Kinetics and Mechanisms of Protein Adsorption and Conformational

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Kinetics and Mechanisms of Protein Adsorption and Conformational Change on Hematite Particles Feng Liu, Xiaoxu Li, Anxu Sheng, Jianying Shang, Zimeng Wang, and Juan Liu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b02651 • Publication Date (Web): 02 Aug 2019 Downloaded from pubs.acs.org on August 2, 2019

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Environmental Science & Technology

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Kinetics and Mechanisms of Protein Adsorption and Conformational

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Change on Hematite Particles

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Feng Liua, Xiaoxu Lia, Anxu Shenga, Jianying Shangb, Zimeng Wangc, Juan Liua,d*

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aThe

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Sciences and Engineering, Peking University, Beijing 100871, China

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bDepartment

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100193, China

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cDepartment

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200433, China

Key Laboratory of Water and Sediment Sciences, College of Environmental

of Soil and Water Sciences, China Agricultural University, Beijing

of Environmental Science and Engineering, Fudan University, Shanghai

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dBeijing

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Beijing 100871, China

Key Laboratory of Mineral Environmental Function, Peking University,

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To be submitted to Environmental Science & Technology

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* Corresponding author:

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Address: College of Environmental Sciences and Engineering, Peking University,

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Beijing 100871, China

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Email: [email protected]

ACS Paragon Plus Environment


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ABSTRACT

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Adsorption kinetics and conformational changes of a model protein, bovine serum

21

albumin (BSA, 0.1, 0.5, or 1.0 g/L), on the surface of hematite (α-Fe2O3) particles in

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39 ± 9, 68 ± 9, and 103 ± 8 nm, respectively, were measured using attenuated total

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reflectance FTIR (ATR-FTIR) spectroscopy. As particle size increases, the amount of

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adsorbed BSA decreases, but the loss in the helical structure of adsorbed BSA increases

25

due to the stronger interaction forces between adsorbed BSA and the larger particles.

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On 39 or 68 nm hematite particles, refolding of adsorbed BSA can be induced by

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protein-protein interactions, when protein surface coverage exceeds certain critical

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values. Two-dimensional correlation spectroscopy (2D-COS) analysis of time-

29

dependent ATR-FTIR spectra indicate that the increase in the amount of adsorbed BSA

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occurs prior to the loss in the BSA helical structure in the initial stage of adsorption

31

processes, whereas an opposite sequence of the changes to BSA conformation and

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surface coverage is observed during the subsequent refolding processes. Desorption

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experiments show that replacing the protein solution to water can quench the refolding,

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but not the unfolding, of adsorbed BSA. A kinetic model was proposed to quantitatively

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describe the interplay of adsorption kinetics and conformational change, as well as the

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effects of particle size and initial protein concentration on the rate constants of

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elementary steps in protein adsorption onto mineral surface.


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INTRODUCTION

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As a major component of dissolved organic matters (DOMs) in aqueous

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environments, proteins tend to adsorb on the surface of colloids and nanoparticles (NPs),

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forming a protein coating known as “protein corona”.1 The presence of the protein

42

corona can dramatically influence aggregation, transport, reactivity, transformation,

43

and bioavailability of colloids and NPs in both natural and physiological

44

environments.2-8 Also, proteins are likely to undergo various changes in their

45

conformation, stability, and enzymatic activity upon adsorption onto particle surface.9-

46

12

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show different catalytic activities from free enzymes.13-15 Extracellular respiration of

48

metal (hydr)oxide minerals by many microorganisms mainly depends on the

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interactions between microbial outer membrane cytochromes and mineral particles.16-

50

18

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affect electron transfer at the mineral-microbe interface.19, 20 In addition, understanding

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the protein-NP interaction is also critical to develop NP-based biomedical and

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environmental applications, such as drug delivery, bioremediation techniques, and

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biosensing.6, 15, 21-23

For example, extracellular enzymes immobilized on the surface of soil minerals will

Conformational change of the cytochromes upon adsorption on mineral surface can

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Physico-chemical properties of particles, such as particle size, shape, coatings, and

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surface charge, may significantly influence the conformation, abundance, and activity

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of proteins adsorbed on particles.21, 24, 25 For example, Lacerda et al. reported that the

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common blood proteins (e.g. albumin, fibrinogen, 𝛾-globulin, histone, and insulin)

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undergo the greater extent of conformational change after adsorption onto a larger gold



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NPs, as the particle size increases from 5 to 100 nm.10 Although this trend was also

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reported in many other studies using different combinations of proteins and NPs,11, 21,

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26

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conformational change of proteins adsorbed on the smaller particles have been reported

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as well.27,

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conformation of adsorbed proteins in a complex manner.13, 26, 30, 31 More studies are

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needed, in order to elucidate how particle size affects protein adsorption. In addition,

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other physicochemical properties of particles, such as hydrophilicity and ζ-potential,

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may also change with the decrease of particle size, which makes it even more

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complicated to reveal the relationship between particle size and protein adsorption.

an irrelevance between protein conformation and particle size or the greater

28 29

The contradicting results suggest that particle size affects the

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Different from small and rigid molecules or ions, proteins may undergo reversible

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or irreversible conformational change upon adsorption onto particle surface,21 which is

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likely to significantly affect the extent and rate of protein adsorption. A variety of

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spectroscopic techniques, like circular dichroism (CD), fluorescence, UV/vis

74

absorption, and fourier transform infrared spectroscopy (FTIR), have been used to

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explore the conformational changes of proteins adsorbed on particle surface.10, 11, 32-34

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However, most studies compared the secondary structure of proteins before and after

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adsorption equilibrium.11,

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dynamics of adsorbed proteins during adsorption processes has been reported.35-37

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Attenuated total reflectance FTIR (ATR-FTIR) spectroscopy is one of few techniques

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that can provide a real-time conformational change of proteins upon binding to NPs.

