Raman spectroscopic study of rice globulin

Raman spectroscopic study of rice globulin

Journal of Cereal Science 43 (2006) 85–93 www.elsevier.com/locate/jnlabr/yjcrs Raman spectroscopic study of rice globulin S.W. Ellepolaa, S.-M. Choib...

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Journal of Cereal Science 43 (2006) 85–93 www.elsevier.com/locate/jnlabr/yjcrs

Raman spectroscopic study of rice globulin S.W. Ellepolaa, S.-M. Choib, D.L. Phillipsc, C.-Y. Mab,* a Institute of Fundamental Studies, Hantana Road, Kandy, Sri Lanka Food Science Laboratory, Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, China c Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, China

b

Received 4 May 2005; revised 22 June 2005; accepted 28 June 2005

Abstract The conformation of rice globulin was studied by Fourier-transform Raman spectroscopy as influenced by different buffer environments and heat treatments. The Raman spectrum of the native protein showed a predominance of a-helical structures as indicated by major amide I and III bands at 1657 and 1270 cmK1, respectively. Highly acidic and alkaline pH conditions induced band shifts and intensity changes in amide I, amide III, and C–H (bending and stretching) vibrations, indicating protein denaturation. Addition of dithiothreitol and b-mercaptoethanol led to changes in S–S stretching vibration, whereas ethylene glycol and urea caused marked changes in tryptophan, tyrosine Fermi doublet and C–H band intensities. Heating at 100 8C resulted in progressive denaturation as indicated by band shifts and intensity changes of major spectral regions. Our results revealed that hydrophobic interactions and disulfide bonds play a major role in stabilizing the conformation and in thermal aggregation of rice globulin. q 2005 Elsevier Ltd. All rights reserved. Keywords: Raman spectroscopy; Rice globulin; Protein conformation.

1. Introduction Rice (Oryza sativa L.) is one of the most extensively cultivated cereals in the world, and is consumed as the staple food for more than half the world’s population and serves as the major source of energy and protein for large populations (Kato et al., 2000). Milled rice has an average protein content of 8.0–13% (Nakase et al., 1996; Villareal and Juliano, 1978) and these proteins are found to be one of the highest in nutritive value among cereal proteins (Bean and Nishita, 1985). Because of the high nutritional value, hypoallergenic properties, and bland taste (Ju et al., 2001), there has been an increased use of rice proteins as potential food ingredients (Chrastil, 1992). However, studies on rice

Abbreviations: DTT, dithiothreitol; EG, ethylene glycol; FT Raman, Fourier-transform Raman; b-ME, b-mercaptoethanol; NEM, N-ethylmaleimide; RSAP, Raman Spectral Analysis Package; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; SH-SS, sulfhydryl-disulfide. * Corresponding author. Tel.: C852 2299 0318; fax: C852 2858 3477. E-mail address: [email protected] (C.-Y. Ma). 0733-5210/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2005.06.006

proteins have been limited when compared to those of the starch component. Generally, the major storage protein fraction in most cereals is prolamin. However, rice protein is unique among cereal proteins in being rich in glutelin and poor in prolamin (Payne and Rhodes, 1982; Simmonds, 1978). Because of its abundance, rice glutelin has been extensively studied in biochemical and molecular genetic investigations (Okita et al., 1989). Globulin, the second major storage protein fraction in rice, accounts for 8–10% of the total endosperm proteins (Juliano, 1985). It has a favorable nutritional profile since it contains high levels of sulfur-containing amino acids and a moderately high content of lysine (Krishnan et al., 1992). The rice globulin fraction is composed of two polypeptides of 23–27 kDa and 16 kDa (Komatsu and Hirano, 1992; Krishnan et al., 1992; Luthe, 1992; Perdon and Juliano, 1978; Tanaka et al., 1980; Yamagata et al., 1982). The 23–27 kDa polypeptide, termed a-globulin, is structurally homologous to wheat grain glutenin (Cagampang et al., 1976; Houston and Mohammad, 1970; Pan and Reeck, 1988). In addition, based on the observation that globulins represent major translational products, these proteins may significantly contribute to the quality of rice seeds and they could be targeted for genetic improvement

