Equilibrium Sorption of Structurally Diverse Organic Ions to Bovine

and sodium azide were purchased from Sigma-Aldrich, Carl Roth or Merck. ...... Gülden , M.; Dierickx , P.; Seibert , H. Validation of a predictio...
0 downloads 0 Views 1MB Size
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