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For example, a recent study used the time-dependent ATR-FTIR spectra to show BSA

32, 33

Very limited information about the conformational


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unfolding upon adsorption onto montmorillonite surface under environmentally

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relevant conditions.36 However, the effect of particle size on the dynamic processes of

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protein conformational change during protein adsorption on particles is still unknown.

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This study presents adsorption behavior and conformational change of a model

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globular protein, bovine serum albumin (BSA), upon adsorption onto hematite (α-

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Fe2O3) particles with mean primary particle sizes of 39, 68, and 103 nm, respectively.

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Hematite is widespread in natural environments, as either nano- or macro-sized

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particles. Using this representative protein-mineral pair, we studied the change of BSA

90

conformation on the surface of hematite particles in three different particle sizes using

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in-situ ATR-FTIR spectroscopy and two-dimensional correlation spectroscopy (2D-

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COS) analysis. Circular dichroism (CD) spectroscopy and atomic force microscopy

93

(AFM) were used to study the BSA conformation after adsorption equilibrium and the

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direct BSA-particle interaction force, respectively. In addition, a kinetic model of BSA

95

adsorption on hematite particles was proposed to describe the correlation between the

96

conformational change of adsorbed proteins and the adsorption kinetics/extents of

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proteins. The present study provides mechanistic insights into the complex effects of

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protein concentration, primary particle size, and contact time on adsorption behavior of

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globular proteins onto the surface of iron oxide minerals.

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MATERIALS AND METHODS

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Synthesis and Characterization of Hematite Particles. Hematite particles in

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three different sizes were synthesized by forced hydrolysis of ferric nitrate according to



104

the method reported by Schwertmann and Cornell.38 Primary particle size and

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crystalline phase of the synthetic particles were determined by transmission electron

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microscopy (TEM) and powder X-ray diffraction (XRD), respectively. More details

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about hematite synthesis and characterization are described in Section S1.

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ATR-FTIR Measurements and Data Analysis. ATR-FTIR measurements were

109

performed to study protein adsorption on particle surface using a Vertex 80v FTIR

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spectrometer (Bruker Corp., Billerica, MA) that was equipped with a horizontal

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attenuated total reflectance (HATR) flow-through cell (PIKE Tech) and a 45° ZnSe

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ATR crystal. The films of hematite particles in different particle sizes were prepared by

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evenly depositing the suspensions of hematite particles on ZnSe surface. Reactive

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surface area of the films was assessed using phosphate as probe ion, because of a high

115

affinity of this anion to hematite surface,39 via both batch adsorption experiments and

116

in situ ATR-FTIR spectroscopy measurements. More details about the film preparation,

117

batch adsorption experiments, and ATR-FTIR measurements are described in Section

118

S2.

119

Adsorption experiment was initiated by flowing a BSA solution with a desired

120

initial BSA concentration (i.e. [BSA]Int.) at 0.1, 0.5, or 1.0 g·L-1 over the hematite film

121

at a flow rate of 0.5 mL·min-1. Control experiments confirmed that the deposition of

122

BSA proteins from the solution to the ZnSe surface was negligible at this flow rate. In

123

all adsorption experiments, FTIR spectra were collected every 3 min from 2000 to 1200

124

cm-1 at a resolution of 4 cm-1, until no changes in the spectra were observed over time.

125

At least three replicates were measured under each experimental condition. Kinetic data


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of BSA adsorption on hematite particles were fitted according to a model proposed in

127

this study that are described in detail below using a Levenberg−Marquardt algorithm in

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MathCAD 15.0 software (PTC Inc.). To study the desorption capability of adsorbed

129

BSA from hematite surface, desorption experiments were conducted on the film of 39

130

nm hematite NPs by replacing the flow of 0.1 g/L BSA solution with Milli-Q water at

131

a flow rate of 0.5 mL·min-1 after 30, 60, or 120 min adsorption, respectively.

132

All ATR-FTIR spectra of adsorbed BSA on hematite particles are generally

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consisted of amide I, II, and III bands at around 1700-1600 cm-1, 1600-1500 cm-1, and

134

1350-1200 cm-1, respectively (Figure S1).40 The adsorption kinetics of BSA onto the

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hematite films were measured from the area of the amine II band over time.40 The

136

conformational change of BSA upon adsorption onto hematite surface was analyzed

137

using the time-dependent changes of the amide I band.40 As shown in Figure S2, the

138

amide I band can be deconvoluted into four peaks that are assigned to α-helices (1650-

139

1655 cm-1), β-sheets (1620-1640 cm-1), β-turns (1660-1690 cm-1), and random coils

140

(1644-1648 cm-1) using the OPUS 7.2 software.12, 34, 40, 41 The relative proportions of

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these secondary structures as a function of time were used to study conformational

142

changes of adsorbed BSA during adsorption processes.

