Difference in Binding of Long- and Medium-Chain Fatty Acids with

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Difference in Binding of Long- and Medium- Chain Fatty Acids with Serum Albumin: the Role of Macromolecular Crowding Effect Tian-Tian Zhu, Yan Zhang, Xing-An Luo, Shen-Zhi Wang, Ming-Qiang Jia, and Zhongxiu Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03548 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 5, 2018

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Journal of Agricultural and Food Chemistry

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Difference in Binding of Long- and Medium- Chain Fatty

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Acids with Serum Albumin: the Role of Macromolecular

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Crowding Effect

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Tian-Tian Zhu, Yan Zhang, Xing-An Luo, Shen-Zhi Wang, Ming-Qiang Jia, and

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Zhong-Xiu Chen*

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Molecular Food Science Laboratory, College of Food & Biology Engineering,

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Zhejiang Gongshang University, Hangzhou, 310018, China

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ACS Paragon Plus Environment


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ABSTRACT

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Fatty acids (FAs) are transported by serum albumin in plasma. Studies have been

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undertaken to address the binding of MCFAs or LCFAs to human plasma albumin

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(HPA) and bovine serum albumin (BSA) by characterizing the binding affinities.

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Previous research on FA binding to serum albumin was usually performed in dilute

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solutions that are not sufficiently concentrated for interpretation the significance of

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the results under normal physiological conditions. How macromolecular crowded

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media affect fatty acids and bovine serum albumin (BSA) binding remains unknown.

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In this paper, we investigated the mechanism of FA-BSA binding in a polyethylene

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glycol crowding environment by using thermodynamic and spectroscopic methods.

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Molecular crowding increased the binding constant for saturated medium-chain fatty

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acids (MCFAs) but significantly decreased the binding constant for unsaturated

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long-chain FAs. The binding sites tended to increase in all the cases. Further

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investigation revealed that crowding media might loosen the structure of BSA,

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facilitating MCFA-BSA binding. This research is useful for understanding the

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transportation of FAs by BSA under physiological conditions and may also help to

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control digestion by the eventual incorporation of macromolecular crowding agents

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into food formulations.

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KEY WORDS:

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Thermodynamics; Fatty acid; Serum albumin; Macromolecular crowding

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INTRODUCTION

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Difference between medium-chain fatty acids (MCFAs) and long-chain fatty

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acids (LCFAs) is the number of hydrophobic carbons and double bonds. Unlike

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LCFA-containing triglycerides, MCFA-containing triglycerides are not major

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constituents of the classical diet.1 The structural differences between these types of

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triglycerides confer distinct metabolic properties to MCFAs. Medium-chain

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triglycerides used as dietary fats has been shown to influence energy balances and

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may therefore promote weight reduction.2 In the circulatory system, serum albumin is

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the carrier for lots of endogenous and exogenous compounds. Investigation of

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digested nutrients, food additives and pharmaceuticals with respect to their binding of

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albumins is important for food chemistry and nutrition sciences;3-5 among these areas,

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the binding of fatty acids with serum albumin has drawn the attention of researchers

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since the early days. Studies have been undertaken to address the binding of MCFAs

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or LCFAs to albumin by characterizing the binding site distributions and binding

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capacities and affinities.6-8 However, previous research on FA binding to serum

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albumin was usually performed in dilute solutions that are not sufficiently

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concentrated for interpretation the significance of the results under normal

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physiological conditions.

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The term “macromolecular crowding” was coined to connote the influence of

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mutual volume exclusion on the thermodynamic, kinetic and structural properties of

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macromolecules in crowded media.9 Proteins are often present in typical crowded

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environments such as blood or cells. However, characterizing the structure change

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and function alternation of proteins in crowded environments is not fully understood.

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It was reported recently that bovine serum albumin (BSA) displayed a more compact

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state due to crowding effect.10 The question then arises: what is the effect of



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macromolecular crowding on the binding of BSA to MCFAs or LCFAs? Is there any

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difference between MCFA and LCFA binding of BSA because of their distinct

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absorption and metabolism efficiencies? Therefore, measuring the quantitative effects

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of crowding in all studies of macromolecular interactions is necessary if these studies

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are to be regarded as physiologically relevant.

