Gamma irradiation as an alternative treatment to abolish allergenicity of lectins in food

Food Chemistry 124 (2011) 1289–1295

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Rapid Communication

Gamma irradiation as an alternative treatment to abolish allergenicity of lectins in food Antônio F.M. Vaz a, Romero M.P.B. Costa a, Luana C.B.B. Coelho a, Maria L.V. Oliva b, Lucimeire A. Santana b, Ana M.M.A. Melo c, Maria T.S. Correia a,* a b c

Departamento de Bioquímica, Universidade Federal de Pernambuco, Recife, Brazil Departamento de Bioquímica, Universidade Federal de São Paulo, São Paulo, Brazil Departamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco, Recife, Brazil

a r t i c l e

i n f o

Article history: Received 8 February 2010 Received in revised form 20 June 2010 Accepted 27 July 2010

Keywords: Food irradiation Lectins Food allergen bis-ANS Misfolded states

a b s t r a c t Food irradiation is the process of exposing food to ionising radiation to eliminate microorganisms or insects. Lectins are the most common agents in food intolerance, and efficient methods to reduce unwanted or intolerant immunological effects of lectins have not yet been described. Sebastiania jacobinensis bark lectin was structurally altered after gamma irradiation. Hemagglutination assays showed that the lectin was stimulated by low doses of radiation (0.1 kGy), while high doses (above 1 kGy) induced a significant loss of activity. The effect of c-radiation on lectin hydrophobicity was measured by intrinsic and bis-ANS fluorescence. High doses of ionising radiation suppressed the intrinsic fluorescence emission and promoted polypeptide fragmentation and hydrophobic surface modification. The results suggested that changes in the hydrophobic surface induced by gamma irradiation led to protein misfolding and, subsequently, to aggregation. The pioneering viewpoints presented on the stability of a food allergen after gamma irradiation might contribute to the development of harmless and more effective methods to reduce or eliminate food allergenicity. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Reliable estimates of the prevalence of food allergies/intolerance or the relative importance of the most common food allergens do not exist. Results from epidemiological studies, combined with knowledge on systemic reactions to contact allergens and celiac disease, have suggested that the prevalence of food allergies/intolerance in the adult European population is approximately 5% (Madsen, 1997), demonstrating the great importance of food allergies/intolerance in the health policy literature. Lectins are proteins or glycoproteins that are ubiquitous in nature and have one or more carbohydrate binding sites that lack catalytic function or immunological characteristics (Sharon & Lis, 2004). Most plants contain lectins that can be toxic, inflammatory or both and cause food intolerances or sensitivities. High levels of lectins are found in grains, legumes (beans and peanuts), dairy products and plants in the nightshade family. The total dietary intake of lectins in vegetarian diets rich in leguminous seeds is about 0.1–10 g/kg (Peumans & Van Damme, 1996). While lectins are present in most

* Corresponding author. Address: Av. Prof. Moraes Rego, 1235, Cidade Universitária, Recife ,PE, CEP: 50670-901, Recife, PE, Brazil. Tel.: +55 81 2126 8574; fax: +55 81 2126 8576. E-mail address: [email protected] (M.T.S. Correia). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.098

foods, those that contain higher levels are problematic for many genetically vulnerable individuals. Previously described methods to reduce unwanted or intolerant immunological effects of lectins have been inefficient (Sathe, Teuber, & Roux, 2005) because many lectins are resistant to cooking and digestive enzymes. These aspects are responsible for reports of aggravation of inflammation, digestive diseases and the emergence of food allergies. Lectins bind to specialised intestinal cells (crypt cells) in the mucosa of the duodenum through the carbohydrate recognition domain (CRD), which causes deleterious effects on digestion (Gupta & Sandhu, 1997). Food irradiation is the process used to eliminate insects, fungi or bacteria that spoil food or cause human disease (Farkas, 1998). Because irradiation kills disease-causing bacteria and reduces the incidence of food borne illnesses, hospitals sometimes use irradiation to sterilise food for immunocompromised patients. Radiation treatments of biological materials have also been applied to various processes, such as the sterilisation of medical supplies. However, high doses of gamma radiation may affect the functional integrity of biomolecules. Protein irradiation promotes extensive structural damage and abolishes biological activity (Davis, Parniak, Kaufman, & Kempner, 1997) through two different mechanisms. First, it splits covalent bonds in target proteins by direct photon energy (Kempner, 2001); second, via water radiolysis, it indirectly produces reactive oxygen species (ROS) responsible for the