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(Krishnan et al., 1992). Previous studies on this protein have mainly focused on its isolation, characterization, and location, whereas studies on its conformation have been limited. Details of the structural and conformational properties of rice globulin are critical to an understanding of its structure-function relationships, and in its application as a functional food ingredient. Many methods have been developed for protein structural analysis, e.g. circular dichroism spectroscopy, but the requirement for dilute and/or non-turbid solutions limits their application. Raman spectroscopy is a potentially suitable technique to study plant proteins with low solubility since it is applicable to samples in solid, liquid or aggregated states (Li-Chan and Qin, 1998). It can also be used to investigate in situ structural changes in proteins during denaturation and aggregation (Li-Chan et al., 1994; Li-Chan, 1996a). Since water has a very weak Raman signal, this technique is applicable to study biological systems such as foods that contains significant amounts of water (Careche and Li-Chan, 1997). Raman spectroscopic analysis is based on the inelastic scattering of photons resulting from vibrational transitions of the functional groups of the molecules. Both the frequency and intensity of molecular vibrations are sensitive to chemical changes and microenvironment of the functional groups, which are reflected by changes in the Raman spectrum (Careche and Li-Chan, 1997). The bands in the Raman spectrum attributed to amide I, amide III, and skeletal stretching modes are useful in characterizing different backbone conformations, while bands attributed to stretching or deformation (bending) vibrational modes could be used to monitor the environment around the side chains (Przybycien and Bailey, 1989; Tu, 1986; Williams, 1983). In addition, the Raman spectrum provides valuable information on the disulfide groups of cystinyl residues, C–H groups of aliphatic residues, and aromatic rings of tryptophanyl, tyrosinyl and phenylalanine residues (Li-Chan and Nakai, 1991). A significant advance in Raman spectroscopy is the development of the Fourier-transform (FT) Raman technique, which can overcome the fluorescence problems in dealing with plant materials containing phenolic compounds (Li-Chan et al., 1994; Schrader et al., 1991). Near-infrared (NIR)-FT Raman spectroscopy has been used to study the conformation on soy proteins (Li-Chan et al., 1994), oat globulin (Ma et al., 2000) and red bean globulin (Meng et al., 2003). In this study, Raman spectroscopy was used to study rice globulin conformation as influenced by different buffer environments and heat treatments.

following the procedure of Ma and Harwalkar (1984). The isolated globulin was freeze-dried and stored at K4 8C. The extracted globulin was purified using the isoelectric precipitation method of Houston and Mohammad (1970) with slight modifications. The crude globulin dissolved in 2% acetic acid at pH 2.5 and 0.5 M NaOH was gradually added with vigorous stirring to bring the pH to 4.5. A white, cloudy precipitate was formed. This precipitate, G-I (pH 4.5 insoluble fraction), was separated by centrifugation at 14,000 g for 20 min and recovered after dialysis against distilled water by freeze-drying. This major globulin fraction (G-I fraction) was further purified by repeating the precipitation at pH 4.5 three times. The three fractions were pooled and recovered after dialysis against distilled water by freeze-drying. The protein contents of crude and purified rice globulin (pooled fractions) were found to be 89.6% and 95.4%, respectively, as determined by the microKjeldahl method (Concon and Soltess, 1973), using a nitrogen to protein conversion factor of 5.95 (Juliano, 1985). The purity of the rice globulin preparations was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970). The purified fraction showed a major band at 26 kDa with 95% homogeneity (estimated by densitometry of the SDS-PAGE pattern), while the crude globulin showed two bands with molecular weights of 26 and 16 kDa, respectively, with the 26 kDa band as the prominent fraction. This is in agreement with a previous report that showed a 23–27 kDa polypeptide (a-globulin) as the major component in rice globulin (Komatsu and Hirano, 1992). 2.2. Sample preparation Rice globulin dispersions (10% w/v of freeze-dried sample) were prepared in distilled water. Salt (NaCl) and protein structure perturbants including sodium dodecyl sulfate (SDS), b-mercaptoethanol (b-ME), dithiothreitol (DTT), ethylene glycol (EG), N-ethylmaleimide (NEM) and urea, were added at the desired concentrations. To study the effect of pH, protein dispersions with desirable pH were prepared by the addition of 0.1 M HCl or 0.1 M NaOH with magnetic stirring (Harwalkar and Ma, 1987). These samples were stirred for an hour to allow pH equilibration. To study the effect of heating, protein dispersions (w2% w/v) in 0.01 M phosphate buffer, pH 7.4 containing 0.7 M NaCl, were heated at 100 8C for different times in a temperature-controlled water bath, followed by rapid cooling in an ice bath for 5 min. The heated samples were dialyzed and recovered by freeze-drying.

