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Molecular Assembly of Wheat Gliadins into Nanostructures: A Small-Angle X-Ray Scattering Study of Gliadins in Distilled Water over a Wide Concentration Range Nobuhiro Sato, Aoi Matsumiya, Yuki Higashino, Satoshi Funaki, Yuki Kitao, Yojiro Oba, Rintaro Inoue, Fumio Arisaka, Masaaki Sugiyama, and Reiko Urade J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02902 • Publication Date (Web): 14 Sep 2015 Downloaded from http://pubs.acs.org on September 17, 2015

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Journal of Agricultural and Food Chemistry

Molecular Assembly of Wheat Gliadins into Nanostructures: A Small-Angle X-Ray Scattering Study of Gliadins in Distilled Water over a Wide Concentration Range Nobuhiro Sato*,†, Aoi Matsumiya‡, Yuki Higashino‡, Satoshi Funaki‡, Yuki Kitao‡, Yojiro Oba†, Rintaro Inoue†, Fumio Arisaka§, Masaaki Sugiyama**†, and Reiko Urade***,‡

Corresponding authors *

**

***

†

Phone: +81 72 451 2499. Fax: +81 72 451 2633. E-mail: [email protected] Phone: +81 72 451 2670. Fax: +81 72 451 2670. E-mail: [email protected] Phone: +81 774 38 3760. Fax: +81 774 38 3765. E-mail: [email protected]

Research Reactor Institute, Kyoto University, 2-1010 Asashiro-nishi, Kumatori-cho,

Sennan-gun, Osaka 590-0494 JAPAN ‡

Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto

University, Gokasho, Uji, Kyoto 611-0011 JAPAN §

Life Science Center, College of Bioresource Science, Nihon University, 1866 Kameino,

Fujisawa 252-0880 JAPAN

E-mail addresses of coauthors A. Matsumiya , Y. Higashino , S. Funaki , Y. Kitao , Y. Oba , R. Inoue , F. Arisaka

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ABSTRACT: Gliadin, one of the major proteins together with glutenin composing

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gluten, affects the physical properties of wheat flour dough. In this study, nanoscale

3

structures of hydrated gliadins extracted into distilled water were investigated

4

primarily by small-angle X-ray scattering (SAXS) over a wide range of

5

concentrations.

6

analyses of SAXS profiles indicate that gliadins are present as monomers together

7

with small amounts of dimers and oligomers in a very dilute solution. The SAXS

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profiles also indicate that interparticle interference appears above 0.5 wt% because of

9

electrostatic repulsion among gliadin assemblies. Above 15 wt%, gliadins form gel-

10

like hydrated solids. At greater concentrations, a steep upturn appears in the low-q

11

region owing to the formation of large aggregates, and a broad shoulder appears in the

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middle-q region showing density fluctuation inside. This study demonstrates that

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SAXS can effectively disclose nanostructure of hydrated gliadin assemblies.

Gliadins are soluble in distilled water below 10 wt%.

Guinier

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KEYWORDS: gliadin; wheat protein; molecular nanostructures; viscoelastic

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properties; SAXS; protein crosslinking

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INTRODUCTION

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The rheological properties of dough are strongly dependent on the physical

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properties of gluten,1 which is formed by mixing wheat flour with water. Gluten

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consists of two component proteins: glutenin and gliadin. The strength, elasticity, and

22

extensibility of dough owe much to the viscoelastic properties of each component

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protein. Glutenin is a polymer linked by intermolecular disulfide bonds, subunits of

24

which are classified into high- and low-molecular-weight ones.2,3 High-molecular-

25

weight glutenin subunits contribute to the elasticity of dough through a network

26

structure connected by intermolecular disulfide bonds.2,4 In contrast, gliadin is a

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monomeric protein that is responsible for the viscosity of dough.5 They are classified

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into α-, γ-, and ω-gliadins according to their primary structure,2,6 and usually are

29

soluble in dilute acids or 60–70% ethanol-water solutions. It has been shown that

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noncovalent intermolecular interactions exert a dominant effect on the viscoelastic

31

properties of hydrated gliadins.7 It has also been demonstrated that gliadins weaken