1290

A.F.M. Vaz et al. / Food Chemistry 124 (2011) 1289–1295

majority of the protein damage (Riley, 1994). ROS interact with biological molecules to produce secondary radicals. The primary forms of damage caused by ROS are hydrogen abstraction from amino acid side chains, rings of aromatic residues, and disulfide bonds (Charlier et al., 2002; Xu & Chance, 2005). The exposure of proteins to radiation produces alterations to their physical and chemical structures by fragmentation, crosslinking, aggregation, unfolding and the formation of new reactive groups, resulting in distortions of the secondary and tertiary structures (Garrison, 1987). These alterations depend upon several factors, such as the protein concentration, the presence of oxygen and the molecular structure. Many protein conformation studies involve intrinsic and extrinsic fluorescence measurements. Denatured proteins often exhibit changes in solvent accessibility to hydrophobic regions after physical treatments. One of the most important probes used to study protein denaturation is 4.40 -bis1-anilinonaphthalene 8-sulphonate (bis-ANS), which is not fluorescent in aqueous media but shows fluorescence when bound to proteins (Farris, Weber, Chiang, & Paul, 1978). The bis-ANS has been used to probe the structure–function relationships of proteins, such as tubulin (Horowitz, Prasad, & Luduena, 1984). Good hygienic practises can reduce the level of contamination but cannot presently eliminate toxic food lectins, particularly from foods that are sold raw. Gamma irradiation is a safe technology that can eliminate pathogens in food; however, it is necessary to elucidate the effect of radiation on lectins in order to establish the feasibility of a large-scale treatment. The United States Department of Agriculture (USDA) has approved the use of low-level irradiation as an alternative treatment to pesticides for fruits and vegetables, and the US Food and Drug Administration (FDA) has cleared the use of radiation to eliminate the residual risk of contamination by virulent Escherichia coli (Kume, Furuta, Todoriki, Uenoyama, & Kobayashi, 2009). Sebastiania jacobinensis bark lectin (SejaBL) has been purified by size exclusion chromatography in milligram quantities. The lectin, a glycoprotein of 52.0 kDa composed of 24 kDa subunits, presents inhibitory activity toward fungi as well as low environmental toxicity. SejaBL has high thermal stability, is resistant to digestive enzymes and has a secondary structure with more alpha helices than beta sheets, which makes it similar to cereal and legume lectins (Vaz et al., 2010). To our knowledge, this paper reports for the first time, the effects of crays on the structure and function of a food allergen and a new and valuable approach for the processing of food by ionising radiation. 2. Materials and methods 2.1. Chemicals The broad-range molecular weight protein was purchased from Sigma Chemical Co., USA. 4.40 -bis-1-Anilinonaphthalene 8-sulphonate (bis-ANS) was purchased from Molecular Probes Inc., USA. All solvents and other chemicals used were of analytical grade from Merck, Germany. All solutions were made with water purified by the Milli-Q system. 2.2. Purification of S. jacobinensis bark lectin (SejaBL) Sebastiania jacobinensis bark was collected from trees in the semi-arid region of the State of Pernambuco, Brazil. S. jacobinensis bark powder was homogenised overnight at 4 °C in 10 mM Tris– HCl buffer (pH 8.5) containing 0.2% (v/v) Triton X-100 detergent. The homogenate was centrifuged at 5000g for 20 min (crude extract), followed by lyophilisation. Cold acetone/water (4:1) was added to the crude extract (2:1), and the mixture was incubated