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Our research group reported that macromolecular crowding affects both catalytic

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reactions that involve small molecules and the thermodynamics of the physiochemical

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binding

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vesicle-catalyzed production of fatty acids during macromolecular crowding.11 In

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crowded media, the binding of FAs to phospholipid bilayers also displays an unusual

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behavior. For instance, macromolecular crowding significantly increases the phase

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transition temperature of lipid vesicles, indicating that the crowded medium

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contributes to the heterogeneity of the bilayers and might repair disturbed

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membranes.12 These results invoked an investigation into the fatty acid binding of

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serum albumin under crowded conditions, which might provide valuable insight into

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the biphasic behavior of this binding in vivo. In this work, BSA was selected because

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bovine and human serum albumins are homologous.13 We used polyethylene glycol

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(PEG) as a crowding agent because it is often used for mimicking physiological

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crowding and modifying proteins in pharmaceutical fields.14-15 Isothermal titration

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calorimetry (ITC) was used to obtain thermodynamic information during the binding.

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Differential scanning calorimetry (DSC), fluorescence and circular dichroism (CD)

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spectroscopy were used to examine the structural and conformational changes of the

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BSA protein when bound by fatty acids. This research is useful for understanding the

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transportation of fatty acids by BSA in the presence of macromolecules and provides

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a useful insight toward the chemical basis for food adsorption manipulation.

process.

Unusual

chain-length

dependence


was

involved

in

the

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MATERIALS AND METHODS

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Materials

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Bovine serum albumin (98%) and polyethylene glycol 2000 (99.5%) were all

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purchased from Aladdin Industrial Corporation and used without further purification.

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Sodium octanoate (C8, >99%), sodium nonanoate (C9, >98%), sodium decanoate

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(C10, >99%), sodium laurate (C12, >99%), sodium oleate (C18:1, >99%) and sodium

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linoleate (C18:2, >99%) were obtained from TCI Development Company Limited.

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Deionized water used in all the experiments.

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Sample preparation

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50 mmol·L-1 Tris-HCl buffer solution (pH 7.40)was prepared by dissolving Tris

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in water and adjusting the pH with HCl. PEG2000 was used as the crowding agent

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and was added into the buffer. After PEG2000 were completely dissolved in the buffer,

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the solution was placed in a shaker for 24 h and used as a stock solution. The

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concentrations of PEG2000 were 1 wt%, 5 wt%, and 10 wt%, respectively.

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Isothermal Titration Calorimetry (ITC)

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Titration of BSA with fatty acids was performed using a VP-ITC instrument

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(Microcal Inc, Northampton, MA) at 310 K. All samples were prepared in 50

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mmol·L-1Tris-HCl buffer. In all the experiments of investigating the interaction of FA

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with BSA in the presence of crowding agents, the “buffer” contains PEG 2000. The

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detailed titration procedure was described previously elsewhere.16

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Steady-State Fluorescence Spectra

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A Hitachi F-7000 fluorescence spectrophotometer was used for fluorescence

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measurements. The excitation wavelength was selected at 285 nm after the

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fluorescence 3D scanning experiment and the emission spectra were collected from

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200 to 800 nm. BSA concentration in all the experiments was kept at 8×10-3 mmol·L-1



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and FAs concentration was varied from 8×10-3mmol·L-1 to 1.2 mmol·L-1.

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Differential Scanning Calorimetry (DSC)

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VP-DSC (Microcal Inc., Northampton, MA, USA) was used to record the

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thermograms of BSA with and without FA. The scanning rate was 1.5 °C·min−1. The

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detailed procedure was described previously elsewhere.12

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Circular Dichroism (CD)

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CD spectra were measured on a JASCO-815 CD spectropolarimeter using a 0.1

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or 1 cm path-length cell according to different sample. The protein concentration was

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kept constant at 3×10-7 mmol·L-1 and FA concentration was 6×10-6 mmol·L-1. The

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spectra were collected a in the range of 190-240 nm. The scanning rate is 100 nm/min.

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The temperature was kept at 37 oC. Results were obtained using the CDPro software

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provided by JASCO.

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RESULTS AND DISCUSSION

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Thermodynamics of the interaction between FAs and BSA in PEG 2000

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macromolecular crowding

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A variety of techniques have been used to characterize the binding of FAs to

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serum albumin, including limited proteolytic digestion,17 electron spin resonance,18

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fluorescence quenching,19 covalent modification20 and nuclear magnetic resonance

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(NMR).21 ITC is a sensitive technique that thermodynamically characterizes

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equilibrium reactions involving a small molecule ligand and biomolecules. By using

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this technique, we have determined the molecular mechanisms behind flavor

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sensations,22 the structural transitions of food proteins,23 and interactions between

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endogenous compounds and food components.16 Systematic investigations of the