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the strength of dough and soften gluten.8-11 Previous studies on the sulfur-rich α- and

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γ-gliadins in dilute acids or ethanol-water solutions have indicated that the C-terminal

34

domains of those gliadins are rich in α-helical content and adopt a compact globular

35

structure stabilized by disulfide bonds.2,12-17 The N-terminal repetitive domains are

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nonglobular and contain elements of a poly-L-proline II structure and β-reverse

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turns.18-20 Because gliadins have known to have poor water solubility,21 analyses such

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as electrophoresis and viscoelastic measurements have been performed on gliadins

39

that were extracted with an ethanol-water solution22 or dilute acetic acid.23,24

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However, it is not known if gliadins in alcohols or acids show the same behavior as

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those in water. In addition, gliadins exist as a hydrated solid in food. It is therefore

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debatable whether data obtained for gliadins in solution are valid for hydrated solids.



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Detailed investigation of the structure of gliadins in relation to water content is

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required to clarify the relationship between superstructures and rheological properties

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of hydrated solid gliadins.

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Classical methods utilizing a 60–70% alcohol or acid solution were primarily

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used to extract gliadins from wheat flour. In 2008, we found that most gliadins could

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be extracted from dough made with 0.51 M NaCl by washing with distilled water.25

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The extracted proteins were primarily found to be gliadin monomers by N-terminal

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amino acid sequencing and analytical ultracentrifugation. These gliadins are free

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from the drawback of higher-order structural changes that may occur through

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treatment with alcohols or acids and, accordingly, are expected to exhibit the same

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structure and properties as gliadins in dough. It was also found that the gliadin

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preparation was soluble in distilled water up to 10 wt% and readily aggregated when

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NaCl or other salts were added.25 Thus, the solubility behavior of extracted gliadins

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needs to be clarified in relation to the structure of the proteins. Small-angle X-ray

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scattering (SAXS) is known to be a powerful method for analyzing the nanoscale

58

structure of various materials. It provides ensemble-averaged structural information

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on the mass distribution and shape of non-crystalline molecules in a non-destructive

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manner. Hence, it has been utilized widely for the nanoscale structural analysis of

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soft matters such as polymer gels, emulsions, and liquid crystals.26,

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characteristic merits of SAXS also can be used effectively for the structural analysis

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of proteins such as gliadins, not only of the gliadin molecule itself, but also of

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aggregated forms at high concentrations. Some results have been reported to date

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concerning the structural analysis on gluten proteins by SAXS. Matsushita et al.

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investigated the structure of high-molecular-weight glutenin subunits by SAXS in 1-

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propanol / acetic acid / water mixed solutions in the concentration range of 1.0–10.0


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The

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mg/mL.28 From Guinier and cross-section Guinier plots, they determined glutenins to

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have a rod-like shape. In another SAXS study, Thomson et al.20, 29 examined α-, γ-,

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and ω-gliadins and high-molecular-weight glutenin subunits in the alcohol/water

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mixed solutions in the concentration range about 1–10 mg/mL and analyzed their

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SAXS profiles by means of Guinier and cross-section Guinier plots. They found that

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gliadins in this concentration range have a prolate ellipsoid shape with a length of 15–

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20 nm and a diameter of about 3.5 nm. These results shed light on the nanostructure

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of gluten protein molecules in dilute solution, but their structure in the alcohol-

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containing water is not necessarily the same as that in alcohol-free water. In addition,

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the structure of gluten proteins at high concentrations is still unclear. In this study, we

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focus on gliadins extracted with distilled water and report on the SAXS analysis of

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their structures over a wide range of concentrations in aqueous solutions. We also

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correlate the results of an electrophoresis measurement and a sedimentation velocity

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experiment with the nanostructure of gliadins established by SAXS.

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MATERIALS AND METHODS Materials.