on ice for 10 min. The solution was centrifuged and the supernatant was concentrated in a vacuum. The residual aqueous solution obtained was precipitated with 60% saturated (NH4)2SO4. The precipitate was dialysed against Tris–HCl buffer and then applied to a CM-Cellulose column. Adsorbed proteins were eluted with 1 M acetic acid, and the fractions (up to 0.100 abs at 280 nm) were pooled, dialysed, concentrated by ultrafiltration (Amicon Ultra15, Mr. 10 000 cut-off) and applied to a Sephadex G-100 column. The first fraction eluted with 150 mM NaCl (SejaBL) was concentrated by ultrafiltration in 10 mM phosphate buffer (pH 7.0) and stored according to Vaz et al. (2010). 2.3. Lectin irradiation The lectin aliquots (0.5 mg/ml) in phosphate buffer (pH 7.0) in borosilicate glass vials (16–125 mm) were frozen and irradiated under atmospheric O2 using a Gammacell 220 Excel 60Co gamma ray irradiator (Ontario, Canada) with doses of 0.020, 0.050, 0.075, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 3.0, 6.0, 12.5, 25 and 35 kGy at a rate of 12.5 kGy/h. Each sample was analysed 1 h, 24 h and 7 days after irradiation by the following methods. 2.4. Hemagglutination activity and protein concentration Hemagglutination activity (HA), which was defined as the lowest sample dilution that showed hemagglutination, was evaluated as described by Correia and Coelho (1995). Specific HA (SHA) corresponded to the relationship between the HA and protein concentration, measured according to Lowry, Rosebrough, Farr, and Randall (1951) using bovine serum albumin (BSA) to construct a standard curve in the range 0–500 lg/ml. The percentage of the remaining SHA (%SHAREM) was calculated according to the equation: %SHAREM = (SHA)GM/(SHA)GO  100, where GM is the lectin SHA of each radiation dose (0.02–35 kGy) and G0 is the SHA of non-irradiated lectin (control). 2.5. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) SDS–PAGE was performed according to Laemmli (1970). Protein samples were mixed with loading buffer (60 mM Tris–HCl, pH 6.8, with 2% SDS, 25% glycerol and 0.1% bromophenol blue), resolved on a 10% separating gel and stained using a silver stain kit (Bio-Rad). The following standard molecular weight markers were used: rabbit muscle myosin (205 kDa), E. coli b-galactosidase (116 kDa), rabbit muscle phosphorylase b (97.4 kDa), rabbit muscle fructose-6phosphate kinase (84 kDa), bovine serum albumin (66 kDa), bovine liver glutamic dehydrogenase (55 kDa), egg albumin (45 kDa), rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (36 kDa), bovine erythrocyte carbonic anhydrase (29 kDa), bovine pancreas trypsinogen (24 kDa), soybean trypsin inhibitor (20 kDa), bovine milk a-lactalbumin (14.2 kDa) and bovine lung aprotinin (6.5 kDa). 2.6. Reverse phase chromatography analysis SejaBL (0.5 mg/ml) was irradiated (0.1, 3 and 35 kGy) and submitted to reverse phase chromatography on a C-18 column (Vydacprotein peptide ultrasphere), performed in an HPLC system (Shimadzu LC-10AD, Kyto, Japan) and monitored at 215 nm. The column was equilibrated with solvent A (0.1% TFA in H2O) at a flow rate of 0.7 ml/min, and a non-linear gradient elution was used with solvent B (90% acetonitrile, 10% H2O, 0.1% TFA) in A, with 5% B at t = 5 min; 70% B at t = 27 min, 80% B at t = 60 min and 100% B at t = 69 min.