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thermodynamic differences between MCFA and LCFA binding to BSA by using ITC

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have not been well documented. Herein, we investigated the calorimetric titration


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profiles of FAs of different chain lengths binding BSA at pH 7.4 and 310 K in the

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presence of PEG2000 crowders. The results are shown in Figure 1. The heat released

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from the interaction between FA and BSA decreases gradually as the concentration of

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FA increases. With increasing PEG2000 concentrations, the binding curves of C8 and

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C9 fatty acids (Figure 1a and 1b) change gradually from typical S-curves to a

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non-S-type curves with only one plateau. In 1 wt% PEG2000, binding reaches

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saturation at an FA/BSA molar ratio of 4.5; in 10 wt% PEG2000, the value increases

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to 10.5, which indicates that the crowding degree affects the interaction of FAs with

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BSA, resulting in more FA molecules being required to saturate BSA binding. The

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enthalpogram for the binding of FAs (C10 and C12) to BSA shows clear inflection

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points separating two plateaus (Figure 1c and 1d), suggesting that multiple binding

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processes are involved. Binding of LCFAs (C18 and C18:1) to BSA displays a sharp

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increase in exothermicity in the enthalpogram during initial binding.

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To further investigate the thermodynamic parameters describing the association

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between FAs and BSA, we fit ITC data to one and two binding site models. Except for

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the cases of C8 and C9 in dilute buffer, whose data are only fit well by the one

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binding site model, ITC data for binding are well fitted by the two classes of

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independent sites model. As was shown in Table 1, the ITC results for both MCFAs

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and LCFAs showed that their binding to BSA is spontaneous and exothermic. The

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values of the △H1 and △S1 indicated that the binding process is dominated by

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electrostatic forces and hydrophobic interactions.24 A similar effect was also observed

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for FAs binding to bovine lactoglobulin.25 There is one type of binding site for

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medium-chain FAs (C8 or C9). For long-chain FAs (C>10), two types of binding sites

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exist; at one binding site, the carboxyl terminal of the fatty acid binds an amino acid

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residue of the serum protein through an electrostatic interaction, and at the other, FAs



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binds through hydrophobic interactions in the hydrophobic cavity of the serum protein.

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In all the cases, the unsaturated fatty acid to BSA molar ratio for binding is greater

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than that of saturated fatty acids, and this result is consistent with Wolfrum's26 report

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that the binding of unsaturated FAs to proteins in the liver has a higher ratio than that

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of saturated FAs. This difference could be attributed to the presence of double bonds,

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which might support the pi-pi interaction for FAs with the hydrophobic binding region

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of BSA.

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During FA binding with the uncharged amino acid side chains of protein, the

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binding sites are pliable to fit the incoming FA.27 Five or six interacting sites was

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found by titrating fatty acid-free HSA with myristate.28. Here, we found that crowding

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increases the total number of the binding sites, which was also found in PEGylated

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BSA.15 Experimental data were fitted to a two-site binding model, which gave a

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stoichiometry of binding less than 1.0 for both sites. These values are close to the

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published studies on lactoglobulin interacting with linoleate.25According to Flory's

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theory, when the macromolecule concentration is less than the critical entangling

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concentration, their chains are farther apart and swollen, independent of each other;

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however, when the concentration of PEG2000 reaches 10 wt%, the viscosity of the

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polymer suddenly increases, making the polymer cross-link between molecules to

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form a supramolecular structure.29 For C18:1 and C18:2 (Figure 1e and 1f), the

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binding plots display an asymmetric U-shaped curve, which indicates that there are

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two different classes of binding sites for these FAs. From the results of the modeling,

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the total number of binding sites may increase upon PEG addition.

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Table 1 also displays the different binding affinities for the binding of LCFA and

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MCFA to BSA in PEG. In diluted buffer, each albumin molecule can bind up to seven

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fatty acids, with affinity constants ranging from 104 to 108 M-1, depending on the


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chain length of the FA. Generally, the binding constant (K1) of the first FA binding

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site of BSA is larger than the binding constant (K2) of the second site, which indicates

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negative cooperativity, i.e., the first binding site does not support the second FA

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binding. Without PEG (the first group in Table 1), the binding constant of short-chain

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fatty acids is of the order of 104, which is in agreement with Spector's results30 but

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slightly different from those of Hamilton and McMenamy6, 31. The reason for this

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difference might be the inability of the NMR method to distinguish whether the two

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sodium caprylate binding sites are the same type of binding site. In dilute buffer, the

