β-casein complex: Characteristics and physicochemical implications

    The folic acid/β-casein complex: characteristics and physicochemical implications Jie Zhang, Yannan Liu, Xiaoming Liu, Yanfang Li, Xundi Yin, Muriel Subirade, Peng Zhou, Li Liang PII: DOI: Reference:

S0963-9969(14)00025-8 doi: 10.1016/j.foodres.2014.01.019 FRIN 5017

To appear in:

Food Research International

Received date: Accepted date:

28 October 2013 9 January 2014

Please cite this article as: Zhang, J., Liu, Y., Liu, X., Li, Y., Yin, X., Subirade, M., Zhou, P. & Liang, L., The folic acid/β-casein complex: characteristics and physicochemical implications, Food Research International (2014), doi: 10.1016/j.foodres.2014.01.019

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ACCEPTED MANUSCRIPT The folic acid/-casein complex: characteristics and physicochemical

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implications

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Jie Zhanga, Yannan Liua, Xiaoming Liua, Yanfang Lia, Xundi Yinb, Muriel Subiradec, Peng Zhoua*, Li Lianga*

State Key Laboratory of Food Science and Technology, School of Food Science and

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a

College of Textiles & Clothing, Jiangnan University, Wuxi, Jiangsu Province 214122,

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b

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Technology, Jiangnan University, Wuxi, Jiangsu Province 214122, China

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c

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China

Chaire de recherche du Canada sur les protéines, les bio-systèmes et les aliments

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fonctionnels, Institut de recherche sur les nutraceutiques et les aliments fonctionnels (INAF/STELA), Université Laval, Québec, QC, Canada

* Author to whom correspondence may be addressed Email: [email protected], Tel/fax: +86 510 85197367 Email: [email protected], Tel/fax: +86 510 85326012

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ACCEPTED MANUSCRIPT Abstract

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Milk proteins are natural vehicles for bioactive molecules. Molecules of casein, the

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principal protein in milk, can reportedly form complexes with bioactive molecules at the molecular level. The interaction of -casein with folic acid (a synthetic form of the B group vitamin known as folates) was studied using fluorescence, absorption

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spectroscopy and circular dichroism. It was found that folic acid bound to -casein by

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hydrophobic contacts with a dissociation constant of  10-5 M. This interaction did not affect changes in -casein structure caused by sodium dodecyl sulfate but did reduce

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the sensitivity of the structure to sodium chloride. Binding to -casein appeared to

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inhibit the photodecomposition of folic acid. These results should provide insight into

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-casein-bioactive-molecule interaction mechanisms and aid the development of

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protein-based carrier systems for the delivery of bioactive molecules.

Keywords: -casein; folic acid; complex; photodecomposition

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ACCEPTED MANUSCRIPT 1. Introduction

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Milk proteins are natural vehicles for bioactive molecules because of several unique

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properties including self-assembly behavior, gelling, emulsification, foaming, as well as formation of complexes with polysaccharides (Livney, 2010). Caseins are major milk proteins and consist of four fractions, called s1-, s2-, -, -casein, accounting

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respectively for 38, 10, 35 and 15 % by weight in bovine milk (Fox, 2003). The

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fractions are quite similar in size, with masses of about 24 kDa. Caseins are amphiphilic in nature and contain distinct hydrophobic and hydrophilic domains along

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the polypeptide chain. They have a strong tendency to associate into micelles, in

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surface (Horne, 2002).