1291

A.F.M. Vaz et al. / Food Chemistry 124 (2011) 1289–1295

highly complex energy landscape of a protein (Frauenfelder, Sligar, & Wolynes, 1991). Abrupt changes in the amplitude and timescale of atomic fluctuations have been shown to initiate structural alterations for a number of proteins (Ferrand, Dianoux, Petry, & Zaccai, 1993). This peculiar behaviour, which was confirmed by the increase in intensity of the maximum fluorescence emission (below), revealed changes in the native tertiary structure of SejaBL and possibly in the carbohydrate recognition domain (CRD). The major function of CRDs is the calcium-mediated recognition of oligosaccharides at the cell surface. The CRD can function independently of the rest of the protein and thus may be employed to assess whether agglutination or clumping is maintained after irradiation (Taylor & Drickamer, 1991). Therefore, the activity of the lectin was not inhibited using irradiation at relatively low doses. Nevertheless, the food allergen showed a significant loss of SHA (p < 0.05) above 1 kGy, due to a direct effect of radiation on its structural stability 1 h after exposure (Fig. 1). In the tertiary structure of proteins, there are four types of bonding interactions between chains, including hydrogen bonding, salt bridges, disulfide bonds, and non-polar hydrophobic interactions, which may be disrupted after irradiation at medium and high doses. Although there was a significant loss of HA, the lectin showed high resistance compared to other previously analysed proteins (Davis et al., 1997). To detect any insoluble aggregates that formed, the precipitate was run on an SDS–PAGE gel after centrifugation, and the supernatant was separated by RP-HPLC. The molecular weight pattern of SejaBL did not change after exposure to lower doses of radiation (0.02–0.075 kGy). The initial degradation of lectin (MW 52 kDa) was observed with doses of 0.2–6 kGy. Degradation of the main band was detected above 3 kGy, indicating that the aggregate formed was composed of fragmented polypeptides (Fig. 2a – arrows). The reverse phase chromatography analysis revealed a loss of the peak area with the structural fragmentation (Fig. 2b). Aggregation was analysed by light scattering (Fig. 2c). Puchala and Schuessler (1995) demonstrated that break points caused by protein radiation occur at fragile bonds in the polypeptide chain. Proteins can be converted to higher molecular weight aggregates due to the generation of inter-protein cross-linking reactions, hydrophobic and electrostatic interactions, as well as the formation of disulfide bonds (Oliveira, Hoz, Silva, Torriani, & Netto, 2006; Xu & Chance, 2005). Therefore, a variety of conditions can induce denaturation, with subsequent precipitation into insoluble amorphous aggregates or structured intermediates that seek alternatively sta-

2.7. Fluorescence spectroscopy The fluorescence emission intensity of the irradiated 0.2 mg/ml SejaBL solution in phosphate buffer at pH 7.0 was measured at 25 °C using a spectrofluorometer (JASCO FP-6300, Tokyo, Japan) in a rectangular quartz cuvette with a 1 cm path length. The excitation wavelengths were 295 and 280 nm; emission spectra were recorded in the range 305–450 nm, and band passes were 5 nm. Light scattering was measured at 90° for the aggregation assays; light scattering values at 320 nm were monitored (300–340 nm). The spectra displayed in the figures are the averages of three scans that were corrected for the solution signal by subtracting the solution spectrum. 2.8. Hydrophobic surface analysis The lectin hydrophobic surface was measured using the same conditions as employed for the intrinsic fluorescence experiment. Samples were transferred to a quartz cuvette and then mixed with 5 lM bis-ANS; fluorescence was measured in the JASCO spectrofluorometer. The fluorescence emission was obtained at 400–600 nm, with an excitation at 360 nm (Bhattacharyya, Mandal, Banerjee, & Roy, 2000). 2.9. Statistical analysis Statistical analysis was performed with the Student’s t test, using the GraphPrismÒ programme (GraphPad Software Inc., San Diego, CA, USA). The significance of the difference between the means was analysed at the level of p < 0.05. 3. Results and discussion Irradiation of a plant lectin in milligram quantities was used to elucidate how this physical method of food processing affects the stability of a food allergen. In order to observe this effect, SejaBL SHA was determined after irradiation. With doses of 0.02– 0.8 kGy, no significant change was observed. Remarkably, a dose of 0.1 kGy significantly increased SHA (p < 0.05) after 1 h and 24 h (Fig. 1). We consider intramolecular motions to be essential for the function of proteins because they are associated with transitions between substrates that represent local minima in the