143

2D-COS Analysis of Conformational Change. To study sequence of the changes

144

to secondary structure features and adsorption amounts of BSA on hematite surface,

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2D-COS analyses of the ATR-FTIR spectra were performed using the 2DShige

146

software (Shigeaki Morita, Japan). The sequential order of the intensity change of two

147

variations at the wavenumbers 𝜈1 and 𝜈2, respectively, was determined from the sign



148

of the synchronous correlation peaks Φ(𝜈1,𝜈2)(indicating the overall similarity or

149

coincidental nature between the two signal variations) and that of the asynchronous

150

correlation peaks Ψ(𝜈1,𝜈2)(representing the out-of-phase or sequentially varying

151

nature of the signals). If Φ(𝜈1,𝜈2) and Ψ(𝜈1,𝜈2) have same signs, the change in the

152

intensity at 𝜈1 occurs prior to that at 𝜈2; otherwise, the change in the intensity at 𝜈1

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occurs after that at 𝜈2. If Ψ(𝜈1,𝜈2) is zero, the changes in the intensity at 𝜈1 and that

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at 𝜈2 happen simultaneously. The detailed procedures for the 2D-COS analysis have

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been described in previous studies.36, 42

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Circular Dichroism (CD) Spectroscopy. The secondary structures of BSA

157

before and after adsorption equilibrium were compared using far-UV CD spectroscopy.

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A desired volume of hematite suspensions, after 5 min sonication, was added into 30

159

mL of 20 or 50 mg·L-1 BSA solution, resulting in a hematite loading of 79.8 mg·L-1.

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Because high particle loading may interference with CD signals, the lower particle

161

loading and BSA concentrations were used in the CD measurements than in the ATR-

162

FTIR experiments. Then, the suspension was incubated in a closed serum bottle, which

163

was continuously shaken at ~150 rpm and 25 ℃ for 2 h. After that, the CD spectra of

164

the suspensions were recorded using a JASCO J-1500-150 spectropolarimeter under a

165

constant stream of nitrogen gas at room temperature. In the observed wavelength range,

166

no CD signals are from hematite particles in the suspensions, so the interference with

167

the CD spectra of adsorbed BSA from hematite particles are negligible.

168

Atomic Force Microscope Measurements. The interaction forces between BSA

169

and hematite particles of different particle sizes were measured using AFM (Asylum


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Research/MFP-3D). BSA was immobilized onto a gold-coated AFM tip with a radius

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of 40 nm (Asylum Research), according to the method reported by Wang et al (more

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details refer to Section S3).43 The hematite films on the ZnSe ATR crystal were

173

prepared using the mentioned method (Section S2). Adhesion force was calculated from

174

the measured retraction force curves, based on the maximum deflection distance of the

175

BSA-coated cantilever during retraction.44, 45 All force measurements were conducted

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in the contact mode in a fluid cell filled with Milli-Q water at room temperature. The

177

spring constant of the BSA-modified tips was fixed using the thermal tuning method.43

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To obtain average interaction forces, at least 100 force-distance curves were collected

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from randomly selected locations on each sample.

180 181

RESULTS AND DISCUSSION

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Characteristics of Synthetic Hematite Particles. The XRD patterns (Figure S3)

183

indicate that only hematite phase is present in all synthetic particles. The representative

184

TEM images and the corresponding size distributions of the particles indicate that the

185

primary particle sizes of the three samples are 39 ± 9 nm (HM39), 68

186

(HM68), and 103

187

exhibit a nearly isotropic shape with similar surface roughness. HM39 shows a

188

rhombohedral shape, which is consistent with previous studies.38,

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potentials of HM39, HM68, and HM103 in Milli-Q water at pH 5.7 are 32.6 mV, 46.6

190

mV, and 54.3 mV, respectively. The inverse correlation between primary particle size

191

and the measured zeta potential is consistent with previously reported results on

± 9 nm

± 8 nm (HM103), respectively (Figure S4). HM68 and HM103


46, 47

The zeta


192

hematite particles in aqueous suspensions at pH 5.7 and low salt concentrations,3, 48

193

which could be related to the change of surface charge behavior or surface reactivity of

194

hematite with the decrease of particle size.49

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Kinetics and Extents of BSA Adsorption on Hematite Particles. Figure S1

196

shows the representative set of ATR-FTIR spectra collected on the film of HM39 under

197

the flow of the 0.1 g/L BSA solution at a flow rate of 0.5 mL·min-1. The intensities of

198

all amide bands progressively increase within the 300 min observation period. Control

199

experiments conducted on the ZnSe crystal without hematite particles under the same

200

BSA flow show that the area of the amide bands in the FTIR spectra is constant with

201

time (data not shown). Thus, the precipitation of BSA from the protein flow onto the

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ZnSe surface is negligible at this flow rate (0.5 mL·min-1). The increasing intensities of

203

the amide bands with time (Figure S1) can be attributed to the increasing amounts of

204

BSA adsorbed on the hematite film over time.