2. Materials and methods

2.3. Raman spectroscopy

2.1. Materials

Rice globulin dispersions were introduced into NMR capillary tubes and spectra were collected on a Bio-Rad FTS-60 FT-NIR Raman spectrometer equipped with an Nd: YAG laser, providing radiation at 1064 nm (Bio-Rad

Globulin was extracted from defatted rice (Oryza sativa var. Xiang mi) grain flour with 0.7 M NaCl (Juliano, 1985),

3. Results and discussion 3.1. Spectral assignment Fig. 1 shows a typical Raman spectrum of 10% rice globulin dispersion in distilled water. Distilled water was used in control sample since water has a rather weak Raman scatter, which does not absorb in the visible spectrum, and hence does not hinder measurements. The Raman spectrum of purified rice globulin (not shown) was identical to that of the crude preparation, and preliminary tests showed that the two globulin preparations behaved similarly in the different buffer environments. Hence, the crude rice globulin was used for subsequent experiments. Tentative assignments of the major bands in the spectrum are presented in Table 1, based on comparison with Raman spectral data obtained from previous studies (Careche and Li-Chan, 1997; Li-Chan and Nakai, 1991; Li-Chan and Qin, 1998; Li-Chan et al., 1994; Nonaka et al., 1993; Peticolas, 1995; Tu, 1986). The location and intensity of

750

1000

1250

1500

Amide I

C-H bending Amide III

500

Phenylalanine

250

Tryptophan Tyrosine Fermi doublet

S-S stretching

Relative intensity

Laboratories, Cambridge, MA). Spectra were recorded with room temperature under the following conditions: laser power, 500 mW; spectral resolution, 4 cmK1; number of scans, 1000. The Raman spectra of the corresponding solvents were also collected under the same conditions. Raman spectra were taken with a laser power of 500 mW, a spectral resolution of 4 cmK1 and the number of scans was 400. The spectra were analyzed using Grams 32 software (Galactic Industries Corp, Salem NH). The spectral data were baseline-corrected and normalized to the intensity of the phenylalanine band at 1004G1 cmK1, in which the phenylalanine intensity was assigned to be 1 (Howell and Li-Chan, 1996; Tu, 1986). The Raman spectra were plotted as relative intensity (arbitrary units) against Raman shift in wavenumber (cmK1). Protein dispersions containing urea were spiked with 0.2 M KNO3 due to vibrational interference of urea in the phenylalanine region, and the KNO3 peak (1046G1 cmK1) was used for normalization. The Raman spectra were corrected for solvent background by subtracting of the spectrum of the solvent. Quantitative estimation of secondary structure of rice globulin fractions under specific conditions was performed using the Raman Spectral Analysis Package (RSAP) Version 2.1 (Przybycien and Bailey, 1991). This software is based on the smoothing, subtraction, normalization and structure estimation algorithms described by Williams (1983) for least-squares analysis of the amide I band. RSAP provides estimates of the ordered and disordered a-helix, parallel and anti-parallel b-sheets, b-reverse turn and random coil contents using constrained superposition of reference proteins with known structures. All analyses were performed at least in duplicate, and the results are reported as the averages of these replicates.

87 C-H stretching

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1750 2800 3000 3200

Wavenumber (cm-1)

Fig. 1. Raman spectrum (300–3200 cmK1) of rice globulin dispersion in distilled water.

