Subscriber access provided by University at Buffalo Libraries
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 31
Environmental Science & Technology
1
Equilibrium Sorption of Structurally Diverse
2
Organic Ions to Bovine Serum Albumin
3
Luise Henneberger,† Kai-Uwe Goss,†,‡ Satoshi Endo,†,§*
4
† Helmholtz Centre for Environmental Research UFZ, Permoserstr. 15, D-04318 Leipzig,
5
Germany
6
‡ University of Halle-Wittenberg, Institute of Chemistry, Kurt-Mothes-Str. 2, D-06120 Halle,
7
Germany
8
§ Osaka City University, Urban Research Plaza & Graduate School of Engineering, Sugimoto 3-
9
3-138, Sumiyoshi-ku, 558-8585 Osaka, Japan
10
ABSTRACT
11
Reliable partitioning data are essential for assessing the bioaccumulation potential and the
12
toxicity of chemicals. In contrast to neutral organic chemicals, the partitioning behavior of
13
ionogenic organic chemicals (IOCs) is still a black box for environmental scientists. Partitioning
14
to serum albumin, the major protein in blood plasma, strongly influences the freely dissolved
15
concentration of many chemicals (including IOCs), which affects their transport and distribution
16
in the body. Because consistent datasets for partitioning of IOCs are rarely available, bovine
17
serum albumin-water partition coefficients (KBSA/w) were measured in this study for 45 anionic
ACS Paragon Plus Environment
1
Environmental Science & Technology
Page 2 of 31
18
and 4 cationic organic chemicals, including various substituted benzoic and naphthoic acids,
19
sulfonates and several pesticides and pharmaceuticals. The results of this study suggest that
20
binding to BSA is substantially influenced by the three dimensional structure of the chemicals
21
and the position of substitutions on the sorbing molecules. For example, we found a difference of
22
> 1.5 log units between isomeric chemicals such as 3,4-dichlorobenzoic acid and 2,6-
23
dichlorobenzoic acid, 1-naphthoic acid and 2-naphthoic acid, and 2,4,6-trimethylbenzoic acid
24
and 2,4,6-trimethylbenzenesulfonate. Conventional modeling approaches (e.g. based on octanol-
25
water partition coefficients) poorly predict log KBSA/w of organic ions (R2 ≤ 0.5), partially
26
because they do not capture the observed steric effects. Hence, alternative modeling strategies
27
will be required for accurate prediction of serum albumin-water partition coefficients of organic
28
ions.
29 30
Introduction
31
Partitioning of ionogenic organic chemicals (IOCs) into biological tissues and their
32
constituents such as various lipids and proteins is of great importance, as it has direct
33
implications for bioconcentration, bioaccumulation, and toxicity of IOCs. However, established
34
models to predict relevant partition coefficients are almost exclusively focused on neutral
35
organic chemicals including the neutral species of ionizable chemicals. For example, a simple
36
regression with log Kow is widely used as a screening model, which sometimes gives a
37
surprisingly
38
Polyparameter linear free energy relationships (PP-LFERs) can give more accurate predictions
39
for broader ranges of chemicals and partition phases.2, 3 These empirical models are convenient
40
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
ACS Paragon Plus Environment
2
Page 3 of 31
Environmental Science & Technology
41
developed specifically for the prediction of biopartitioning of IOCs in general.4 One reason for
42
the absence of models may be the lack of experimental data that are measured under consistent
43
conditions for partitioning of charged chemicals.
44
Bioaccumulation models used for risk assessment often assume that the dominant sorption
45
phase in organisms is the total lipid fraction. For IOCs storage lipids are expected to play a minor
46
role while phospholipid membranes and the protein fraction of an organism are supposed to
47
contribute significantly to the overall partitioning process.5 Serum albumin, the most abundant
48
protein in blood plasma, is likely an important sorption phase for IOCs, because research on
49
pharmaceuticals and endogenous chemicals has long shown that serum albumin binds a broad
50
spectrum of chemicals, especially hydrophobic anions of medium size.6-8 Binding to serum
51
albumin has major impact on transport and distribution of a target chemical in the body, because
52
it increases the sorption capacity of the blood and decreases the free, unbound concentration of
53
the chemical.9,
54
serum albumin binding often determines the bound and the unbound fractions of the test
55
chemical in the medium.11 Furthermore, serum albumin is often considered a generic protein to
56
represent various properties of the bulk protein fraction of organisms, including sorption
57
properties,12 although this assumption may not always be valid and thus needs careful
58
evaluation.13
59 60
10
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
ACS Paragon Plus Environment
3
Environmental Science & Technology
Page 4 of 31
61
where Cfree [mol/Lwater] is the freely dissolved molar concentration of the chemical, Cbound the
62
molar concentration of the chemical bound to BSA, and [BSA] the free molar concentration of
63
BSA in the solution. The extent of binding to BSA can also be expressed as BSA-water partition
64
coefficient (KBSA/w [Lwater/kgBSA]), which is preferably used by environmental scientists and is
65
defined as the ratio of the concentration of the target chemical in BSA (CBSA [mol/kgBSA]) to the
66
freely dissolved concentration of the chemical: ⁄ = ⁄ 2
67 68
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
69
Large amounts of data are available in the literature for binding of pharmaceuticals (including
70
also IOCs) to human serum albumin. However, reliability and comparability of these data are
71
difficult to evaluate. As reviewed by Nilsson et al.,15 data for protein binding are often published
72
without carefully controlling all the factors that can influence the partitioning process, e.g.,
73
temperature; the pH value; concentrations of the test chemical, salt, and the protein; non-specific
74
adsorption to the used equipment; equilibrium disturbances; and leaking of dialysis membrane.