205

Compared to the amide I band, the amide II band is less susceptible to

206

conformational change or aggregation of proteins on particle surface, so the amide II

207

band area was monitored over time to study the amounts of BSA adsorbed on hematite

208

surface under different experimental conditions.12, 36, 40 Figure 1 shows the amide II

209

band area vs. time in the adsorption experiments of BSA (the initial BSA concentration

210

[BSA]Int.=0.1, 0.5 or 1.0 g/L) on HM39, HM68, and HM103, respectively. All kinetic

211

profiles show a similar trend that the amount of adsorbed BSA gradually increases with

212

time until reaching a plateau. However, the adsorption rates and extents are obviously

213

different at varying particle sizes or initial BSA concentrations. When the initial BSA


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concentration is same, the extent of BSA adsorption at the end of experiments

215

significantly decreases with the increase of particle size (Figure S5). Because all

216

hematite films were evenly deposited on the ZnSe crystal with the same size (width 1

217

cm × length 7 cm), the calculated surface area of all hematite films on the crystal is

218

similar, independent on the primary particle size (the calculation of the geometrical

219

surface area of the films refers to SI Section S4 and Figure S6). Also, as shown in Figure

220

S7 and SI Section S4, the results of batch experiments and in situ ATR-FTIR

221

spectroscopy measurements of phosphate adsorption on the films of different hematite

222

particles confirm that the reactive surface area of all films are similar. Therefore, the

223

different extents of BSA adsorption on these films are not due to the different specific

224

surface area of these hematite particles.

225

As the initial BSA concentration increases from 0.1 to 1.0 g/L, the rates and extents

226

of BSA adsorption on HM39 and HM68 significantly increase with the increasing

227

[BSA]Int., but the values are unchanged on HM103 (Figure 1a-1c). These results suggest

228

that not only the primary particle size but also the initial protein concentration can

229

influence the adsorption kinetics and extents of BSA on hematite particles. As

230

mentioned above, BSA may undergo conformational changes upon adsorption onto

231

hematite surface, resulting in the change of surface area occupied by each BSA

232

molecule and the decrease of available surface sites for the adsorption of incoming BSA.

233

The commonly used adsorption kinetic models are usually developed for rigid, small

234

molecules or ions, so they are not applicable for fitting the adsorption kinetic data

235

(Figure 1a-1c) of BSA proteins that undergo conformational changes to different



236

extents. In order to derive kinetic parameters from the measured profiles, an appropriate

237

kinetic model considering both the time-dependent adsorption amount and the

238

conformational change of adsorbed BSA needs to be developed. Thus, the adsorption

239

kinetics of BSA on hematite particles will be discussed further below, together with the

240

changes of BSA secondary structure features over time.

241

Conformational changes of adsorbed BSA on hematite particles. The relative

242

proportions of secondary structure components, including α-helices, random coils, β-

243

sheets, and β-turns, in free BSA and adsorbed BSA on different hematite particles after

244

210 min adsorption are compared in Table S1. Relative to free BSA in the bulk solution,

245

adsorbed BSA generally possesses the lower α-helix content but the higher proportion

246

of random coils. When [BSA]Int. is same, a greater loss in the helical structure and a

247

more significant increase in the content of random coils for adsorbed BSA are observed

248

on the larger hematite particles. On the other hand, the change of [BSA]Int. affects the

249

secondary structure of adsorbed BSA differently for the particles in different particle

250

sizes. As [BSA]Int. increases from 0.1 to 1.0 g·L-1, the α-helix content of BSA adsorbed

251

on HM68 increases from 34.1% to 52.1%, but the values of BSA adsorbed on HM39

252

and HM103 are almost unchanged. Thus, the adsorption processes of BSA onto

253

hematite particles may result in a perturbation of its secondary structure, mainly

254

including a loss of the helical structure and an increase in random coils, to different

255

extents, but the degree of BSA conformational deformation varies with particle size and

256

initial BSA concentration.

257

The relative proportions of α-helices, random coils, β-sheets, and β-turns of BSA


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([BSA]Int. = 0.1-1.0 g/L) adsorbed on HM39, HM68, and HM103, respectively, as a

259

function of time are shown in Figure S8 – S10. In all cases, the proportions of α-helices

260

and random coils evidently change with time, but the contents of β-sheet and β-turn

261

show no substantial changes over time. Moreover, the proportions of α-helices and

262

random coils change synchronously but in the opposite directions. Thus, α-helix% of

263

adsorbed BSA over time was used to study the conformational change of BSA during

264

adsorption processes.

265

Figure 1d shows that, at a low BSA concentration ([BSA]Int. = 0.1 g/L), α-helix%

266

of BSA adsorbed on HM39 decreases from ~58% to ~35% within 60 min and then

267

slowly increases back to ~63% after ~270 min. However, α-helix% of adsorbed BSA

268

decreases from ~55% to ~33% on HM68 and to ~35% on HM103 within about 30 min.

269

No refolding of adsorbed BSA occurs in these two cases. When [BSA]Int. is increased

270

to 0.5 g/L, a slight decrease in α-helix% (~5%) of BSA adsorbed on HM39 is observed

271

within ~30 min and then α-helix% slowly increases back to ~63%. On the surface of

272

HM68, α-helix% decreases from ~55% to ~35% within ~40 min and then reverts to ~45%

273

after ~75 min. The similar unfolding-refolding processes are observed on HM68 in the

274

experiments with the high [BSA]Int. of 1.0 g/L (Figure 1e), but the minimum α-helix

275

content in this case is ~40%, indicating the less extent of BSA unfolding. However, α-

276

helix% of BSA adsorbed on HM39 in the 1.0 g/L BSA solution fluctuates around 63%

277

throughout the 270 min experiment. Moreover, the time-dependent conformational

278

change of BSA adsorbed on HM103 is nearly same in all solutions with different

279

[BSA]Int. values. No refolding of adsorbed BSA occurs on HM103 under the conditions



280

used in this study. These results suggest that the conformational change of BSA

281

adsorbed on hematite particles is dynamic and involves multiple states. The extent of

282

BSA conformational change depends not only on particle size, but also on [BSA]Int. and

283

interaction time.