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which s-casein and -casein make up core structures stabilized by -casein at the

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Study of the mechanism underlying interaction between caseins and bioactive molecules is important for the development of effective carrier systems. All casein proteins are rich in proline residues and are therefore less ordered in structure and more flexible than typical globular proteins (Swaisgood, 1993). The formation of casein complexes with vitamins or polyphenols has been studied at the molecular level (Forrest, Yada, & Rousseau, 2005; Hasni, Bourassa, Hamdani, Samson, Carpentier, & Tajmir-Riahi, 2011; Bourassa, & Tajmir-Riahi, 2012; Bourassa, N’soukpoe-Kossi, & Tajmir-Riahi, 2013a). Affinities of folic acid and tea polyphenols for -casein appear to be greater under certain conditions, due to the

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ACCEPTED MANUSCRIPT greater hydrophobic character of this protein (Hasni et al., 2011; Bourassa et al., 2012). Proline residues are reportedly important for interactions of proteins and

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polypeptides with polyphenols (Siebert, Troukhanova, & Lynn, 1996). -Casein

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wraps itself around epigallocatechin gallate by hydrophobic contact between the compound phenolic rings and proline residues in the protein structure ( Jobstl, Howse, Fairclough, & Williamson, 2006). Curcumin has been loaded into the core of casein

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micelles (Yazdi & Corredig, 2012), and complexes such as these could provide

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alternative drug formulations for the delivery of bioactive molecules in cancer

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chemotherapy (Sahu, Kasoju, & Bora, 2008).

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Folate refers to a group of heterocyclic compounds based on the pteroic acid structure

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conjugated with one or more L-glutamic acid molecules linked through the -carboxyl group (Eitenmiler, & Landen, 1999). It is also known as the water soluble vitamin B9,

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present mainly in leafy green vegetables and legumes. In vivo, it is reduced to form biologically active tetrahydrofolate, which acts as a coenzyme in one-carbon transfer reactions required in the biosynthesis of DNA and RNA (Erbe, & Wang, 1984). Folate reduces the risk of neural tube defects and influences the likelihood of developing vascular

diseases

and

some

cancers

(Lucock,

2000).

Folic

acid

(4-[(pteridin-6-ylmethyl)amino] benzoic acid) is a synthetic and more stable form of folate and is commonly used for nutritional fortification and for formulation of pharmaceuticals (Eitenmiler et al., 1999). However, folic acid is sensitive to ultraviolet (UV) light, which causes its decomposition to inactive photoproducts (Off et al.,

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ACCEPTED MANUSCRIPT 2005).

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Interactions of folic acid with various proteins including human and bovine serum

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albumin, -lactoglobulin, -lactalbumin and caseins have been reported (Zhang, & Jia, 2006; Liang, & Subirade, 2010; Bourassa, Hasni, & Tajmir-Riahi, 2011; Bourassa et al., 2012; Liang, Zhang, Zhou, & Subirade, 2013). Bourassa and Tajmir-Riahi studied

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the binding mode of folic acid to -casein using the double-logarithmic Stern-Volmer

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equation (Bourassa et al., 2012), which is the most widely used to analyze ligand-induced quenching of protein fluorescence. However, it has been point out that

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the double-logarithmic plotting linearizes non-linear data, resulting in a false

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impression of a very good correlation (van de Weert, & Stella, 2011). In this study,

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the interaction between folic acid and -casein was further investigated using fluorescence quenching. The impact of the -casein/folic acid interaction on the

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sensitivity of the protein to the physicochemical environment and on the vitamin photosensitivity is also discussed. The photodecomposition of folic acid can be inhibited completely in the presence of -casein, suggesting that casein might be a more suitable carrier for folic acid than are other globular proteins that have been shown to delay the photodecomposition to some extent but not to prevent it (Vorobey, Steindal, Off, Vorobery, & Moan, 2006; Liang et al., 2010).

2. Experimental section

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ACCEPTED MANUSCRIPT 2.1. Materials

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β-Casein from bovine milk (Purity  90%, 98%, C6905) and folic acid ( 98%,

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F7876) were purchased from Sigma-Aldrich Chemical Co. Sodium chloride (NaCl), sodium dihydrogen phosphate (NaH2PO42H2O) and disodium hydrogen phosphate (Na2HPO412H2O) were obtained from SinoPharm CNCM Ltd. (Shanghai, China) and

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sodium dodecyl sulfate (SDS) was purchased from Sangon Biotech Co., Ltd.