75
Moreover, pharmaceuticals are often multifunctional molecules with complex structure. A
76
collection of data for such diverse chemicals is difficult to interpret in terms of structural
77
influences on the binding constant. Systematically measured data for a series of chemicals with
78
incremental changes in the substructure units (e.g., the number of Cl on the benzene ring) or data
79
for a pair of chemicals that differ by only one structural feature may shed additional light on the
80
binding mechanisms of IOCs to serum albumin.
81
This study aims to elucidate how the molecular structure of organic ions (e.g., different basic
82
structures, substitutions and charged functional groups) influences their partitioning to serum
ACS Paragon Plus Environment
4
Page 5 of 31
Environmental Science & Technology
83
albumin and which mechanisms underlie the sorption process. To this end, we investigated the
84
partitioning of systematically selected organic anions and cations to BSA, starting from simple
85
compounds onwards to more complex structures. Particularly, many varyingly substituted
86
benzenes and naphthalenes with an anionic functional group were included to study the
87
influences of molecule structures on serum albumin binding. All measurement conditions were
88
thoroughly controlled. Additionally, influence of pH value, dependence on the concentration of
89
inorganic ions, and reversibility of BSA binding were determined experimentally. Finally, the
90
data obtained were correlated with various descriptors to gain an insight into the requirements of
91
successful modeling for serum albumin binding.
92
Materials and methods
93 94
Materials
95
No. A3803) and used without further purification. BSA was chosen because of its good
96
availability and comparability to the previous publication for neutral compounds.14 Water
97
purified with a Milli-Q Gradient A10 system from Millipore was used. Methanol (Suprasolv)
98
was obtained from Merck and acetonitrile (gradient grade) from Sigma Aldrich. Unless
99
otherwise noted below, all sorption experiments were performed using Hanks’ balanced salt
100
solution (HBSS, without phenol red and sodium bicarbonate, Sigma Aldrich) buffered with 10
101
mM tris(hydroxymethyl)aminomethane (Tris) from Carl Roth. After addition of sodium
102
bicarbonate and Tris, the pH value of HBSS was adjusted to 7.4 using 1 N HCl or NaOH
103
solution from Merck. The exact salt concentrations of HBSS and a comparison with human
104
plasma are shown in Table S3, SI. Ammonium acetate, formic acid, orthophosphoric acid, bis(2-
105
hydroxyethyl)amino-tris(hydroxymethyl)methane
Bovine serum albumin (essentially fatty acid free) was purchased from Sigma Aldrich (Product
(Bis-Tris),
sodium
bicarbonate,
sodium
ACS Paragon Plus Environment
5
Environmental Science & Technology
Page 6 of 31
106
chloride, sodium sulfate and sodium azide were purchased from Sigma Aldrich, Carl Roth or
107
Merck.
108
The chemicals used for the binding experiments were different organic acids and bases, or salts
109
of them, all of which have only one ionizable functional group and are more than 99 % ionized at
110
pH 7.4. To investigate the influence of different substitutions on the binding constant, a series of
111
benzoic acids, naphthoic acids and sulfonates were chosen for the dataset. More complex
112
compounds (i.e., pesticides and pharmaceuticals) were included, because of their environmental
113
relevance. All test chemicals are listed in Table 1 and had purity of at least 98%. More details on
114
the test chemicals (e.g., CAS number, provider, analytical method used for quantification,
115
chemical structure, pKa values and recovery from control experiments) can be found in the
116
Supporting Information Table S1. In this work, weak acids and bases are always denoted with
117
the chemical names of their neutral species (e.g., 4-chlorobenzoic acid) because they are more
118
common than the names for the ionic species (e.g., 4-chlorobenzoate), despite the fact that weak
119
acids and bases in our test solutions are always predominantly in their ionic form and that KBSA/w
120
reported in this work is thus for the ionic species.
121 122
Dialysis experiments
123
detail.16 In short: a custom-made dialysis cell that consists of two half-cells (total volume approx.