284

It is worth mentioning that multilayer adsorption of BSA molecules on hematite

285

films is not feasible under the conditions of the present study. The assembly of protein

286

molecules usually results in an increased level of nonnative intermolecular β-sheet

287

structures,50 which was not observed in all time-dependent FTIR spectra (Figure S8-

288

S10). Thus, the rebounce of α-helix% cannot be attributed to multilayer adsorption of

289

BSA molecules or the attachment of more native BSA molecules to pre-adsorbed BSA.

290

Although driving forces and mechanisms for the conformational changes of

291

proteins adsorbed on particle surface are still not well understood, it is widely

292

recognized that both protein-surface interactions and the interactions between

293

neighboring proteins on surface can influence protein adsorption behavior and

294

conformational change.35 The AFM force measurements (Figure S11) show that the

295

average adhesion forces between BSA and hematite particles in Milli-Q water are in

296

the order: HM103 > HM68 > HM39. The protein-particle interaction force increases

297

with the increase of particle size, which agrees well with the previously reported

298

results.26, 32, 51, 52 The lower surface curvature of large particles can provide more contact

299

sites for the adsorption of globular proteins, such as BSA, resulting in the greater

300

interaction forces. The stronger protein-particle interaction forces on the surface of the

301

larger particles may lead to the faster conformational change of adsorbed BSA. Because


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the unfolded BSA has a more open secondary structure, the faster unfolding of adsorbed

303

BSA may lead to more surface sites occupied by the adsorbed BSA and inhibit the

304

adsorption of incoming proteins. For example, in the 0.1 g/L BSA solution, α-helix%

305

of adsorbed BSA decreases to the minimum value (~35%) at ~30 min on HM103, but

306

it takes ~60 min on HM39 to reach the minimum α-helix content (~35%). However,

307

within the first 30 min, the amount of adsorbed BSA is much more on HM39 than on

308

HM103 (Figure S5a). In addition, the stronger protein-particle interaction forces can

309

also counteract the protein-protein interaction forces among neighboring proteins on

310

particle surface and inhibit the refolding of adsorbed BSA. That could be the reason

311

why the refolding of adsorbed BSA occurs on HM39, but not on HM68 or HM103, in

312

the presence of 1.0 g/L BSA.

313

In this study, all adsorption experiments were conducted at pH = 5.7 that is the pH

314

value of Milli-Q water without buffers and above the isoelectric point (pH = ~4.76) of

315

BSA.53 Neighboring proteins adsorbed on particle surface may have the domains

316

bearing a net charge of equal sign, resulting in repulsive protein-protein interactions.

317

Once certain surface coverage of BSA is exceeded, the lateral interactions between

318

neighboring proteins may exert substantial influence on the secondary structure of

319

adsorbed proteins. On the surface of HM39, the refolding of adsorbed BSA occurs from

320

~60 min in the 0.1 g/L BSA solution and from ~30 min in the 0.5 g/L solution. At these

321

turning points, the corresponding amide II band area is 0.155 in 0.1 g/L BSA and 0.149

322

in 0.5 g/L BSA solution, respectively. The similar refolding process of BSA adsorbed

323

on HM68 occurs from ~40 min, when the amide II band area decreases to 0.133 in the



324

0.5 g/L BSA solution or to 0.156 in the 1.0 g/L BSA solution. Although the refolding

325

starts from different time points in these experiments, the amount of adsorbed BSA (or

326

the amide II band area) at the turning points is similar in all cases. As mentioned above,

327

the films of hematite particles in different particle sizes have the similar surface area

328

for BSA adsorption due to the fixed size of the ZnSe crystal. The similar amount of

329

adsorbed BSA at the turning points might correspond to the surface cover for forming

330

a monolayer of unfolded BSA on the films. The interactions between neighboring

331

proteins on particle surface or between adsorbed and incoming proteins can drive the

332

refolding of adsorbed proteins. However, the protein-particle interactions tend to cause

333

conformational change of adsorbed BSA and counteract the protein-particle

334

interactions. Thus, competition between these two kinds of interaction forces may result

335

in the different conformational changes under these conditions.

336

The conformational changes of adsorbed BSA after adsorption equilibrium were

337

studied using far-UV CD spectroscopy (Figure S12). A relatively lower mineral loading

338

was used in the CD measurements to minimize the interference of suspended particles

339

with CD signals. Correspondingly, the lower initial BSA concentration (20 or 50 mg/L)

340

was used in the CD measurements than in the ATR-FTIR experiments. However, in

341

both of the experiments, the initial BSA concentration was much more than the

342

calculated protein concentration needed to form a monolayer on the surface of all

343

particles. After adsorption equilibrium (~ 2h), the α-helix content in adsorbed BSA was

344

calculated from the ellipticity at 222 nm in the CD spectra (the details for CD data

345

analysis refer to SI Section S5). In the 20 mg/L BSA solution, the loss of α-helix content


Page 16 of 35

Page 17 of 35


346

is 31.8 % on HM68 and 55.5% on HM103, respectively, relative to the value in free