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(Shanghai, China).

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2.2. Sample preparation

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Stock solution of β-casein (100 M) in 10 mM phosphate buffer at pH 7.4 was prepared, based on absorbance around 280 nm using a molar extinction coefficient of

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11 000 M-1 cm-1 (Bourassa et al., 2013a), and stored at 4 °C until use. This solution was mixed with phosphate buffer in order to obtain experimental concentrations. Stock solution of folic acid (200 M) was prepared freshly for each experiment by dissolving in 10 mM phosphate buffer at pH 7.4 and diluted to experimental concentrations in the same buffer. Experimental mixtures of β-casein and folic acid were prepared by adding stock solutions to phosphate buffer in varying proportions. NaCl or SDS (1.0 M) was added to these mixtures about 1 hour after preparation. All samples were homogenized by gently shaking and then incubated for at least 1 hour at room temperature prior to analysis.

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2.3. Fluorescence Measurements

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Steady-state fluorescence with a spectral resolution of 2.5 nm for both excitation and emission was measured in 10-mm quartz cuvettes using a FluoroMax®-4 fluorescence spectrophotometer (Horiba Jobin Yvon Inc., Edison, NJ). Protein

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intrinsic fluorescence was measured at 1 or 10 M -casein in the presence of 0, 1, 5,

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10 and 20 M folic acid. Emission spectra were recorded from 300 to 550 nm with an excitation wavelength of 295 nm. Buffer and folic acid backgrounds were subtracted

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from the raw spectra. Fluorescence intensity was normalized relative to that of 10 M

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-casein at the emission maximum. The fluorescence intensity of 10 M folic acid at

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about 455 nm (excitation  = 348 nm) was recorded after UV irradiation for up to 240 min in the presence of -casein at various concentrations. Buffer and -casein

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backgrounds were subtracted from the raw spectra. Fluorescence intensity was normalized relative to that of 10 M folic acid after 240 minutes of UV radiation at the emission maximum.

2.4. Absorbance Measurements

Absorption spectra of 10 M folic acid in the absence and presence of 10 M -casein in 10 mm quartz cuvettes were recorded from 240 to 420 nm on a CARY 50 UV-Vis spectrophotometer (Varian Inc.). Buffer and -casein backgrounds were

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ACCEPTED MANUSCRIPT subtracted from the raw spectra.

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2.5. Circular dichroism (CD) measurements

Far-UV CD spectra of -casein in the absence and presence of folic acid in 1 mm quartz cuvettes were recorded from 190 to 250 nm at a speed of 100 nm/min and with

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1.0 nm bandwidth using a MOS-450/AF-CD spectropolarimeter (n = 3 replications).

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The concentrations of -casein and folic acid were respectively 5 M and 10 M. The

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2.6. Radiation Procedure

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buffer background was subtracted from the raw spectra.

Samples were exposed to UV light (peak  = 365 nm, UVA range) using an ultraviolet

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lamp (UVL-21, VWR Scientific) with the fluence rate set at 1 mWcm-2. Samples were analyzed every 10 minutes for up to 240 minutes.

3. Results and discussion

3.1. Binding properties of folic acid with -casein

-Casein contains one tryptophan residue, located at position 143 at the surface of the molecule. This residue is believed to be responsible for the fluorescence emitted with

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ACCEPTED MANUSCRIPT a maximum (max) at 340 nm upon excitation at 295 nm (Bourassa, Bariyanga, & Tajmir-Riahi, 2013b). At both 1 and 10 M -casein (Fig. 1A and 1B), the peak

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intensities decreased as the concentration of folic acid increased up to 100 M. In the

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case of 1 M -casein, with fluorescence intensity at max being about 1/10 of that at 10 M, a new peak appeared around 445 nm, attributed to fluorescence of folic acid pterin moieties (Thomas, Lorente, Capparelli, Pokhrel, Braun, & Oliveros, 2002),

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when the folic acid concentration exceeded 5 M. Since the folic acid background

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was subtracted from raw spectra, these results suggest that the fluorescence of folic acid bound to -casein is stronger than that of free folic acid, and that the molecule

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thus transfers to a more hydrophobic environment after binding to -casein.