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binding process is exothermic, and the binding constant increases with increasing

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carbon chain length. With increasing crowding (PEG concentrations from 0 to 5 wt%),

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the BSA-MCFA (C8,C9 and C10) binding constants increase; however, significant

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decreases in binding constants were observed for LCFAs (C18:1 and C18:2) with

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increased crowding. Whether in dilute solutions or in crowded environments, negative

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∆H and positive ∆S values were obtained during the binding of different FAs with

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BSA, indicating that electrostatic forces and hydrophobic interactions dominate the

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interaction. By comparing the ∆H and T∆S values for the first set of binding sites, we

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conclude that in dilute solutions, the electrostatic forces dominated for most of the

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FAs; however, the addition of PEG2000 led hydrophobic interactions to tend to

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dominate the binding process, and the absolute value of T∆S was larger than that of

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∆H. Moreover, the differences between BSA-FA association constants (K1 and K2) are

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much larger in dilute buffers than in PEG2000, suggesting that the crowded media

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reduced the availability of or binding affinity discrepancy between different sites,

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especially for long-chain unsaturated FAs. Early research mentioned that the protein

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concentration affect the binding isotherms for butanal and butanone to soy proteins.32

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Protein-protein interactions at higher concentrations were assumed to result in



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decrease in protein-ligand interactions. We speculated that PEG-protein complex

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might form in which PEG is coiled on the protein surface.33 This hypothesized

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behavior might explain the changes in the binding properties of BSA upon crowding.

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Actually, in a control experiment (See Figure S4 in supporting information), we found

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a little heat was released during the interaction of PEG and BSA in the absence of FA.

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PEG-BSA interaction is non-specific and weak, but might affect the interaction sites

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and thus weaken LCFA-BSA affinities. In fact, the high-affinity sites of BSA are

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completely inactivated by PEGylation.15 However, PEG exerts a macromolecular

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crowding influence on the interaction between MCFAs and BSA, increasing their

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effective concentrations and thus resulting in increased binding constants.

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Binding of FAs to the fluorescent sites of BSA during macromolecular crowding

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BSA contains tryptophan residues with natural fluorescence. The configurational

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adaptability that accompanies FA binding to these sites alters the environment of

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buried tryptophan residues and leads to progressive quenching.34 Therefore, the

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fluorescence changes reflect the microenvironment changes around the amino acid

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residues. Figure 2 shows the fluorescence emission spectra of BSA in the presence of

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FAs with and without PEG2000. The fluorescence intensity of BSA decreased

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gradually with increasing FA concentration. In dilute buffers, the emission maximum

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undergoes a blueshift of 4 nm (from 341 to 337 nm) with increasing concentrations of

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FA, suggesting a less polar environment for the relevant tryptophan residue.35 A

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variety of molecular interactions result in quenching. Two quenching mechanisms

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including dynamic or static quenching can be deduced from Stern-Volmer plots.

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Binding constants of different FAs binding to BSA in dilute and crowded solution was

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calculated according to literature (See supporting information).36-39 Generally, the

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maximum value of collision quenching rate constant (kq) is 1010 L· mol-1· s-1.38 In the


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present study (Table 2), the values of kq were greater than the maximum value. This

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result suggested that the fluorescence quenching was a consequence of static

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quenching. The binding constant was obtained by the intercept and slope of fits to the

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data (see Figure S5 in supporting information). As was shown in Table 2, the binding

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constant increases with the length of the carbon chain, indicating the gradually

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increasing stability of the complexes. The fluorescence data herein suggests that FA

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binds a single binding site of BSA, but the ITC data suggests two binding sites. A

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similar result was reported for the association of vitamin C with HSA40 and the

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interaction of matrine with BSA.41 The reason lies in information about the protein

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regions that were far away from tryptophan residues cannot be obtained by

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fluorescence measurements. Figure 2 also shows that FAs can quench the

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fluorescence of BSA in crowded environments, causing a blueshift (from 341 to 334

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nm) of the maximum wavelength of BSA fluorescence and suggesting that the

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tryptophan residues on the BSA surface were in a more hydrophobic environment.

