To control the pH value in the experiments, 10 mM Bis-

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Tris (pKa = 6.5) were added for solutions at pH 6 and 10 mM Tris (pKa = 8.06) for pH 7 and 8

To test the reversibility of binding and the mass conservation, the following experiment was

The partitioning of IOCs to proteins may be influenced by pH value and salt concentration. In


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(for more details see SI). Salt concentration dependence was investigated by measuring the BSA-

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water partition coefficient of 2,6-dichlorobenzoic acid at Cl- concentrations of 10, 50, 300 and

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500 mM (adjusted with NaCl) and at a SO42- concentration of 163 mM (added as Na2SO4; the

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same ionic strength as 500 mM Cl-). These salt solutions contained 10 mM Tris and 10 mM HCl

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and pH was adjusted to 7 with 0.1 N NaOH solution.

180 181

Instrumental analysis

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either a UV detector (UV-970 M, JASCO) or a fluorescence detector (RF-10AXL, Shimadzu).

183

For chemicals that needed a sensitive quantification method, LC-MS/MS measurements were

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performed with two different instruments: an Acquity UPLC system from Waters with a Xevo

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TQ mass spectrometer and an UPLC system from Agilent Technologies (1290 Infinity Series)

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equipped with a 6400 Triple Quad mass spectrometer. Details on the instrumental analysis are

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presented in the Supporting information.

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Results and discussion

189 190

Reversibility tests

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presented in Table S6, SI. No significant difference between the partition coefficients determined

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after three and six days was found (difference between the mean values was

Equilibrium Sorption of Structurally Diverse Organic Ions to Bovine

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