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The folic-acid-induced decrease in the intensity of -casein fluorescence was analyzed using the most generally valid equation, as expressed below (van de Weert

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et al., 2011; Neamtu, Mic, Bogdan, & Turcu, 2013):

(1)

Where F0, F and Fc are the fluorescence respectively of -casein, -casein in the presence of folic acid, and -casein saturated with folic acid; [L]a and [P]t are respectively the concentrations of added folic acid and -casein; Kd is the dissociation constant. From the plots of F as a function of [L]a at 1 and 10 M -casein in the insets of Figures 1A and 1B, Kd were calculated at 1.02 ( 0.98) × 10-5 and 3.44 ( 0.86) × 10-5 M respectively. The affinities (1/Kd) were comparable to the value of 7.0

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ACCEPTED MANUSCRIPT ( 0.9) × 104 M-1 reported previously using the double-logarithmic Stern-Volmer equation (Bourassa et al., 2012). Fc was 7 ( 7) at a concentration of 1 M, suggesting

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that -casein fluorescence could be almost totally quenched by folic acid. The ratio of

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-casein fluorescence emission intensities without and with folic acid versus the concentration of folic acid in Stern-Volmer equation shows a linear plot (data not shown), suggesting that folic-acid-induced tryptophan fluorescence quenching is

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attributed to static (complex formation) quenching. In the case of 10 M, Fc was -31

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( 13), which is a physically meaningless signaling potential discrepancy in interpreting the obtained fluorescence data (Neamtu et al., 2013). An upward

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curvature was observed in Stern-Volmer equation (data not shown), indicating both

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dynamic (collisional) and static quenching is occurring in the system (Bourassa et al.,

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2012). It is thus speculated that negative Fc value might be attributed to the presence

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of dynamic quenching at 10 M.

3.2. Influence of folic acid binding on the sensitivity of -casein structure to the surrounding environment

Beta-casein can be considered as an amphipathic diblock-like copolymer with the N-terminal portion containing anionic phosphoserine residues and the C-terminal portion composed primarily of hydrophobic residues (Forrest et al., 2005). The protein tends to associate by intra-molecular and/or inter-molecular hydrophobic interactions to form core/shell micelles, which results in displacement of the

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ACCEPTED MANUSCRIPT tryptophan residue to a more hydrophobic environment (Gangnard et al., 2007). The critical micelle concentration of -casein is reportedly between 20 and 80 M,

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varying with environmental factors such as temperature, pH and ionic strength

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(Livney, Schwan, & Dalgleish, 2004).

Protein structure can be investigated using protein intrinsic fluorescence, since the

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photo-physicochemical character of tryptophan is sensitive to the polarity of its

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surrounding environment. Figure 2 shows the fluorescence emission spectra of -casein at various concentrations of NaCl. Addition of 5 or 10 M NaCl induced a

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similar increase in the intensity of -casein fluorescence. As the salt concentration

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was increased to 200 M, the fluorescence intensity at max instead decreased

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gradually down to about 78 % of that of the protein alone. No change in max was observed at the same time. The decrease in the fluorescence intensity might therefore

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result from internal quenching induced by certain residues, due to the formation of a more compact state of -casein before micelle formation (Gangnard et al., 2007), since Na+ binds to phosphoserine groups, which are negatively charged above the isoelectric point at pH 4.6 (Strange, van Hekken, & Holsinger, 1994). However, addition of NaCl did not affect the fluorescence of -casein in the presence of folic acid (Fig. 2B), indicating that formation of complexes with folic acid can decrease the sensitivity of the protein structure to salt.