the phenylalanine band was reported to be insensitive to conformation or microenvironment, hence, the sharp band at 1004G1 cmK1 may be used as an internal reference for normalization of the Raman spectra of proteins (Li-Chan, 1996a). Quantitative estimation of the secondary structure composition of rice globulin using the RSAP program is summarized in Table 2. Locations of the amide I (1656 cmK1) and III peaks (1270–1300 cmK1) (Fig. 1) and the estimation of secondary structure composition (Table 2) demonstrate the predominance of a-helical structures in the native rice globulin. The relatively high amide I/amide III ratio further supports the predominance of a-helical structures (Li-Chan, 1996a). Previous studies also showed that native globulins of cultivated rice (Pan and Reeck, 1988) and those of amaranth and quinoa seeds (Gorinstein et al., 1996) exhibited typical a-helical patterns, and that the major rice globulin fraction was composed of mainly a-helical structures. The bands near 1240 and 1249 cmK1 in the amide III region suggest the presence of b-sheets and random coils, respectively. The Raman spectrum in the region 507–540 cmK1 indicates that rice globulin contains disulfide bonds in three different conformations (Fig. 1). The major conformation was gauche–gauche–gauche as indicated by the band at 512 cmK1, which is the most preferred conformation in many naturally occurring proteins with disulfide bonds (Tu, 1982). The minor bands at 525 and 540 cmK1 have been assigned to gauche–gauche–trans and trans– gauche–trans conformations, respectively (Kitagawa et al., 1979; Li-Chan et al., 1994; Nakanishi et al., 1974). The present data are in agreement the previous study of Komatsu and Hirano (1992) which indicated that there is one intra-molecular disulfide bond in rice globulin which carries two cysteine residues. Other studies suggested that C-H bending vibration of the aliphatic side chains of

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Table 1 Tentative assignment of selected bands in the Raman spectrum of rice globulin (in distilled water) Wavenumber (cmK1)

Assignment

Structural information

515, 511 525,530 544

S–S stretch S–S stretch or C–C–C deformation S–S stretch or Tryptophan Tryptophan Tyrosine Fermi doublet

Gauche-gauche-gauche Gauche–gauche–trans Trans–gauche–trans Aliphatic side chains Sharp intense line for buried structure State of phenol-OH (exposed or buried, H donor or acceptor) a-helix a-helix Antiparallel b-sheet Disordered structure Microenvironment, polarity a-helix Antiparallel b-sheet Disordered structure Microenvironment, polarity

755 830, 854 940 1260–1300 1235G5 1245G4 1450 1655G5 1670G3 1665G3 2800–3000

Amide III Amide III Amide III C–H bending Amide I Amide I Amide I C–H stretching

tryptophan also contribute to minor bands near 540 cmK1 (Careche and Li-Chan, 1997; Li-Chan and Nakai, 1991). The C–H stretching band, particularly in the range of 2934–2945 cmK1, is mainly characteristic of high abundance of sulfur-containing and hydrophobic amino acids such as methionine, serine, cysteine, arginine, glutamic acid, proline, and aspartic acid. This is in agreement with the amino acid profile of rice globulin (Perdon and Juliano, 1978; Steenson and Sathe, 1995). 3.2. Effect of pH Fig. 2 shows the Raman spectra of rice globulin dispersions (10%) at different pHs. There was a shift in the amide I and III vibrations, which suggests a transition from a-helical structure near neutral pH to anti-parallel b-sheets and disordered structures at highly acidic (pH 3) and alkaline (pH 11) conditions. Extreme pHs also shifted the C-H vibrations to higher wave numbers, suggesting the formation of random coil structures associated with protein denaturation. Increases in intensities of amide I, C–H bending and C–H stretching vibrations at extreme pHs (pH 3 and 11) also indicate protein denaturation (Fig. 3). Increases in C–H bending band intensity may be attributed to increased exposure of hydrophobic groups to a more polar

environment, whereas increases in C–H stretching band intensity may be due to increasing polarity at highly acidic and alkaline pH conditions (Bouraoui et al., 1997). Estimation of the secondary structure composition in rice globulin using the RSAP program (Table 2) also shows a marked decrease in a-helical content and an increase in random coil and b-sheet structures under highly alkaline condition, whereas the changes in secondary structures under acidic condition were less pronounced. The intensity of tryptophan band (755 cmK1) was stable between pH 5 and 9, but increased at extremes of pH (Fig. 3). This indicates decreased exposure of tryptophan aromatic residues when the solution changes from neutral to acidic or alkaline pH (Li-Chan, 1996a,b). Moreover, the drop in intensity ratio of the tyrosine Fermi doublets (854/ 830 cmK1) at extreme pHs (Fig. 3) suggests either a decrease in exposure or an increase in involvement of some tyrosine residues as strong hydrogen bond donors (Li-Chan, 1996a, b). The S–S vibration was prominent at alkaline pH with a marked intensity increase and shifting of the band position from 512 to 540 cmK1. The data suggest a conformational transition of disulfide bonds from gauche– gauche–gauche to the trans-gauche-trans conformation under highly alkaline pH. However, the increased intensity of Raman band near 530 cmK1 could also be attributed to