110

Relative Integral SHA (%)

100

*

90

*

*

80

* *

70

*

60 50 40 30

*

20 10

*

0 C

0.02 0.05 0.075 0.1 0.2 0.4

0.6 0.8

1

3

6

12.5 25

35

Dose (kGy) Fig. 1. Effect of c-radiation on lectin activity. The percentage of remaining specific hemagglutination activity, %SHAREM, is represented at (N) 1 h, (s) 24 h and (h) 7 days after irradiation. Error in the determination of %SHAREM for the different doses was approximately ±1%, which is less than the size of the symbols. *Significant difference (p < 0.05) compared to non-irradiated lectin.

1292

A.F.M. Vaz et al. / Food Chemistry 124 (2011) 1289–1295

a

MW

P

1

2

3

4

5

6

7

8

9

10

205 116 97.4 84 66 55 45 36

29 24

20

14.2 6.5

b

c

Fluorescence intensity (A.U.)

Absorbance at 215 nm (mAbs)

900

25 kGy

750 600 450 300 150

Control

0 300

310 320 330 Wavelength (nm)

340

Time (min) Fig. 2. SDS–PAGE and chromatographic profile from irradiated SejaBL. (a) SDS–PAGE was performed in a discontinuous system with 10% separating and 5% stacking gels. (MW) Molecular weight; (P) non-irradiated; (1) 100 Gy; (2) 200 Gy; (3) 400 Gy; (4) 600 Gy; (5) 800 Gy; (6) 1 kGy; (7) 3 kGy; (8) 6 kGy; (9) 12.5 kGy and (10) 25 kGy. Arrows indicate the formation of degraded lectin after a high dose of gamma radiation. (b) Reverse phase chromatography in an HPLC system: ( ) control and irradiated lectins at ( ) 0.1 kGy, ( ) 3 kGy, and ( ) 35 kGy. (c) Light scattering for the aggregation assays; excitation (320 nm) and emission (300–340 nm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

ble but poorly defined conformations. From a practical point of view, there are three general applications and dose categories that

are referred to when foods are treated with ionising radiation (Table 1). To our surprise, the agglutination or clumping of cells

1293

A.F.M. Vaz et al. / Food Chemistry 124 (2011) 1289–1295

by lectin was not inhibited using low irradiation doses, which are usually effective against microorganisms and insects. Gamma irradiation can affect the atomic fluctuations of the CRD in lectins, and it is possible to estimate these changes by fluorescence spectroscopy. The intrinsic fluorescence emission increased (at 0.02–0.1 kGy) and decreased (at 0.2–35 kGy) without changing the kmax, at approximately 330 nm for hydrophobic residues

(Fig. 3a–e). Tryptophan residues that are exposed to water have a maximal fluorescence at wavelengths around 340–350 nm, whereas completely buried residues fluoresce at about 330 nm. From an unfolded state, the quantum yield may either increase or decrease during folding. Accordingly, a folded protein can have either a higher or a lower fluorescence than the unfolded form (Tsonev & Hirsh, 2000). Because they act indirectly, all aliphatic

Table 1. General application and dose categories that are referred to when foods are treated with ionising radiation. Dose categories

Doses (kGy)

Low-dose irradiation Medium-dose irradiation

Up to 6 1 kGy 1–10 kGy

Sprout inhibition; Reduction in numbers of spoilage microorganisms;

General application

High-dose irradiation

Above 10 kGy

Reduction in numbers of microorganisms to the point of sterility

Insect disinfestation; Reduction in numbers or elimination of non-sporeforming pathogens;

Parasite inactivation Reduction of the agglutination or clumping of cells by Food allergen (Lectin)a

Source: Kume et al. (2009). a Current study.