SDS is an anionic surfactant consisting of a hydrophobic tail attached to a sulfate

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ACCEPTED MANUSCRIPT group. Chakraborty and Basak reported the interaction of SDS with casein at surfactant concentrations greater than 100 M (Chakraborty, & Basak, 2008). Figure

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3 shows fluorescence emission spectra of -casein at SDS concentrations up to 200

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M. Addition of 5 M SDS induced a blue shift of max to 331 nm and an increase in the intensity of -casein fluorescence (Fig. 3), indicating the transfer of tryptophan to a more hydrophobic environment. The hydrophobic tail of SDS interacted with

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specific hydrophobic regions of -casein, leading to relocation of the tryptophan

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residue to the inside of the partially folded polypeptide chain (Chakraborty et al., 2008). Increasing the SDS concentration beyond 5 M did not affect max but instead

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caused the fluorescence intensity to drop to below that of protein alone until 100 M

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and then remain constant. The pattern was very similar in the presence of folic acid,

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indicating that formation of complexes with folic acid does not affect the sensitivity of

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the protein structure to SDS.

Inter-molecular interactions are known to be sensitive to agents that cause dissociation. The insensitivity of the -casein structure to NaCl in the presence of folic acid suggests that neither electrostatic attraction nor hydrogen bonding is a major driving force stabilizing the -casein/folic acid interaction. SDS is known to disrupt hydrophobic interaction (Zhang, Liang, Tian, Chen, & Subirade, 2012). The sensitivity of the -casein structure to SDS in the absence or presence of folic acid suggests that hydrophobic interaction is important for casein/folic acid binding. This is consistent with Bourassa and Tajmir-Riahi report about hydrophobic contact

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ACCEPTED MANUSCRIPT between heterocyclic rings of folic acid and hydrophobic pockets in casein by Fourier

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Transform Infrared spectroscopy (Bourassa et al., 2012).

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3.3. Influence of -casein on the photosensitivity of folic acid

Free pterin emits fluorescence with a max near 455 nm and a quantum yield of about

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0.30 when excited at 348 nm (Thomas et al., 2002). However, pterin moieties of folic

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acid have an extremely low quantum yield of fluorescence (< 0.005), which has been attributed to internal quenching (Thomas et al., 2002; Off et al., 2005). Figure 4 shows

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the liberation of pterin moieties from folic acid (measured as fluorescence intensity at

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455 nm) as a function of UV irradiation time in the presence of increasing

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concentrations of -casein. A marked increase in intensity was observed when FA alone was irradiated, suggesting the release of pterin moieties. The addition of

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-casein reduced this fluorescence, in fact to negligible levels at a concentration of 10 M. These results indicate that -casein can inhibit the decomposition of folic acid induced by UV irradiation.

Figure 5 shows absorption spectra of folic acid in the absence and presence of -casein as a function of UV irradiation time. The spectrum of folic acid has a weak peak at 348 nm due to the pterin moiety and a strong peak around 281 nm due to both the pterin and p-aminobenzoyl glutamate moieties, with a shoulder of unknown origin around 300 nm (Vorobey et al., 2006). The absorbance of bands at 280 nm and 300 13

ACCEPTED MANUSCRIPT nm decreased over time to form a broad band around 275 nm, while the absorbance of the band near 348 nm increased with a concomitant red shift, reaching 354 nm until

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160 minutes followed by a blue shift down to 348 nm after 240 minutes of UV

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irradiation. These results suggest a two-step photodecomposition process, consistent with previously reported results showing that the C9-N10 bond of folic acid breaks to yield 6-formylpterin and p-aminobenzoylglutamate, followed by conversion of

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6-formylpterin to pterine-6-carboxylic acid (Thomas, Suarez, Cabrerizo, Martino, &

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Capparelli, 2000; Vorobey et al., 2006). In the presence of -casein at 1 M, only a slight decrease at 300 nm and a slight increase at 348 nm were observed (Fig. 5B).

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The photo-induced change in the folic acid absorption spectra was inhibited

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completely at 10 M -casein (Fig. 5C). These results support that the protein could

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be used to protect folic acid against photodecomposition.