124
10 mL) and dialysis membranes from Spectrum Laboratories (type Spectra/Por 4 RC with a
125
molecular weight cutoff of 12-14 kD) was used. One half-cell of the dialysis unit received 5 mL
126
HBSS buffer and the other half-cell 4.9 mL BSA solution (1-50 g/L). All samples were spiked
127
with 100 µL of a dilution of the test chemical in HBSS, which was prepared from a concentrated
128
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
ACS Paragon Plus Environment
6
Page 7 of 31
Environmental Science & Technology
129
dialysis cells were equilibrated at 37°C. Three to four replicates were prepared. Aliquots of 100
130
µL were taken from the buffer side of the BSA samples after two and three days (with no
131
significant difference observed between the two time points). The concentration of the test
132
chemicals (Cfree) was quantified in all samples as described in the instrumental analysis section,
133
CBSA was obtained from the mass balance calculation, and log KBSA/w was calculated using eq 2.
134
Test chemicals for which the fraction bound to BSA was less than 20 % were excluded from the
135
dataset. At high concentrations BSA possibly causes a colloid osmotic pressure that leads to a
136
volume shift in the dialysis cell. However, this was not observed in our experiments. In
137
preliminary experiments we also determined the amount of proteins that passes the dialysis
138
membrane using the Bradford assay. Only 0.01 % of the total protein were found to diffuse
139
through the membrane, which is not expected to influence the determination of KBSA/w in our
140
experiments. Control samples without BSA were also prepared and measured in parallel. If the
141
recovery for a test chemical from the control was consistently below 95 % or above 105 %, the
142
concentration in the BSA samples was corrected according to the recovery. This correction is
143
justified, because a consistent deviation from 100 % recovery was found to result from the first
144
dilution step of the methanolic stock solution in HBSS, which should cause exactly the same
145
error in the dose amounts for BSA and control samples. Test chemicals with recoveries below 90
146
and above 120 % were excluded from the dataset. The average recovery from the control
147
samples for the remaining test chemicals was 91-117 %. All sorption experiments were
148
performed for individual chemicals and not with mixtures. Additionally, in all experiments the
149
amount of the bound test chemicals was kept well below the amount of BSA (i.e., ≤ 0.1 mol/mol
150
at equilibrium, see also Table S2, SI), to avoid saturation of the binding sites of BSA. More
151
details on the dialysis experiments are listed in Table S2, SI (e.g., concentration of stock
ACS Paragon Plus Environment
7
Environmental Science & Technology
Page 8 of 31
152
solutions in methanol, initial water phase and measured equilibrium water phase concentrations
153
of all test chemicals, concentration of BSA solution used for the dialysis experiments).
154 155
Reversibility of BSA binding
156
performed for a subset of the test chemicals. Dialysis cells with BSA were prepared as described
157
above, but additionally with 300 mg/L sodium azide to prevent microbial activity. This was
158
necessary to extend the experimental time without causing precipitation of BSA. Additional
159
experiments showed no significant influence of sodium azide on the partitioning of benzoic acid,
160
2-phenoxyacetic acid and 2-methoxy-1-naphthoic acid to BSA (data not shown). After
161
equilibrium was established (three days) the buffer-containing half-cell was emptied completely
162
and 5 mL of fresh buffer were added. Additional three days were given for re-equilibration and
163
the buffer was sampled again. With both equilibrium buffer concentrations after three and six
164
days, log KBSA/w was calculated, assuming the mass conservation. For samples taken after six
165
days, the removal of test chemical due to clearance of buffer after three days was considered in
166
the mass balance calculation. If binding to BSA was fully reversible and no mass loss occurred,
167
the determined KBSA/w after three and six days should be the same. If either (or both) of the
168
conditions is not fulfilled, KBSA/w calculated for the six days sample should become larger than
169
that for the three days sample.
170 171
Dependence of BSA binding on pH value and salt concentration
172
this study sorption of 2,6-dichlorobenzoic acid to BSA was measured at pH 6, 7 and 8 at a
173
constant concentration of Cl- (150 mM). To control the pH value in the experiments, 10 mM Bis-
174
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
ACS Paragon Plus Environment
8
Page 9 of 31
Environmental Science & Technology
175
(for more details see SI). Salt concentration dependence was investigated by measuring the BSA-
176
water partition coefficient of 2,6-dichlorobenzoic acid at Cl- concentrations of 10, 50, 300 and
177
500 mM (adjusted with NaCl) and at a SO42- concentration of 163 mM (added as Na2SO4; the
178
same ionic strength as 500 mM Cl-). These salt solutions contained 10 mM Tris and 10 mM HCl
179
and pH was adjusted to 7 with 0.1 N NaOH solution.
180 181
Instrumental analysis
182
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
184
performed with two different instruments: an Acquity UPLC system from Waters with a Xevo
185
TQ mass spectrometer and an UPLC system from Agilent Technologies (1290 Infinity Series)
186
equipped with a 6400 Triple Quad mass spectrometer. Details on the instrumental analysis are
187
presented in the Supporting information.
188
Results and discussion
189 190
Reversibility tests
191
presented in Table S6, SI. No significant difference between the partition coefficients determined
192
after three and six days was found (difference between the mean values was