Table 2 Secondary structure composition of rice globulin under selected conditions estimated by Raman Spectral Analysis Package (RSAP) Treatment Control (pH 6.7) pH 3 pH 7 pH 11 Heating 10 min 30 min 60 min a

a-Helix

b-Sheet

b-Turn

Random

0.41G0.04 0.26G0.02 0.42G0.04 0.21G0.02

0.22G0.02 0.35G0.01 0.22G0.02 0.32G0.03

0.20G0.01 0.14G0.01 0.18G0.02 0.18G0.06

0.17G0.01 0.12G0.01 0.18G0.02 0.29G0.03

0.35G0.04 0.28G0.02 0.20G0.01

0.27G0.02 0.32G0.04 0.31G0.03

0.19G0.01 0.10G0.00 0.12G0.01

0.19G0.02 0.30G0.03 0.37G0.04

a

Average of duplicate determinationsGstandard deviations.

S.W. Ellepola et al. / Journal of Cereal Science 43 (2006) 85–93 2.4 540

1249 x3

755 x3

1453

89

755 cm-1 (Trp) 854 cm-1 / 830 cm-1 (Tyr)

1668

1.6

e 0.8

1450 1660

1274 1241

0.0

758 x3

515

2

4

6

8

10

12

6

8

10

12

8

10

12

1450 757 512

1240

1657

1270

x3

c 1450 758 x3

512

1658

1240 1271

Normalized intensity

Relative intensity

d

2

1453

1450 cm-1 (C-H bending) 2936 cm-1 (C-H stretching)

1672

8

a

800

4

10

x3

757 x3

600

1.3

1.0 1241

400

1.6

b

1271

505

1657 cm-1 (Amide I)

1.9

1000

1200

1400

1600

6 4

1800

Wavenumber (cm-1)

2

Fig. 2. Raman spectra of rice globulin under different pH conditions. (a) pH 3; (b) pH 5; (c) pH 7; (d) pH 9; (e) pH 11.

3.3. Effect of ionic strength The Raman spectra of rice globulin dispersions in different concentrations of NaCl showed amide I and III peaks located at 1657 cmK1 and 1270–1275 cmK1, respectively (data not shown), indicating the predominance of a-helical structure. There were no marked changes in the intensities of the amide and C–H vibrations, suggesting that the conformation of rice globulin was not affected by increasing ionic strength.

4

6

pH Fig. 3. Effect of pH on normalized intensity of several regions in Raman spectra of rice globulin. Error bars represent standard deviations of the means.

The intensity of the tryptophan band gradually increased and the tyrosine Fermi doublet band decreased with increasing NaCl concentration (Fig. 4). These results suggest decreased exposure of tryptophan and tyrosine aromatic residues and changes in hydrophobic interactions. Addition of salts can influence electrostatic interactions in 0.5

Normalized intensity

changes in hydrophobic interactions of the aliphatic and tryptophan residues as reported by previous workers (Bouraoui et al., 1997; Li-Chan and Nakai, 1991). No marked conformational changes were observed in the Raman spectrum in the pH range 5–9. The isoelectric pH range of rice globulin was found to be pH 4.3 and 7.9 (Ju et al., 2001), and the native conformation could hence be stabilized between pH 5 and 7. Most proteins remain in a native, and more stable, state near their isoelectric pH when the repulsive forces are extremely low. At pHs far from the isoelectric point, large net charges are induced and proteins would be partially unfolded due to intra-molecular side chain charge repulsion leading to rupture of hydrogen bonds and a breakup of hydrophobic interactions (Morrissey et al., 1987).

2

3.0

755 cm-1 (Trp) 854 cm-1 / 830 cm-1 (Tyr)

2.5

0.4

2.0

0.3 1.5

0.2 1.0

0.1

0.5

0.0 0.0

0.1

0.3

0.5

0.7

1.0

0.0

NaCl concentration (M) Fig. 4. Effect of sodium chloride concentration on normalized intensity of tryptophan and tyrosine Fermi doublet bands. Error bars represent standard deviations of the means.