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Figure 6 shows far-UV CD spectra of -casein in the absence and presence of folic acid before and after UV irradiation. The spectrum of -casein shows a negative minimum around 203 nm, consistent with that previously reported by Farrell Jr. et al., suggesting mainly random-coiled structure and possibly the presence of proline Ⅱ structure (Farrell Jr., Wickham, Unruh, Qi, & Hoagland, 2001). The spectra of the protein in the presence of folic acid and/or UV irradiation show similar profiles with only a slight change in the ellipticity around 203 nm. The spectra shown here are averages of two replicate samples. The changes were not statistically significant, based on analysis using the online GraphPad QuickCalcs free t test calculator 14

ACCEPTED MANUSCRIPT (GraphPad Software Inc., San Diego, CA, USA). It can thus be concluded that neither

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addition of folic acid nor UV irradiation changed the structure of -casein.

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Many proteins including human and bovine serum albumin, -lactoglobulin and -lactalbumin have been reported to delay the photodecomposition of folic acid to

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some extent, which has been attributed mainly to the interactions of the proteins with folic acid photodecomposition products and indirect photo-oxidation of proteins

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sensitized by folic acid photoproducts in a competitive manner with folic acid itself

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(Vorobey et al., 2006; Liang et al., 2013). The interactions result in protein structural

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transition and possibly even protein degradation or aggregation (Pattison, Rahmanto, & Davies, 2012; Liang et al., 2013). At the highest concentration tested, -casein

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appeared to inhibit the photodecomposition of folic acid completely (Fig. 4 and 5). Under conditions of UV irradiation, the formation of complexes with folic acid did

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not cause a significant change in the structure of -casein (Fig. 6). It can therefore be speculated that the protective effect of -casein against the decomposition of folic acid does result from the formation of protein-vitamin complexes. These results also suggest that -casein may be better than globular whey proteins as a carrier of folic acid.

4. Conclusions

-Casein can interact with folic acid by hydrophobic contacts with a dissociation 15

ACCEPTED MANUSCRIPT constant of  10-5 M. Complex formation with folic acid does not affect SDS-induced changes in the structure of -casein but does decrease the sensitivity of the protein to

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the

higher

protein

concentration,

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inhibits

folic

acid

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more

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NaCl. Binding to -casein appears to reduce the photodecomposition of folic acid,

photodecomposition completely at 10 M. Beta-casein may thus be considered as a carrier material suitable for folic acid delivery, and folic-acid--casein complex

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formation as a useful model of the interaction between proteins and bioactive

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molecules.

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Acknowledgments

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

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(NSFC Projects 31201291).

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ACCEPTED MANUSCRIPT References Bourassa, P., Hasni, I., & Tajmir-Riahi, H. A. (2011). Folic aid complexes with human

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and bovine serum albumins. Food Chemistry, 129, 1148-1155.

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Bourassa. P., & Tajmir-Riahi, H. A. (2012). Locating the binding sites of folic acid

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with milk α- and β-caseins. Journal of Physical Chemistry B, 116, 513-519. Bourassa P., & Tajmir-Riahi, H. A. (2013a). Binding sites of resveratrol, genistein, and curcumin with milk - and -caseins. Journal of Physical Chemistry B,

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117, 1287-1295.

Bourassa, P., N’soukpe-Kossi, C.N. & Tajmir-Riahi, H. A. (2013b). Binding of

MA

vitamin A with milk - and -caseins. Food Chemistry, 138, 444-453. Chakraborty, A., & Basak, S. (2008). Effect of surfactants on casein structure: A spectroscopic study. Colloids and Surfaces B: Biointerfaces, 63, 83-90.