Gliadins used in this study were prepared by the following

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procedure, the details of which are described in our previous paper.25 Dough was

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prepared by mixing and kneading 100 g of wheat flour (Super KingTM, Nisshin Flour

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Milling, Inc.; 13.8% protein, 0.42% ash, 14% water) with 67 mL of 0.5 M NaCl in a

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mixer (KN-200, Taisho Denki, Tokyo, Japan) for 20 min, and then repeatedly

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kneaded with one hand in 500 mL of distilled water for 15 min at 15 °C. The first and

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second wash solutions were discarded. The third to sixth washing solutions were

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collected and centrifuged at 18,000 × g for 10 min at 20 °C. After NaCl was added to

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the supernatant to adjust its concentration to 0.5 M, the precipitated gliadins were



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collected by centrifugation at 18,000 × g for 20 min at 20 °C, suspended in 300 mL of

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distilled water, dialyzed five times against 3 L of distilled water for 8 h at 4 °C, and

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finally freeze-dried. The gliadins were also extracted from wheat flour with 70%

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ethanol according to the method described by Tatham and Shewry.13 Protein was

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assayed with an RC.DC protein assay kit from Bio-Rad Laboratories (Hercules, CA).

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Sedimentation Velocity Experiments. Sedimentation velocity experiments

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on 0.02 wt% gliadins dissolved in distilled water were performed with an Optima XL-

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I unit (Beckman Coulter, Inc. Brea, CA, USA) using an An50Ti rotor at 20°C.

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Charcoal-filled Epon double sector cells were used with the reference sectors filled

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with distilled water.

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successive scans.

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obtained by use of the SEDFIT program.30, 31 The partial specific volume of gliadin

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was set to 0.73, which is the default value.32 A protocol for calculating the molecular

106

weight corresponding to each peak using the Svedberg equation was implemented in

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SEDFIT, where a common frictional ratio, f/f0, was assumed for all molecular species.

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Electrophoretic Analysis of Gliadin Preparations. Gliadins extracted into

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distilled water or 70% ethanol were subjected to 2D-PAGE isoelectric focusing and

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SDS-PAGE.33 ,34 Gliadins were treated with a 2D clean-up kit (GE Healthcare UK

111

Ltd.) and applied to 7-cm Ready-Strip IPG Strips (Bio-Rad Laboratories). Isoelectric

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focusing was performed using a Protean IEF Cell (Bio-Rad Laboratories). The strips

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were then subjected to SDS-PAGE and stained with Coomassie Brilliant Blue R-250.

All data were acquired without time intervals between

The sedimentation coefficient distribution function, c(s), was

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SAXS Experiments. The SAXS experiments were carried out at the beam

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line BL-10C of Photon Factory, a synchrotron radiation facility of Institute of

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Materials Structure Science, High Energy Accelerator Research Organization (KEK).

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Dilute solution samples were measured in a 1-mm thick aluminum cell with 20-µm


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thick quartz windows. Viscous hydrated solids were measured in a 1-mm thick PTFE

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sandwich cell with windows of 7.5-µm thick Kapton® films (TORAY-DuPont). The

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X-ray wavelength was 0.1488 nm, and the observed range of scattering vector

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(denoted by q) was 0.06–2.5 nm−1 as calibrated with silver behenate. The scattered

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beam was detected with two-dimensional area detectors. The typical X-ray exposure

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time was 300 s. Samples were used for SAXS measurements at least four days after

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preparation. Measured two-dimensional data were converted into one-dimensional

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scattering profiles followed by the standard procedure of data correction for cell and

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background scatterings, transmission, and beam intensity.

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Crosslinking Experiments.

Gliadin solutions were incubated with and

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without 5 mM 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) from Dojindo

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(Tokyo Japan) at 25 °C for 60 min. After addition of N-ethylmaleimide (20 mM), the

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solutions were incubated for 10 min at room temperature and then freeze-dried.

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Laemmli's SDS sample buffer31 without reducing reagent was added to the gliadin

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solutions, which were then boiled for 5 min. The suspension was centrifuged at

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10,000 × g for 40 min at room temperature. The supernatant (30 µg protein) was

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subjected to SDS-PAGE under non-reducing conditions.