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proteins and change the hydrophobic interactions by modifying water structure (Damodaran and Kinsella, 1982). With an increase in NaCl concentration, more ions will compete with the protein molecule for water and this may lead to the formation of a compact structure (Morrissey et al., 1987). The previously exposed hydrophobic residues could become buried inside the protein structure, as demonstrated by changes in tyrosine Fermi doublet and tryptophan band intensity.

changes in tertiary structure and in the exposure of buried tryptophan and tyrosine residues (Li-Chan, 1996b). In general, NEM did not elicit any marked changes in the positions and intensities of the major Raman vibrations. SDS is an anionic detergent/surfactant which can bind to protein by non-covalent forces and cause ionic repulsion and protein unfolding (Steinhardt, 1975). Due to the amphiphilic character of these surfactants, their interaction with protein is expected to be strongly dependent on their concentration. In the presence of 10 mM SDS, there were no marked changes in the major Raman bands (data not shown), suggesting that protein conformation was not affected by low concentration of SDS. However, at a higher SDS concentration (40 mM), the Raman data indicate protein denaturation. Low SDS concentrations have been shown to stabilize protein against denaturation by highly specific interactions between the cationic groups of proteins and the anionic groups of SDS (Tanford, 1970). The binding of protein to SDS at higher concentrations often results in considerable structural deformation/denaturation (Steinhardt, 1975). DTT and b-ME are reducing agents which can break up the disulfide bonds and interrupt tertiary structure, leading to destabilization of proteins. EG, an organic solvent, can weaken the non-polar interactions between the protein molecules and enhance hydrogen-bonding and electrostatic interactions (Damodaran and Kinsella, 1982;

3.4. Effect of physical and chemical perturbants of protein structure In the presence of SDS (40 mM), DTT (30 mM), b-ME (10%), EG (40%) and urea (6 M), the amide I and III bands were shifted from 1656 to 1660 and 1270–1250 cmK1, respectively, and in EG or urea C–H bending vibration shifted to higher wave numbers (data not shown), while the intensity of the C–H stretching band was increased by all the perturbants except NEM (Fig. 5). The results show transitions from a-helical to disordered conformation, an indication of protein denaturation. The presence of DTT or b-ME caused a decrease in the intensity of the S–S stretching vibration (510–540 cmK1) (Fig. 5) which could be attributed to a reduction or breakage of the disulfide bonds. Changes in the tryptophan and tyrosine Fermi doublet band intensities by the perturbants (Fig. 5) suggest 0.5

4

755 cm-1 (Trp)

854 cm-1 /830 cm-1 (Tyr)

0.4

3

0.3 2

Normalized intensity

0.2 1

0.1 0.0

1.00

control SDS DTT NEM

EG

ME

Urea

0 control SDS DTT NEM

EG

ME

Urea

ME

Urea

12

510-540 cm-1 (S-S stretching)

2936 cm-1 (C-H stretching)

9 0.75

6

0.50

3

0.25

0

0.00 control SDS DTT NEM

EG

ME

Urea

control SDS DTT NEM

EG

Protein structure perturbants Fig. 5. Effect of protein structure perturbants on normalized intensity of several regions in Raman spectra of rice globulin. Control, no additive; SDS, 40 mM sodium dodecyl sulfate; DTT, 30 mM dithiothreitol; NEM, 10 mM N-ethylmaleimide; EG, 40% ethylene glycol; ME, 10% b-mercaptoethanol; urea, 6 M urea. The error bars represent standard deviations of the means.