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Eitenmiler, R. R., & Landen W. O. (1999). Folate. In B. Raton (Ed.), Vitamin Analysis

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for the Health and Food Sciences (pp.411-465).USA:CRC Press. Erbe, R. W., & Wang, J. C. C. (1984). Folate metabolism in humans. American

CE P

Journal of Medical Genetics, 17, 277-287. Farrell H.M., Wickham, E. D., Unruh, J. J., Qi, P. X., & Hoagland, P. D. (2001). Secondary structural studies of bovine caseins: temperature dependence of

AC

-casein structure as analyzed by circular dichroism and FTIR spectroscopy and correlation with micellization. Food Hydrocolloids, 15(4), 341-354.

Forrest, S. A., Yada R. Y., & Rousseau, D. R. (2005). Interactions of Vitamin D3 with Bovine -Lactoglobulin A and -Casein. Journal of Agriculture and Food Chemistry, 53, 8003-8009. Fox, P. F. (2003). Milk proteins: general and historical aspects. In Fox, P. F., & Mcsweeney, P. L. H.(Eds.), Advanced dairy chemistry (3rd ed.). Proteins, Part A(pp.1-48). New York: Kluwer Academic/Plenum Publishers. Gangnard, S., Zuev, Y., Gaudin, J.-C., Fedotov, V., Choiset, Y., Axelos, M. A.V., Chobert, J.-M., & Haertle, T. (2007). Modifications of the charges at the N-terminus of bovine -casein: Consequences on its structure and its 17

ACCEPTED MANUSCRIPT micellisation. Food Hydrocolloids, 21, 180-190. Hasni, I., Bourassa, P., Hamdani, S., Samson, G., Carpentier, R., & Tajmir-Riahi, H. A. (2011). Interaction of milk - and -caseins with tea polyphenols. Food

IP

T

Chemistry, 126, 630-639.

Horne, D. S. (2002). Casein structure, self-assembly and gelation. Current Opinion in

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Colloid and Interface Science, 7, 456-461.

Jobstl, E., Howse, J. R., Fairclough, J. P., & Williamson M. P. (2006). Noncovalent cross-linking of casein by epigallocatechin gallate characterized by single

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molecule force microscopy. Journal of Agriculture and Food Chemistry, 54, 4077-4081.

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Liang, L., & Subirade. M. (2010). Beta-lactoglobulin/folic acid complexes: formation, characterization, and biologicalimplication. Journal of Physical Chemistry B,

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114, 6707-6712.

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Liang, L., Zhang, J., Zhou, P., & Subirade, M. (2013). Protective effect of ligand-binding proteins against folic acid loss due to photodecomposition.

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Food Chemistry, 141,754-761. Livney, Y. D. (2010). Milk proteins as vehicles for bioactives. Current Opinion in Colloid & Interface Science, 15, 73-83.

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Livney, Y. D., Schwan, A. L., & Dalgleish, D. G. (2004). A study of β-casein tertiary structure by intramolecular crosslinking and mass spectrometry. Journal of Dairy Science, 87, 3638-3647.

Lucock, M. (2000). Folic acid: Nutritional biochemistry, molecular biology, and role in disease processes. Molecular Genetics and Metabolism, 71(1-2), 121-138. Neamtu, S., Mic, M., Bogdan, M., & Turcu, L. (2013). The artifactual nature of stavudine binding to human serum albumin. A fluorescence quenching and isothermal titration calorimetry study. Journal of Pharmaceutical and Biomedical Analysis, 72(18), 134-138. Off, M. K., Steindal, A. E., Porojnicu, A. C., Juzeniene, A., Vorobey, A., Johnsson, A., & Moan, J. (2005). Ultraviolet photodegradation of folic acid. Journal of Photochemistry and Photobiology B: Biology, 80, 47-55. 18

ACCEPTED MANUSCRIPT Pattison, D. I., Rahmanto, A. S., & Davies, M. J. (2012). Photo-oxidation of proteins. Photochemical & Photobiological Sciences, 11(1), 38-53. Sahu A., Kasoju N., & Bora U. (2008). Fluorescence study of the curcumin-casein

IP

T

micelle complexation and its application as a drug nanocarrier to cancer cells. Biomacromolecules, 9, 2905-2912.