347

BSA (Table S2). When [BSA]Int. increases from 20 to 50 mg/L, the loss of α-helix

348

content obviously decreases on HM68 but is nearly unchanged on HM103. The loss of

349

α-helix content in BSA adsorbed on HM39 is negligible in both 20 and 50 mg/L BSA

350

solution, which is consistent with the result of the FTIR spectra in the solution of the

351

high [BSA]Int. (0.5 and 1.0 g/L) on the film of HM39 (Figure 1d). Although the CD

352

spectra (Figure S12) generally agree well with the trend shown in the FTIR spectra

353

(Figure 1 and Table S1), the CD spectra only show the secondary structure of adsorbed

354

BSA after adsorption equilibrium. No information about the transient states of adsorbed

355

BSA and the sequence of BSA conformational changes over time can be provided by

356

the CD spectra.

357

Sequence of changes to conformation and surface coverage of adsorbed BSA.

358

The key advantage of in-situ ATR-FTIR is its capability to characterize both time-

359

dependent conformational changes and kinetics of protein adsorption. To study the

360

sequential order of changes to secondary structure and amount of adsorbed BSA during

361

the adsorption processes, 2D-COS analysis of the time-dependent FTIR spectra of

362

adsorbed BSA on HM39 in the solution with [BSA]Int. = 0.1 g/L was conducted (Figure

363

2 and Table S3). The plot of α-helix% vs. time (Figure 1d) suggests that, in this case,

364

the adsorption processes can be divided into two stages: (1) Stage I (0-60 min): α-helix%

365

of adsorbed BSA continuously decreases with time; (2) Stage II (60-300 min): α-helix%

366

gradually increases back to the initial value. Accordingly, the 2D-COS analysis was

367

conducted using the FTIR spectra collected in Stage I and Stage II, respectively. The



368

IR vibration frequencies and assignment of BSA secondary structure elements that are

369

shown in the cross peaks of 2D-COS results are listed in Table S3.

370

In the Stage I, a positive cross peak at (1546, 1656) is shown in the both

371

synchronous and asynchronous correlation contour map (Figure 2a and 2b). The peak

372

around 1546 cm-1 corresponds to the amide II band, which is related to the amount of

373

adsorbed BSA. The intensity of the feature at 1656 cm-1 is correlated to the content of

374

α-helices in adsorbed BSA. The same sign of the cross peak at (1546, 1656) on both

375

the synchronous and asynchronous plots suggests that the increase in the amount of

376

adsorbed BSA occurs before the changes of BSA secondary structure in the Stage I of

377

the adsorption processes. The other asynchronous cross peaks at (1546, 1679), (1656,

378

1680), and (1637, 1656) are not resolved in the synchronous plot. This phenomenon is

379

quite common in previously reported 2D-COS results, which can be attributed to peak

380

positon shift or bandwidth change in adsorption spectra during reactions.54 The

381

appearance of these asynchronous cross peaks probably suggests that the relative

382

proportions of these secondary structural components change at different rates during

383

adsorption processes. However, the cross peaks that are not observed in both

384

synchronous and asynchronous plots cannot give the sequential order of the changes to

385

these features, so they will not be discussed further in the present study.

386

In the Stage II, the positive cross peak at (1546, 1656) is also observed in the

387

synchronous correlation contour map (Figure 2c), but the cross peak at (1546, 1656) in

388

the asynchronous plot is negative (Figure 2d). The opposite signs of the cross peaks on

389

the synchronous and asynchronous plots indicate that, in this stage, conformational


Page 18 of 35

Page 19 of 35


390

changes of adsorbed BSA predominantly occur prior to the further adsorption of BSA

391

onto hematite surface. In the stage I, the surface coverage of proteins is relatively low,

392

so there is enough surface area for the adsorption of incoming proteins, as well as for

393

the conformational change of adsorbed BSA. In the stage II, protein coverage is much

394

higher, and most surface sites have been occupied by unfolded BSA. If the protein-

395

protein interactions between neighboring proteins on surface or between incoming and

396

adsorbed proteins can drive the refolding of adsorbed BSA, more surface sites become

397

available for the subsequent BSA adsorption. Otherwise, the adsorption of incoming

398

proteins can be inhibited due to the limited number of available surface sites. The 2D-

399

COS results confirm the different sequences of the changes to secondary structure and

400

the adsorption amount of BSA during the two adsorption stages.

401

Desorption study. It is widely recognized that protein adsorption on particle

402

surface can be reversible or irreversible. To investigate the capability of adsorbed BSA

403

to detach from hematite surface, desorption experiments on HM39 were conducted by

404

replacing the flow of the 0.1 g/L BSA solution with Milli-Q water at the same flow rate

405

after adsorption for 30 min (Stage I), 60 min (at the minimum α-helix%), and 120 min

406

(Stage II), respectively (Figure 3). HM39 was selected for the desorption experiments,

407

because it has the weaker adhesion force with BSA and accordingly the higher

408

possibility to detach from surface, compared to HM68 and HM103 (Figure S11). Figure

409

3a shows that the amount of adsorbed BSA is constant after changing the BSA solution

410

to Milli-Q water at any of the time points, indicating that no measurable desorption of

411

BSA occurs from HM39 surface in all cases. However, replacing the BSA solution with



412

Milli-Q water at different time points significantly influences the conformational