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3.5. Effect of heat treatments

0.8

512 cm-1 (S-S) 540 cm-1 (S-S)

0.6

0.4

0.2

0

20

30

40

50

60

755 cm-1 (Trp)

0.8

0.6

0

10

20

30

40

50

60

70

40

50

60

70

6.5

2936 cm-1 (C-H stretching)

6.0

5.5

5.0 0

Effect of heating at 100 8C on the Raman spectral characteristics of rice globulin was studied using freezedried solid samples since these samples gave a better spectral resolution than the dispersions. There were shifts in the amide I peaks to higher wave numbers and amide III peaks to lower wave numbers with prolong heating (data not shown). The peak of S–S vibration was shifted from 512 to 542 cmK1 (data not shown), suggesting a transition from gauche–gauche–gauche to trans–gauche–trans conformation with increasing heating time. Table 2 shows the changes in secondary structure composition of rice globulin heated for different times. The results indicate a progressive conformational transition from a-helix to anti-parallel b-sheets and disordered structures upon protein denaturation. Fig. 6 shows the intensity changes in several Raman spectral bands during heating. There were progressive increases in the intensities of the C–H stretching vibration suggesting involvement of hydrophobic interactions during heat aggregation (Howell and Li-Chan, 1996). The intensity of tryptophan band decreased progressively on heating

10

1.0

Normalized intensity

Tanford, 1962). Urea markedly disrupts the hydrogenbonded structure of water and permits the apolar residues into the protein, resulting in the weakening of hydrophobic interactions and loss of native structure (Franks and England, 1975; Kinsella, 1982). All these perturbants cause protein denaturation and changes in conformation to anti-parallel b-sheets and/or disordered structures. It is believed that the unfolding of helical structures followed by the formation of anti-parallel sheet structures could occur through intermolecular interactions between exposed hydrophobic residues (Bouraoui et al., 1997). The Raman data show that the addition of NEM did not cause pronounced structural changes in rice globulin. NEM is a sulfhydryl-blocking agent, which could prevent sulfhydryl–disulfide (SH–SS) interchange reaction in the protein molecules. In the presence of NEM, the conformation of protein molecules shifted to a state that contributes much less to the unfolding transition (Gorinstein et al., 1996). NEM was also found to reduce the amount of aggregation in rice bran globulin (Yuno-Ohta et al., 1994). The present data show that some protein structure perturbants caused marked changes in rice globulin conformation. The changes could be attributed to the perturbation of the tertiary and/or quaternary structures of the oligomeric protein by destabilizing the covalent (disulfide bonds) and non-covalent (electrostatic, hydrogen and hydrophobic bonds) chemical forces. DTT and b-ME influenced the disulfide stretching region whereas the influence of urea, SDS and EG were mainly on the polarity of the microenvironment. Hence, it is apparent that both covalent and non-covalent forces play important roles in stabilizing the secondary structure of rice globulin.

91

10

20

30

Heating time (min) Fig. 6. Effect of heating (at 100 8C) on normalized intensity of several regions in Raman spectra of rice globulin (freeze-dried sample). Error bars represent standard deviations of the means.

(Fig. 6) indicating increased exposure of previously buried residues to the protein surface and involvement of these surface residues as strong hydrogen bond acceptors (Bouraoui et al., 1997; Ogawa et al., 1995). The intensity of the S–S vibration at 512 cmK1 was progressively decreased whereas that of 540 cmK1 band was increased (Fig. 6). The data suggest progressive formation of intermolecular disulfide linkages during heating, with the newly formed disulfide bonds in the trans–gauche–trans conformation, after transition from the gauche–gauche– gauche conformation in the unheated protein. Our results show that thermal denaturation of rice globulin is accompanied by major structural changes. There were progressive increases in the formation of disordered structures, and previously buried hydrophobic

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residues were exposed. Disulfide bond formation seems to play a major role in the thermal aggregation of rice globulin. The formation of disulfide linkages during heating is a frequently observed phenomenon in many plant proteins, where they serve to stabilize the aggregates (Kitagawa et al., 1979).

4. Conclusion The present data show that the native conformation of rice globulin exhibits a predominance of a-helical structures, and is influenced by pH, ionic strength, and by some protein structure perturbants and heat treatments. The study reveals that both non-covalent and covalent forces are involved in stabilizing the protein conformation. Both disulfide bonds and hydrophobic interactions play a major role in heat-induced aggregation of rice globulin. Since the structure/conformation is the critical determinant of protein functionality, these studies will be useful for predicting the structure-function relationships of rice globulin. Confocal Raman microspectroscopy has been used to study the spatial distribution of proteins in rice seed (Piot et al., 2000), and the technique might be extended to measure rice grain protein composition and conformation in situ. Such data would be of value in assessing the technological properties of rice protein as a food ingredient.

Acknowledgements The work described in this paper was fully supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. HKU 7200/01M).

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