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supernatant (100 µg protein) was subjected to SDS-PAGE with a NativePAGETM 3–

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12 % gel (Invitrogen, Carlsbad, CA) and electrophoresed with 50 mM bis(2-

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hydroxyethyl)amino-tris(hydroxymethyl)methane-HCl (Bis-Tris HCl) buffer having a

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pH of 6.8, including 50 mM tricine and 0.1 % SDS as running buffer, under

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nonreducing conditions. After electrophoresis, the sample lane was cut from the gel

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and immersed in Laemmli's SDS sample buffer with 5% 2-mercaptoethanol for 30

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min and subjected to SDS-PAGE under reducing conditions. The proteins were

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stained with Coomassie Brilliant Blue R-250.


Another part of the


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RESULTS AND DISCUSSION

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Gliadin-rich proteins extracted from NaCl-containing dough with

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distilled water. Previously we found that gliadins can be extracted with distilled

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water from dough prepared with wheat flour and a 0.51 M NaCl aqueous solution.25

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When the dough made with 100 g wheat flour was washed with 500 mL distilled

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water, starch, albumins, and globulins were eluted from the dough into the first and

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second wash solutions. However, gliadins were eluted into the third to sixth wash

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solutions when the NaCl content in the dough becomes less than 5 mg/100 g dough.25

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In the present study, we also observed abundant gliadins in the third and subsequent

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extracts by repeating the extraction procedure. The amount of extracted proteins

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(2.63 g from 100 g flour) was almost same as that of gliadins extracted with 70%

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ethanol (2.53 g from 100 g flour). 2D-PAGE shows that the composition of proteins

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extracted by our procedure was almost same as that of gliadins extracted with 70%

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ethanol except for a minor spot at 75 kDa in distilled water, which is probably due to

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contamination (Figure 1).

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The gliadins obtained by our procedure can be dissolved in distilled water to

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yield a transparent solution at concentrations of 10 wt% or less, while they form gel-

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like hydrated solids at concentrations of 15 wt% or more. Gliadins resting in a plastic

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tube at 40 wt% or more do not flow even upon turning the tube upside down, which

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suggests the presence of a network structure.

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Molecular weight distribution. A sedimentation velocity analysis revealed

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the molecular weight distribution of the gliadin in 0.02% distilled water solutions as

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shown in Figure 2. The distribution curve shows the largest peak at a sedimentation


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coefficient corresponding to a molecular weight of 25.7 kDa indicating that most of

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the extracted gliadins are in the monomeric form. A small, broad peak found at a

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molecular weight of ca. 103 kDa suggests that associated gliadins exist in addition to

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monomeric gliadins.

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Nanostructure of gliadins in distilled water as revealed by SAXS. Figure 3

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shows SAXS profiles of gliadins in distilled water at various concentrations: (a)

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solution components of low concentration samples (≤ 10 wt%) and (b) high

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concentration gel-like solid samples (≥ 15 wt%). Because of a large difference in the

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physical state between the two regions, we discuss each concentration region

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separately.

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In the low concentration region, the scattering intensity increases gradually

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with increasing concentration. A small shoulder appears near q = 0.3 nm−1 at 0.5 wt%

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and gradually shifts to 0.13 nm−1 at 10 wt% with increasing intensity. The emergence

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of this shoulder indicates the presence of considerable interparticle interference.

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Therefore, it is required that the behavior of isolated gliadin molecules be observed at

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concentrations below 0.5 wt%. We first attempted to evaluate the size of gliadin

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molecules at concentrations below 0.5 wt% by means of a Guinier analysis of the

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scattering curves. However, the results could not be well fitted with a simple Guinier

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relationship, I = A exp(−Rg2 q2 /3), indicating a polydispersity of scatterers. Thus, we

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employed the three-component Guinier equation expressed as I(q) = A1 exp(−Rg12 q2

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/3) + A2 exp(−Rg22 q2 /3) + A3 exp(−Rg32 q2 /3). As shown in Figure 4a, the scattering

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profiles are well fitted by this equation; the fitting parameters are summarized in

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Table 1. The value of the smallest radius of gyration ranges from 4.10 to 6.46 nm.