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Higher

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microenvironments. Independent research on the interaction of BSA with FAs or PEG

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has proven that the fluorescence quenching of BSA in the crowded environment is

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caused by FAs and not by PEG. It can also be seen from Table 2 that the FA statically

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quenches BSA fluorescence in crowded media, which is the same type of quenching

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observed in dilute solution.

concentrations

of

PEG2000

resulted

in

less

polar

amino

acid

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In 1 wt% PEG2000, the quenching constants, kq, for C8, C9 and C10 FAs are

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slightly higher than those in dilute buffer, but those for C18:1 and C18:2 are lower,

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which is similar to ITC results. However, in 5 or 10 wt% PEG 2000, the average kq for

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all of the FA binding BSA decreased, suggesting that the presence of crowded

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environments hinders the binding of FA to the fluorescent groups in the BSA. In the



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same crowded environment, the quenching constant, kq, increases with chain length,

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indicating that longer chain lengths are favorable for the binding of FAs to the

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luminescent amino acid residue. By comparing the ITC and fluorescence quenching

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results, we conclude that the effect of PEG2000 crowding was not significant when

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only changes in the microenvironments of fluorescent sites of BSA due to FA binding

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are considered. Conformational structural changes of BSA must therefore be

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investigated.

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Secondary structure changes of BSA during its binding with FA in

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macromolecular media

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In studying the thermodynamics of the FA-BSA binding process in crowded

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environments, we found that both the hydrophobic carbon chain length of FAs and the

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crowded environment affected binding. To further study the structural changes during

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serum protein binding in the crowded environment, we used circular dichroism (CD)

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to characterize the secondary structure changes of serum protein. Figure 3 shows that

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BSA has two α-helix negative bands at 208 and 222 nm. With the addition of FAs, the

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molar ellipticity (ɵ) of BSA decreases, indicating that the molecular hydrophobicity

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increases and the peptide chain becomes more compact. In the presence of Fas, the

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shape of the CD spectra of BSA did not change, indicating that α-helix dominates the

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structure.42 The helical content of the BSA structure in the crowded environment is

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shown in Table 3. In the presence of 1 wt% PEG 2000, the addition of saturated

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medium-chain FAs (C8 and C10) to the system increased the α-helix content of BSA

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(from 41.8 to 51.9%) and decreased its β-turn content. This phenomenon is consistent

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with α-helix content changes in dilute solution. However, the helical content of BSA

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in crowded environments (1 and 5 wt%) is lower than that of BSA in dilute solution,

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indicating that crowding decreases the protein helix content and causes the structure


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to become slightly less stable. Figure 3c shows the effect of FAs with different chain

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lengths on the secondary structure of BSA in 5 wt% PEG2000, which is consistent

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with the CD curve of the sample at 1 wt%. In all cases, the addition of FAs made the

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α-helix content in BSA increase from the pure BSA value, suggesting that whether in

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the PEG2000 crowded environment or not, the structure of BSA is stabilized by FA.

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This result confirms that in macromolecular media, BSA underwent conformational

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changes resulting in decreased helical content, weakened structural stability, and

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altered fluorescence properties. Based on these results, we propose that the protein

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suffered conformational changes during its binding of FAs. The process can be

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regulated by the crowding environment, which is evidenced by the different

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parameters of fluorescence quenching and the different enthalpies obtained from ITC.

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The increase in the helical content indicated that more hydrogen bonds were formed

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between the amino acid residues of the BSA under those conditions. The structural

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change at 10 wt% of PEG2000 is different from that at other concentrations, similar to

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the ITC experimental results. Together, the crowded environment (1 and 5 wt%

310

PEG2000) slightly decreases protein helix content, resulting in the decreased stability

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of BSA, while FA binding affords BSA stability with a magnitude that depends on the

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saturation degree and chain length of the FA. The combined effect of the crowded

313

environment and fatty acids is evidenced in Table 3.

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Thermostability of BSA during its binding with FAs in macromolecular media

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DSC was used to study the thermostability of BSA because of the

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conformational change that occurs upon FAs binding in macromolecular media.

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Figure 4 shows the effect of FAs on the thermostability of BSA in 1, 5, and 10 wt%

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PEG2000. The solid lines represent fatty acid binding to serum proteins in dilute

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solutions, while the corresponding dotted lines represent the calorimetric curves of



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fatty acids and serum proteins binding in the crowded environment. When different

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chain lengths of FAs are added to BSA, the DSC curve gradually shifts to the right.