413

changes of adsorbed BSA over time. If the BSA solution is changed to water at 30 min,

414

α-helix% of adsorbed BSA continues to decrease at the same rate, until the minimum

415

value of ~35% is reached. Then, the α-helix% value fluctuates around this value over

416

time, and no recovery of secondary structure is observed. If the BSA solution is changed

417

to Milli-Q water at 60 or 120 min, the rebounce of α-helix% is significantly inhibited,

418

and the extent of BSA conformational deformation is kept afterwards. No increase of

419

α-helix% after the change of the BSA solution with water confirms that protein-protein

420

interaction forces are the driving force for the refolding of adsorbed BSA on hematite

421

surface. However, in the stage I, the loss of the helical structure in adsorbed BSA is

422

mainly driven by the protein-surface interactions. Changing the BSA concentration in

423

bulk solution can barely affect the rate or extent of BSA unfolding on particle surface,

424

so α-helix% continues to decrease after changing the solution.

425

Toward a kinetic model for BSA adsorption on hematite particles in different

426

particle sizes. In the light of the above-mentioned results, we developed a mathematical

427

model to describe the adsorption kinetics and conformational changes of BSA on

428

hematite particles, based on a classic two-state model proposed by McGuire.55 As

429

shown in Figure 4, the adsorption processes of BSA onto hematite surface consist of

430

three steps: (1) transport of proteins from the bulk solution to the near-surface region:

431

BSA in the bulk solution diffuses into a stagnant layer in close proximity to particle

432

surface at a rate constant of 𝑘1. Accordingly, the state of BSA changes from free BSA

433

in the bulk solution (BSAbulk) to that in the stagnant layer (BSAT). The stagnant layer


Page 20 of 35

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434

has a different protein concentration from that in the bulk solution due to a large number

435

of the loosely bound proteins around particle surface. The reverse process, i.e. BSA in

436

the near-surface region diffuses back to the bulk solution, occurs at a rate constant of

437

𝑘2.56; (2) adsorption/desorption of proteins onto/from the surface: a portion of

438

proteins close to particle surface may overcome the energy barrier for adsorption and

439

attach to the surface at a rate constant of 𝑘3. As a result, the state of BSA changes from

440

BSAT to adsorbed BSA without conformational deformation (BSAO), which is a

441

transient state. The desorption experiments (Figure 3a) indicate that BSA desorption

442

from hematite surface is negligible under the conditions of this study. Thus, the rate

443

constant of the reverse reaction, i.e. protein desorption from surface, is not included in

444

this model; (3) conformational changes/refolding of adsorbed proteins: As shown in

445

Figure 1, adsorbed proteins undergo a gradual loss of ordered secondary structure

446

within the first 30-60 min at a rate constant of 𝑘4, resulting in the change of the protein

447

state from BSAO to an unfolded state (BSAU). When the surface coverage of BSA

448

exceeds a critical value, the α-helix content of adsorbed BSA starts to revert at a rate

449

constant of 𝑘5, resulting in the change of its state from BSAU back to BSAO. Because

450

the values of 𝑘4 and 𝑘5 depend on protein surface coverage (or the number of

451

occupied surface sites) that changes over time, 𝑘04 and 𝑘05 were used to denote the rate

452

constants of protein unfolding and refolding, respectively, independent on protein

453

surface coverage. Equations S11 and S12 in the supporting information Section S6

454

describe the relationship between the apparent rate constants (𝑘4 or 𝑘5) and the

455

coverage-independent rate constants (𝑘04 or 𝑘05). More details of the model and



456

calculation are also described in SI Section S6.

457

The calculated and experimental kinetic data of BSA adsorption under different

458

experimental conditions are compared in Figure S13. The results suggest that all

459

measured kinetic data are fitted well with this model (R2 ≥ 0.91). The rate constants

460

derived from the model are listed in Table 1. The values of 𝑘1 are in the same order of

461

magnitude for all hematite particles in different particle sizes, but 𝑘2 becomes three

462

orders of magnitude slower as particle size increases from 39 to 103 nm. As mentioned

463

above, 𝑘1 is the rate constant of protein diffusion from the bulk solution into the

464

stagnant layer, which is barely affected by particle surface due to the limited force range

465

of the protein-particle interactions. On the contrary, the rate constant (𝑘2) of protein

466

diffusion from the near-surface region back to the bulk solution can be significantly

467

influenced by the interaction forces between proteins and particle surface. The zeta

468

potential of BSA (0.5 g/L) in Milli-Q water at pH 5.7 is -27.4 mV. At pH 5.7, the zeta

469

potentials of hematite particles increase from 32.6 to 54.3 mV, as particle size increases

470

from 39 to 103 nm. Thus, the electrostatic attraction force between proteins and the

471

larger particles is relatively higher, resulting in the significantly lower value of 𝑘2 for

472

HM103. In the second step, proteins need to overcome an energy barrier for the BSA

473

attachment onto hematite surface, which can be affected by many factors, such as the

474

steric and electrostatic forces between proteins and particle, as well as the osmotic

475

repulsion from a water layer on particle surface.57, 58 As shown in Table 1, the values

476

of 𝑘3 is independent on particle size, suggesting that particle size is not the

477

predominant factor controlling the energy barrier. In the third step, the conformational


Page 22 of 35

Page 23 of 35


478

change of adsorbed BSA is mainly driven by the protein-surface interactions. As shown