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Thomson et al.20 previously reported Rg values of 3.55, 3.80, and 4.60 nm for α-, γ-,



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and ω-gliadins in 70% ethanol, respectively. By comparing their data with the Rg1

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values of our data, we can say that gliadin molecules are present as monomers in the

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0.025 wt% solution, although some molecules with larger Rg values such as gliadin

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dimers and oligomers are also present. The presence of these larger molecules is

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consistent with the results of the sedimentation velocity experiment described above.

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The values of Rg1 increase slightly with increasing concentration indicating that some

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gliadin monomers associate to dimers at higher concentrations. The parameters Rg2

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and Rg3 in Table 1 are much larger than that of gliadin monomers and likely

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correspond to larger aggregates or a high-molecular-weight contaminant (e.g.

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glutenin).

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When anisotropic particles, such as those possessing a rod-like shape, are

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examined by SAXS, a cross-section Guinier plot may be used to obtain information

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about the cross-section size. Figure 4b shows the cross-section Guinier plots for the

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three low concentration samples. The scattering curves in the low-q region were well

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fitted with straight lines obeying the relationship qI(q) = A exp(−Rc2 q2 /2). The

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resulting values for the cross-sectional radius of gyration, Rc, are listed in Table 2.

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These values are more than twice as large as those reported by Thomson et al.20 The

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reason for this is due in part to the aggregation of gliadin molecules, but also may be

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ascribable to structural differences between gliadins in ethanol and in water. The rod

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length, Λ, obtained from the equation, Rg2 − Rc2 = Λ 2 / 12, is also presented in Table 2.

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The radii and length become larger at 0.5 wt%. However, we think that this change is

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due to an increasing number of aggregated gliadins rather than to a change of

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molecular size itself; accordingly, the average value of each dimension becomes

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larger.

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In the 1–10 wt % concentration range, Guinier plots do not yield reasonable

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physical values due to interparticle interference. Instead, the correlation length, L,

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was obtained from the Bragg equation, L = 2π /qs, where qs is the magnitude of the

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scattering vector of the shoulder appearing above 1 wt%. As shown in Table 3, L

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increases with increasing concentration, which suggests that the size of aggregated

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gliadins becomes larger and that the distance between aggregates increases under

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these conditions.

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At concentrations above 15 wt%, gliadins are insoluble in water and form gel-

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like hydrated solids. The SAXS profiles in this region are displayed in Figure 3b.

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The scattering curve at 15 wt% is similar to that of the 10 wt% solution. This fact

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indicates that the nanostructure of these two samples are similar despite the difference

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in water solubility. It is suggested that only intermolecular association occurs in this

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concentration range without a significant change in the size and distribution of

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aggregated gliadins. As concentration increases further, the shoulder near 0.13 nm-1

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diminishes while the upturn in the low-q region grows and a broad shoulder appears

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in the higher-q region. When the low-q changes are examined closely, the 0.13 nm-l

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shoulder shifts to the even lower q and finally protrudes out of the observable q-range.

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This transition suggests the evolving formation of gliadin aggregates.

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concentration increases, isolated gliadin aggregates coalesce to form larger aggregates

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resulting in the growth of the low-q upturn. Concurrently, the density fluctuation

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inside the aggregates also increases, which is manifested as a broad peak in the high-q


As


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region. The characteristic peaks observed in this region are summarized in Table 3.

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When the concentration reaches 70 wt%, the broad shoulder is almost unrecognizable,

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and only the upturn in the low-q region remains.

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Association of gliadins examined by crosslinking experiments. The SAXS

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analysis described above indicates noncovalent association of gliadins in solution.