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Both the phase transition temperature (Tm) and the phase transition enthalpy (∆H)

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(Table S1 in supporting information) increase gradually, which is consistent with the

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increased helix content of BSA protein. The Tm of BSA is lower in the crowded

325

environment than in dilute solution. This result is consistent with the decreased

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thermodynamic stability of the protein, which is occasionally covalently attached to

327

PEG15. As the concentration of PEG2000 increases from 1 to 10 wt%, the Tm of pure

328

BSA decreases, that is, its thermodynamic stability decreases. The phase transition

329

peak of BSA simultaneously becomes sharper than that in dilute solution, indicating

330

that the cooperativity of the system gradually increases with crowding. The Tm of

331

BSA increases more significantly with increased chain lengths of saturated FAs. To

332

some extent, the addition of FAs offsets the influence of crowding on BSA, making

333

BSA more stable than in crowded environments without FA, displaying positive

334

synergy. The addition of C8, C9 and C10 FAs increased the Tm of BSA greater than

335

the Tm of BSA was decreased by PEG2000. However, the addition of FAs to

336

concentrated PEG2000 was not enough to offset the influence of crowding on BSA.

337

The evidence for this conclusion is that the Tm of BSA bound with C12, C18:1 and

338

C18:2 FAs is lower in a crowded environment at all PEG2000 concentrations than in a

339

dilute buffer.

340

In summary, the thermodynamic results show that with increased crowding degree,

341

the BSA-MCFA (C8, C9 and C10) binding constant tends to increase; however, BSA

342

binding LCFA (C18:1 and C18:2) exhibited significantly decreased binding constants

343

with increased crowding. By tracking the fluorescence spectrum changes, which

344

reflect the microenvironment of BSA fluorescence sites during their interaction with


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FAs, we found that BSA fluorescence was quenched through a static quenching

346

mechanism. Slightly crowded media increase the binding of FAs to the fluorescent

347

groups in the BSA, but concentrated PEG 2000 hinders the binding process. The

348

crowded environment slightly decreases protein helix content, resulting in the

349

decreased stability of BSA, but FA binding compensates for the stability loss of BSA,

350

with the strength of the compensation depending on the saturation degree and chain

351

length of the FA. The addition of C8, C9 and C10 FAs increased the Tm of BSA by a

352

value greater than the Tm was decreased by PEG2000. However, in concentrated

353

PEG2000, the addition of FAs was not enough to offset the influence of the crowded

354

environment on BSA. These results suggest that the crowding media might perturb

355

and loosen the structure of BSA, increasing the affinity of BSA for medium-chain

356

saturated FAs, which is indicated by higher thermodynamic binding and effective

357

fluorescence quenching constants. However, the reduced exclusion size of PEG at

358

high PEG concentrations resulted in complex binding behavior. This research

359

disclosed structural changes of BSA in the presence of FA in a semi-physiological

360

environment, as well as the different thermodynamic binding properties of BSA with

361

medium- and long-chain FAs in the presence of macromolecular crowding. It provides

362

useful information for understanding the transport of fatty acids by BSA in the

363

presence of biomacromolecules under physiological conditions.

364

ASSOCIATED CONTENT

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Supporting Information description

366

The raw isothermal titration curves of FA binding to BSA in buffer and in PEG, the

367

Stern-Volmer Plot of FA with BSA in diluted buffer and in PEG2000, the

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thermodynamic parameters of phase transition in thermal unfolding process of BSA in

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dilute solution with different FAs. This material is available free of charge via the



370

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

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AUTHOR INFORMATION

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Corresponding Author

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*E-mail [email protected] ; phone +86-571-28008980; fax +86-571-28008900.

374

Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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The authors are grateful to the National Nature Science Foundation of China (NSFC,

378

No.21673207) and the Zhejiang Provincial Top Key Discipline of Food Science and

379

Biotechnology (JYTSP20141012) for financial support.

380

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Page 22 of 30

Table 1. Thermodynamic parameters for the interaction of FA with BSA obtained from ITC in different concentrations of PEG crowders at pH 7.4 and 310K.

PEG2000

0 wt%

1 wt%

5 wt%

10 wt%

△H1 (kJ·mol-1)

△S1 (J·mol-1· K-1)

N2

K2(×104 L·mol-1)

△H2 (kJ·mol-1)

△S2 (J·mol-1·K-1)

FA

N1

K1(×104 L·mol-1)