479

by the AFM results (Figure S11), the BSA-hematite interaction force increases, as

480

particle size increases. The stronger particle-protein interaction forces may induce the

481

faster unfolding rate constant (𝑘04) of adsorbed BSA on the larger particles. On the other

482

hand, the refolding rate (𝑘05) of adsorbed BSA on HM103 is three orders of magnitude

483

slower than that on HM39. As discussed above, the recovery of BSA secondary

484

structure on hematite surface is mainly driven by protein-protein interactions, but strong

485

protein-surface interaction forces may counteract the protein-protein interactions and

486

inhibit the refolding of adsorbed BSA. Thus, the increased 𝑘04 but decreased 𝑘05

487

values are observed with the increase of particle size. This new model allows us to

488

quantitively describe the dynamic interplay between adsorption kinetics and

489

conformational changes of proteins on particle surface during adsorption processes.

490

Further studies using different combinations of proteins or particles are needed to test

491

the applicability of this model and ultimately to establish a general adsorption model of

492

proteins on particles.

493 494

Environmental Significance

495

This study reveals the dynamic change of the amount and conformation of adsorbed

496

BSA on hematite surface as response to primary particle size, protein concentration in

497

the bulk solution, adsorption time/surface coverage. The unfolding or refolding of

498

adsorbed BSA result from the competition between protein-particle and protein-protein

499

interactions. The former is mainly affected by primary particle size, but the latter



500

depends on the protein surface coverage that is related to adsorption time and protein

501

concentration in the bulk solution. The findings suggest that, although adsorption

502

behavior of proteins on particle surface can be affected by many factors, the adsorption

503

kinetics and conformational change of proteins are essentially controlled by the protein-

504

particle and protein-protein interactions. Understanding how these factors influence the

505

two kinds of interactions is helpful for us to manipulate the amount and conformation

506

of proteins immobilized on particle surface for biomedical applications. Moreover, this

507

study presents the dynamic conformational changes of proteins on common soil mineral

508

particles, when protein concentration in the bulk solution or protein-particle contact

509

time is changed. Thus, in addition to the inherent properties of proteins and minerals,

510

the alternation in solution conditions and contact time need to be considered in order to

511

estimate the activity of immobilized extracellular enzymes in the natural environment.

512

On the other hand, the different adsorption behavior and conformational changes of

513

proteins on the surface of mineral nanoparticles/colloids under different conditions may

514

also influence particle-particle interaction forces, leading to complex aggregation

515

behavior of NPs/colloids or membrane fouling phenomena.

516 517 518

ASSOCIATED CONTENT

519

Supporting Information. Additional figures and details for Materials and Methods and

520

Results and Discussion are presented. This material is available free of charge via the

521

Internet at http://pubs.acs.org.

522


Page 24 of 35

Page 25 of 35


523

AUTHOR INFORMATION

524

Corresponding Author

525

*Phone: (+86)010-62754292-808; email: [email protected]; address: College of

526

Environmental Sciences and Engineering, Peking University, Beijing 100871, China

527 528

ACKNOWLEDGEMENTS

529

This work was financially supported by National Natural Science Foundation of China

530

(91751105 and 41820104003). We also thank Dr. Chuanyong Jing and Dr. Wei Yan at

531

Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences for

532

the advice on the ATR-FTIR measurements.



533

534

Page 26 of 35

Table 1 Rate constants of elementary steps in BSA adsorption onto hematite particles

Sample

𝒌𝟏

𝒌𝟐

𝒌𝟑

𝒌𝟎𝟒

𝒌𝟎𝟓

HM39 HM68 HM103

7.283 8.723 10

2.715 0.023 0.0063

0.234 0.952 0.122

16.148 50.188 90

0.219 0.085 0.00018

*The

Goodness of fit (R2) 0.1 g/L* 0.5 g/L* 1.0 g/L*

initial BSA concentration in solution ([BSA]Int.)


0.997 0.925 0.98

0.973 0.965 0.983

0.994 0.912 0.987

Page 27 of 35


535

Figure 1. Adsorption profiles of 0.1 (black), 0.5 (red), and 1.0 (blue) g/L BSA on

536

HM39(a), HM68(b), and HM103nm(c), respectively, and the corresponding changes in

537

the α-helix% of BSA adsorbed on HM39(d), HM68(e), and HM103 (f), respectively,

538

during the adsorption processes.



539

Figure 2. Synchronous (a, c) and asynchronous (b, d) correlation contour maps of the

540

dynamic FTIR spectra collected in the first stage (0-60 min, a and b) and the second

541

stage (60-300min, c and d), respectively, of BSA adsorption onto HM39 ([BSA]Int. =

542

0.1 g/L).

543


Page 28 of 35

Page 29 of 35


544

Figure 3. The amide II band area (a) and the α-helix% (b) of BSA adsorbed on HM39

545

as a function of time in the desorption experiments. The flow of the BSA solution

546

([BSA]Int.= 0.1 g/L) was changed with water after 30 (red), 60 (blue), and 120 (green)

547

min, respectively. The data of the control experiment without the change of the BSA

548

solution are shown in black.



549 550

Figure 4. Schematic conceptual illustration of BSA absorption onto hematite surface.


Page 30 of 35

Page 31 of 35

551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594


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Kinetics and Mechanisms of Protein Adsorption and Conformational

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