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Thus, crosslinking experiments were performed with DTSSP, a crosslinking molecule

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that is cleavable with reducing agents after crosslinking, to examine the specificity of

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association between gliadin molecules. By applying nonreducing SDS-PAGE to the

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soluble fraction after centrifugation, it is found that the gliadin bands disappear almost

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entirely from the DTSSP-treated samples at concentrations of 8 wt% and above

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(Figure 5a). This suggests that gliadins crosslink to form large polymers at 8 wt% and

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above and become insoluble in an SDS solution. Monomeric and crosslinked gliadins

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are detected in the supernatant for 0.5–7 wt% samples treated with DTSSP. The most

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prominent bands of crosslinked gliadins correspond to 50–75 kDa in size. Gliadins at

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7 wt% were analyzed by 2D-PAGE (Figure. 5b). By the first nonreducing SDS-

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PAGE with bis-Tris-HCl buffer containing tricine, crosslinked gliadins were detected

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as ~55–65 kDa (i), ~65–75 kDa (ii), ~90–120 kD (iii), ~140–180 kDa (iv) in addition

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to the large smear bands (Figure 5b, panel 1). The crosslinked gliadins were cleaved

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to monomers in the gel after the first nonreducing SDS-PAGE by reduction with 2-

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mercaptoethanol and separated by the second reducing SDS-PAGE. The crosslinked

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~55–65 kDa bands on the first nonreducing SDS-PAGE are composed of 30–35 kDa

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gliadins, which have been shown to be a mixture of α- and γ-gliadins by N-terminal

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amino acid sequence analysis.25 The 65–75 kDa bands on the first nonreducing SDS-

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PAGE are composed of ~37 and ~40 kDa gliadins, which have been identified as α-

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gliadins.25 These results suggest that some gliadins associate specifically in distilled


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water. The crosslinked ~55–65 kDa and ~65–75 kDa bands are predicted to be

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dimers based upon their sizes. In addition, the 90–120 and140–180 kDa smear bands

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on the first nonreducing SDS-PAGE were shown to be composed of 30–35, ~37, and

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~40 kDa gliadins, suggesting that these bands originate from the formation of

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oligomers.

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Plausible model for gliadin association in distilled water. From above

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results, we present the model shown in Figure 6 for the nanoscale structures of

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gliadins in distilled water. In the 0.025–0.5 wt% concentration range, gliadins are

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dispersed in water as isolated particles (Figure 6a), because no shoulder peak is found

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due to interparticle interference.

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populations of dimers and oligomers are also present based on the results of the

279

sedimentation velocity measurement and the SAXS analyses.

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reported that α-, γ-, and ω-gliadins in 70% ethanol show Rg values of 3.55, 3.80, and

281

4.60 nm, respectively, at concentrations below 1 wt%.

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obtained in this study are slightly larger. This suggests that the structures of gliadin

283

monomers in distilled water differ somewhat from those in ethanol. The cross-

284

sectional radius of gyration and the length of gliadins at the lowest concentration are

285

2.50 nm and 11.3 nm, respectively, which is consistent with a rod-like shape.

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Although some gliadins form dimers and oligomers at the lowest concentration, the

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shape of the associated particles is rod-like. The frictional ratio of 1.52 obtained from

288

the sedimentation velocity analysis supports the rod-like model.

Most of the particles are monomers, but small

Thomson et al.

However, the Rg values

289

In the 0.5–10 wt% concentration range, gliadin molecules self-assemble and

290

form small soluble aggregates (Figure 6b) by noncovalent interactions. Interparticle

291

interference is apparent, but gliadins remain soluble in distilled water up to at least 10



292

wt%. This interparticle interference arises from the electrostatic repulsion between

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small aggregates, which is confirmed by the fact that the SAXS shoulder peak

294

vanishes when NaCl is added to the solution at a concentration of 10 mM (data not

295

shown). The correlation length among small aggregates gradually increases with

296

increasing concentration indicating that the interparticle distance is becoming greater.

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At 15–20 wt%, gliadins become insoluble in distilled water but the SAXS

298

profiles are similar to those at 10 wt% concentration (Figure 6c). We suggest that the

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small aggregates become more closely spaced and come in contact with one another

300

to form a continuous network without significantly changing aggregation structure. In

301

the SAXS profiles at concentrations above 30 wt%, the shoulder near 0.13 nm-1

302

disappears and broad peaks at 0.3–1.0 nm-1 become noticeable. In this region, the

303

space is almost entirely filled with gliadin molecules and a definite correlation length

304

disappears; however, density fluctuation is present inside the aggregated gliadins

305

(Figure 6d). Above 50 wt% (Figure 6e), the broad peaks diminish and only the upturn

306

in the low-q region remains, suggesting that gliadins form large aggregates of more

307

than 100 nm in size.