C8

2.07

3.65±0.46

-15.07±0.17

42.30

/

/

/

/

C9

1.50

5.68±0.38

-17.04±0.37

32.50

/

/

/

/

C10

0.40

100.00±0.48

-22.14±1.67

43.50

5.60

4.10±0.53

-12.77±0.25

47.31

C12

3.50

1290.00±53.7

-10.71±0.12

101.7

3.50

20.5±7.39

-4.06±0.33

88.34

C18:1

0.58

12400.00±0.02

-29.68±0.37

59.46

4.83

9.29±0.29

-56.10±0.33

-85.41

C18:2

4.40

59200.00±0.00

-24.99±0.58

87.50

0.60

8.49±0.60

-34.88±0.50

-18.00

C8

2.00

7.12±0.43

-14.32±0.12

46.89

/

/

/

/

C9

1.50

26.2±0.07

-16.04±0.59

71.17

6.00

8.34±2.0

-2.14±0.17

87.50

C10

0.40

110.00±38.10

-26.67±1.63

29.81

6.30

2.62±0.45

-11.35±0.33

48.15

C12

3.50

884.00±48.90

-12.44±1.92

92.95

3.50

9.15±4.40

-4.19±0.29

81.64

C18:1

5.07

256.00±12.70

-27.84±2.68

32 .99

0.62

14.0±0.85

-49.07±0.41

-59.46

C18:2

0.60

10.00±3.430

-13.86±0.79

51.08

4.44

2.18±0.43

-271.4±31.8

-793.3

C8

4.00

50.20±2.91

-18.30±0.46

50.24

2.00

1.67±0.95

-2.34±0.33

73.27

C9

1.30

257.0±13.6

-19.13±0.46

61.13

6.00

5.71±3.23

-2.51±0.21

82.90

C10

0.80

711.0±65.3

-19.63±0.83

67.82

6.20

3.37±0.78

-12.06±0.38

47.73

C12

4.50

505.0±12.9

-14.99±0.08

79.97

3.50

3.08±0.05

-7.12±0.17

63.22

C18:1

0.40

23.40±5.11

-15.87±5.65

51.50

2.40

6.58±0.50

-419.5±43.5

-1260.3

C18:2

3.25

110.00±7.95

-13.94±2.51

70.76

3.50

10.6±1.03

-36.3±0.88

-20.73

C8

2.00

25.10±13.30

-22.15±0.63

31.99

6.00

1.05±0.56

-4.65±0.5

61.96

C9

0.60

75.90±3.02

-32.91±1.80

6.57

7.00

6.54±2.85

-3.35±0.25

81.23

C10

0.60

1720.0±98.4

-17.42±0.79

82.48

6.00

10.8±2.92

-7.66±0.17

71.59

C12

3.20

340.0±42.3

-14.28±0.13

90.44

4.20

10.3±2.82

-4.98±0.17

79.97

C18:1

0.40

16.60±5.35

-16.66±3.43

54.06

2.00

6.96±2.07

-45.22±17.3

-136.49

C18:2

0.30

69.20±4.21

-37.98±1.21

-10.55

3.40

2.75±0.39

-52.79±1.61

-161.62


Page 23 of 30


Table 2 The Stern–Volmer quenching constant and the binding constants of different fatty acids to BSA in dilute and crowded solution at the temperature of 310K and pH 7.4.* Rate constant of quenching PEG

0 wt%

1 wt%

5 wt%

10 wt%

Binding constants K 3 (×10 L·mol-1)

n

R2

0.9848 0.9773 0.9931 0.9583 0.9919 0.9832

0.02 0.24 4.23 7.31 19.0 13.5

0.62 0.76 0.93 0.94 1.02 0.98

0.9970 0.9963 0.9969 0.9974 0.9989 0.9989

2.35 19.2 84.7 128.6 128.2 82.2

0.9683 0.9844 0.9917 0.9835 0.9853 0.9966

0.02 0.10 4.25 3.27 15.2 57.0

0.65 0.67 0.92 0.89 1.00 1.17

0.9881 0.9832 0.9877 0.9876 0.9941 0.9863

C8 C9 C10 C12 C18:1 C18:2

1.74 17.6 61.1 87.7 94.1 138.6

0.9825 0.9847 0.9904 0.9818 0.9419 0.9744

0.02 0.23 10.9 22.7 24.1 10.3

0.67 0.76 1.05 1.07 1.06 0.99

0.9791 0.9996 0.9729 0.9949 0.9848 0.9907

C8 C9 C10 C12 C18:1 C18:2

3.63 15.8 74.1 110.7 134.6 128.5

0.9887 0.9888 0.9909 0.9877 0.9771 0.9789

0.01 0.28 16.2 31.0 34.9 34.0

0.53 0.80 1.08 1.09 1.08 1.10

0.9921 0.9955 0.9906 0.9967 0.9717 0.9877

FA

kq 10 (×10 L·mol-1s-1)

R

C8 C9 C10 C12 C18:1 C18:2

2.53 17.9 82.5 128.8 152.8 156.2

C8 C9 C10 C12 C18:1 C18:2

2

* R2 is coefficient of Determination. n is the number of binding sites.