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Siebert, K. J., Troukhanova, N. V., & Lynn, P. Y. (1996). Nature of polyphenol-protein interactions. Journal of Agriculture and Food Chemistry, 44, 80-85. Strange, E. D., van Hekken, D. L., & Holsinger, V. H. (1994). Effect of sodium

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chloride on the solubility of caseins. Journal of Dairy Science, 77, 1216-1222. Swaisgood, H. E. (1993). Symposium: genetic perspectives on milk proteins:

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comparative stud1 es and nomenclature review and update of casein chemistryl. Journal of Dairy Science, 76, 3054-3061.

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Thomas, A. H., Lorente, C., Capparelli, A. L., Pokhrel, M. R., Braun, A. M., &

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Oliveros, E. (2002). Fluorescence of pterin, 6-formylpterin, 6-carboxypterin and folic acid in aqueous solution: pH effects. Photochemical &

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Photobiological Science, 1, 421-426. Thomas, A. H., Suarez, G., Cabrerizo, F. M., Martino, R., & Capparelli A. L.(2000). Study of the photolysis of folic acid and 6-formyl pterin in acid aqueous

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solutions. Journal of Photochemistry and Photobiology A: Chemistry, 135, 147-154.

Van de Weert, M., Stella, L. (2011). Fluorescence quenching and ligand binding: A critical discussion of a popular methodology. Journal of Molecular Structure, 998, 144-150. Vorobey, P., Steindal, A. E., Off, M. K., Vorobery, A., & Moan, J. (2006). Influence of human serum albumin on photodegradation of folic acid in solution. Photochemical & Photobiological Science, 82, 817-822. Yazdi, S.R., Corredig, M. (2012). Heating of milk alters the binding of curcumin to casein micelles. A fluorescence spectroscopy study. Food Chemistry, 132, 1143-1149. Zhang, A. M., & Jia L. P. (2006). Spectroscopic study of the interaction between folic 19

ACCEPTED MANUSCRIPT acid and bovine serum albumin. Spectroscopy Letters, 39, 285-298. Zhang, J., Liang, L., Tian, Z., Chen, L., & Subirade, M. (2012). Preparation and in vitro evaluation of calcium-induced soy protein isolate nanoparticles and their

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formation mechanism study. Food Chemistry, 133(2), 390-399.

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ACCEPTED MANUSCRIPT Figure captions Fig. 1. Fluorescence emission spectra of 1 (A) and 10 (B) M -casein at pH 7.4 in 10

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mM phosphate buffer in the presence of 0, 1, 5, 10, 20, 50 and 100 M folic acid.

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Inset: plot of F versus [L]a (equation 1).

Fig. 2. Fluorescence emission spectra of 10 M -casein in the presence of various

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concentrations of NaCl (A) and in the presence of NaCl and 10 M folic acid (B).

Fig. 3. Fluorescence emission spectra of 10 M -casein in the presence of various

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concentrations of SDS (A) and in the presence of SDS and 10 M folic acid (B).

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Fig. 4. Fluorescence intensity of 10 M folic acid around 455 nm in the presence of 0,

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1, 2, 5 and 10 M -casein as a function of UV irradiation time.

Fig. 5. Absorption spectra of 10 M folic acid alone (A) and in the presence of 1 and 10 M -casein (B and C respectively) as a function of UV irradiation time. For clarity, only typical curves are shown.

Fig. 6. Far-UV CD spectra of -casein in the absence and presence of folic acid before (a, b) and after (c, d) UV irradiation for 4 h.

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ACCEPTED MANUSCRIPT Highlights > -Casein interacts with folic acid by hydrophobic contacts with Kd of  10-5 M-1.

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> Folic acid doesn’t affect SDS-sensitivity of -casein but reduces NaCl-sensitivity.

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> Binding to -casein could inhibit the photodecomposition of folic acid.

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