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Finally the influence of NaCl on the structure of associated gliadins should be

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noted. In this study, NaCl acts as a water-solubilizer of gliadins in dough, but it is

311

well known that NaCl also causes the precipitation of gliadin in aqueous solutions.25

312

Because these two observations appear to be contradictory, it is of interest to clarify

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the effect of NaCl addition on the state of association of gluten proteins in relation to

314

the intermolecular interactions among them. This issue will be addressed in our next

315

paper, which will focus on the difference between the association states of highly


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concentrated gliadins hydrated with distilled water and the precipitates produced by

317

NaCl addition.

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In conclusion, SAXS analyses have clarified the state of association of

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gliadins in distilled water over a wide concentration range. At low concentrations (≤

320

0.5 wt%), gliadin molecules are present as monomers plus a minor amount of dimers

321

and oligomers. At greater concentrations, gliadin molecules begin to coalesce (0.5–1

322

wt%) and then show interparticle interference among aggregated molecules (1–10

323

wt%). In these regions, the correlation length increases as concentration increases,

324

because the aggregates grow in size and their mutual separation distance increases.

325

Crosslinking experiments indicate that association of gliadin molecules at 0.5–10 wt%

326

occurs in a specific manner. Both dimers and oligomers are formed, but some are

327

preferentially composed only of α-gliadins instead of a mixture of α- and γ-gliadins.

328

Gliadin becomes insoluble at 15 wt%, although the SAXS profile does not show a

329

significant difference from that at 10 wt%.

330

continuous network of aggregated gliadins. As concentration increases further, the

331

space is filled with gliadin molecules while a density fluctuation remains at ~40 wt%,

332

and finally the large aggregates are formed.

This is due to the formation of a

333 334

Abbreviations Used

335

SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; Bis-Tris,

336

bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane;

337

dithiobis[sulfosuccinimidylpropionate]; SAXS, small-angle X-ray scattering.

338 339

Acknowledgment


DTSSP,

3,3'-


340

This work was supported in part by JSPS KAKENHI Grant Number 26660111 (R.U.),

341

15H02042 (M.S.), and 24310068 (M.S.), MEXT KAKENHI Grant Number 26102524

342

(M.S.), and a grant from the Tojuro Iijima Foundation for Food Science and

343

Technology (R.U.). This work has been performed under the approval of the Photon

344

Factory Program Advisory Committee (Proposal No. 2014G127 and 2014G162).

345 346

References

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Figure Captions Figure 1. Two-dimensional PAGE of gliadin fractions. (a) Gliadin extracted from dough made with wheat flour and a 0.5 M sodium chloride solution by washing with distilled water. (b) Gliadin extracted from wheat flour with 70% ethanol.

Figure 2. Sedimentation velocity analysis of 0.02 wt% gliadins in distilled water. (a) Raw data of sedimentation velocity scans with best-fit curves based on the distribution shown in b.

(b) Concentration distribution versus the sedimentation

coefficient, s, obtained from the fitting result of a. The peaks appearing at 2.06 and 4.76 S correspond to molecular weights of 25.7 and 103 kDa, respectively.

Figure 3. SAXS profiles of gliadins in distilled water. (a) Solution components at low concentrations, (b) paste-form samples at high concentrations. The curves are vertically shifted for clarity.

Figure 4. (a) Guinier plots for dilute aqueous solutions of gliadins. Scattering profiles are fitted by a three-component Guinier equation (thick lines). Small arrows indicate the points at which the relationship qRg1 = 1.3 holds (Rg1: smallest radius of gyration). (b) Cross-section Guinier plots for aqueous solutions of gliadins. Small arrows indicate the points at which the relationship qRc = 1 holds (Rc: cross-sectional radius of gyration).

Figure 5. Detection of the self-assembled gliadins in solution. Gliadin solutions (0.5, 1, 3, 5, 6, 7, 8, 9, 10 wt% in distilled H2O) were incubated at 25˚C for 60 min in the



absence (-) or presence of 5 mM DTSSP (+). After NEM-treatment for 10 min, the solutions were boiled in Laemmli’s SDS sample buffer without reducing agent. The soluble fraction was separated by centrifugation and then subjected to nonreducing SDS-PAGE (a).