Page 24 of 30

Table 3. Secondary structural changes of BSA caused by FA binding in PEG2000 at pH 7.4 in 310K. PEG2000

0 wt%

1 wt%

5 wt%

10 wt%

Sample

α-helix

β-turn

Random

BSA

45.8%

4.80%

49.4%

BSA+C8

48.0%

5.90%

46.1%

BSA+C10

53.5%

2.00%

44.5%

BSA+C18:1

47.7%

0.80%

51.5%

BSA

41.8%

20.8%

37.4%

BSA+C8

48.0%

16.9%

35.1%

BSA+C10

51.9%

10.9%

37.2%

BSA+C18:1

44.3%

18.7%

37.0%

BSA

33.2%

1.10%

65.8%

BSA+C8

41.1%

0.50%

58.4%

BSA+C10

34.9%

0.00%

65.1%

BSA+C18:1

43.2%

0.5%

56.3%

BSA

67.6 %

17.6 %

14.8 %

BSA+C8

75.0 %

23.6 %

1.40 %

BSA+C10

71.0 %

16.9 %

12.1 %

BSA+C18:1

58.0 %

7.60%

34.4 %


Page 25 of 30


Figure captions Figure 1. Isothermal Titration Calorimetry for FA binding with BSA in different concentrations of PEG crowders at pH 7.4 and 310K. (a) C8; (b) C9; (c) C10; (d) C12; (e) C18:1; (f) C18:2. Figure 2. Fluorescence emission spectra of BSA in the presence of FA. (a, a1) C8; (b, b1) C9; (c, c1) C10; (d, d1) C12; (e, e1) C18:1; (d, d1) C18:2. a-f, in diluted buffer; a1-f1, with 1wt% of PEG2000. F0 and F are the fluorescence intensities of BSA in the absence and presence of the quencher, respectively. [Q] is the concentration of the quencher (FA). Plots of F0/F against [Q] at 310K are shown in the upper right corner of the figure. Figure 3. CD spectra of FA-BSA in the presence of PEG2000 at pH 7.4 and 310K. (a) in diluted buffer; (b) in 1wt% PEG2000; (c) in 5 wt% PEG2000 (d) in 10 wt% PEG2000. BSA concentration was 3×10-7 mmol·L-1 and FA concentration was 6×10-6 mmol·L-1. Figure 4. DSC thermograms (a, b, c) and the changes of the phase transition temperature (∆Tm) (d, e) of BSA during its binding with different chain-length of FA in the presence of PEG2000. (The solid lines represent BSA or BSA-FA in buffer and the broken lines represent BSA or BSA-FA in PEG2000).



Figure 1. Isothermal Titration Calorimetry for FA binding with BSA in different concentrations of PEG crowders at pH 7.4 and 310K. (a) C8; (b) C9; (c) C10; (d) C12; (e) C18:1; (f) C18:2.


Page 26 of 30

Page 27 of 30


Figure 2. Fluorescence emission spectra of BSA in the presence of FA. (a, a1) C8; (b, b1) C9; (c, c1) C10; (d, d1) C12; (e, e1) C18:1; (d, d1) C18:2. a-f, in diluted buffer; a1-f1, with 1wt% of PEG2000. F0 and F are the fluorescence intensities of BSA in the absence and presence of the quencher, respectively. [Q] is the concentration of the quencher (FA). Plots of F0/F against [Q] at 310K are shown in the upper right corner of the figure.



Figure 3. CD spectra of FA-BSA in the presence of PEG2000 at pH 7.4 and 310K. (a) in diluted buffer; (b) in 1wt% PEG2000; (c) in 5 wt% PEG2000 (d) in 10 wt% PEG2000. BSA concentration was 3×10-7 mmol·L-1 and FA concentration was 6×10-6 mmol·L-1.


Page 28 of 30

Page 29 of 30


Figure 4. DSC thermograms (a, b, c) and the changes of the phase transition temperature (∆Tm) (d, e) of BSA during its binding with different chain-length of FA in the presence of PEG2000. (The solid lines represent BSA and BSA-FA in buffer and the broken lines represent BSA and BSA-FA in PEG2000)



Graphic for table of contents


Page 30 of 30

Difference in Binding of Long- and Medium-Chain Fatty Acids with

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