Gliadins from the 7 wt% solution treated with DTSSP were

separated by nonreducing SDS-PAGE on 3-12% gel with Bis-Tris running buffer (b, panel 1), reducing SDS-PAGE (b, panel 2) and 2D-PAGE of nonreducing SDS-PAGE on 3-12% gel with Bis-Tris running buffer and reducing SDS-PAGE (b, panel 3). Proteins were stained with Coomassie Brilliant Blue.

Figure 6.

Schematic illustrations of the nanostructures of gliadin assemblies in

distilled water over a wide range of concentrations. (a) Gliadins are present as watersoluble isolated monomers and a few dimers at 0.025–0.5 wt%. (b) Gliadin molecules self-assemble to form small aggregates (dashed line circles) at 0.5 –15 wt%. The distance between the aggregates is estimated to be ~40 nm. (c) Gliadins begin to form continuous networks at 15–20 wt%.

(d) Gliadin molecules fill the space, but

condensed regions due to density fluctuation (dashed line circles) appear above 30 wt%. The distance between dense domains is estimated to be 14 nm at 40 wt%. (e) Above 50 wt% the density fluctuation almost vanishes, but large aggregates over 100nm in size are formed.


Page 22 of 30

Page 23 of 30


Tables

Table 1. Radii of gyration calculated by fitting with a three-component Guinier equation. conc / wt% Rg1 / nm Rg2 / nm Rg3 / nm 0.025 4.10±0.74 13.7±1.1 33.2±0.9 0.1 5.68±0.20 16.4±0.7 34.9±1.2 0.5 6.46±0.05 16.7±0.1 40.0±0.3

Table 2. Radius of gyration (Rg1), cross-sectional radius of gyration (Rc), and rod length (Λ) calculated from Guinier and cross-section Guinier plots. conc / wt% Rg1 / nm Rc / nm Λ / nm 0.025 4.10±0.74 2.65±0.05 10.8±3.4 0.1 5.68±0.20 2.71±0.01 17.3±0.8 0.5 6.46±0.05 3.59±0.003 18.6±0.2

Table 3. Correlation lengths (L) calculated from Bragg's law. conc / wt% qs / nm-1 1 0.210±0.003 3 0.169±0.002 5 0.165±0.001 10 0.139±0.003 15 0.150±0.004 20 0.144±0.002 25 0.145±0.001 30 0.147±0.002 35 0.304±0.005 40 0.434±0.001 50 0.665±0.002 60 0.741±0.008


L 29.9 ±0.4 37.2 ±0.4 38.0 ±0.2 45.3 ±0.8 41.9 ±1.0 43.4 ±0.5 42.7 ±0.4 40.7 ±0.4 20.6 ±0.3 14.5 ±0.05 9.45 ±0.04 8.48 ±0.09


Figures

Figure 1


Page 24 of 30

Page 25 of 30


Figure 2.



Page 26 of 30

Figure 3

b

2

10

10 wt% 5 3 1 0.5 0.1 0.025

1

10

0

10 Intensity

-1

10

-2

10

-3

15 20 35 25 40 30 50 60 70 wt%

Intensity

a

10

-4

10

-5

10

-6

10

0.1

q / nm

-1

1

0.1


q / nm

-1

1

Page 27 of 30


Figure 4

a

b 0.5 0.1 0.025 wt%

qRc = 1 qI

Intensity

0.5 0.1 0.025 wt%

qRg1 = 1.3 0.05

0.10 2

q / nm

0.15

0.05 0.10 0.15 0.20 0.25 0.30

-2

2

q / nm


-2


Figure 5


Page 28 of 30

Page 29 of 30


Figure 6

a

b

d

e

c



TOC Graphics

15 wt%

Gliadin 10 wt% 0.025

Intensity

70

aqueous solutions 0.1

q / nm

-1

hydrated solids 1

0.1

q / nm

-1

1


Page 30 of 30

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