Fluorescence intensity (A.U.)

Relative integral intensity (%)

60 50 40 30

a 20

400

c

350

Control

300 250 200 150

35 kGy

100 50 0 305 335 365 395 425

10

Wavelength (nm)

0 C 0.02 0.05 0.07 0.1 Control 0.05 0.1

0.2 0.4

0.8 0.4 0.6 0.8 Dose (kGy)

1

33

6 12.5 25

12.5

35 35

50

350 300

d

0.1 kGy

Control

250 200 150 100

35 kGy

50 0

40 Fluorescence intensity (A.U.)

Relative integral intensity (%)

60

Fluorescence intensity (A.U.)

400

30

b 20 10 0 C

0.02 0.05 0.07 0.1 0.2 0.4

Control 0.05

0.1

0.4

0.6

0.8

0.8

Dose (kGy)

1

3

3

6 12.5

12.5

25

35

35

305 335 365 395 425 Wavelength (nm) 400 350 300

Control

e

250 200 150 100

35 kGy

50 0 305 335 365 395 425 Wavelength (nm)

Fig. 3. SejaBL intrinsic fluorescence. (a) Total intrinsic fluorescence. The relative intensity (%) of lectin at (N) 1 h, (s) 24 h and (h) 7 days after irradiation. (b) Tryptophan fluorescence; error in fluorescence determination from the relative peak area of integral intensities to different doses was approximately ± 0.5%, which is less than the size of the symbols. (c–e) Fluorescence of lectin at 1 h, 24 h and 7 days after irradiation, respectively. Lectin excitation (295 nm) and emission (305–450 nm); ( ) non-irradiated and lectin irradiated with (- - -) 0.02–0.075 kGy, (—) 0.02–6 kGy and (. . .) 12.5–35 kGy.

1294

A.F.M. Vaz et al. / Food Chemistry 124 (2011) 1289–1295

carbohydrates and some lectins, including the galectins and certain bacterial toxins, such as E. coli lytic toxin and non-lectin proteins (Rini, 1995). The bis-ANS fluorescence was weakly observed in the control non-irradiated SejaBL compared with the buffer; this indicates that the hydrophobic surface is exposed to the solvent (Vaz et al., 2010). After irradiation, bis-ANS fluorescence increased at all doses, with a maximum blue shift to 490 nm at doses above 0.8 kGy (Fig. 4a–e). Hydrophobic surfaces in proteins are readily attacked by hydroxyl radicals and, in this case, changes in the protein hydrophobicity are considered as the prevailing factor for structural collapse. Hydrogen abstraction from aromatic amino acids might promote greater exposure of the hydrophobic surfaces to the solvent, due to an increase in the surface apolarity, leading to greater binding of bis-ANS (causing a blue shift in the emission) via the formation of the misfolded state(s). ANS binds strongly to the misfolded state(s), and the emission intensity of the blue shift of ANS upon binding to a misfolded protein is more than twice that observed with a protein in its native form or a completely unfolded protein (Kathir, Kumar, Rajalingam, & Yu, 2005). An increased affinity of bis-ANS for the protein and a stronger fluorescence enhancement, as compared to ANS, have been described for several proteins (Pastukhov & Ropson, 2003; Rosen & Weber, 1969). Aggregation is primarily attributed to attractions between

amino acids are potential targets of modification (Garrison, 1987) by hydroxyl radicals generated after water radiolysis (Franzini, Sellak, Hakim, & Pasquier, 1993). Aromatic amino acid residues are particularly sensitive, and formylkynurenine from tryptophan, 3.4-dihydroxyphenylalanine (DOPA) from tyrosine, and O-tyrosine from phenylalanine have been recognised as the major oxidation products (Shi, Dong, Zhao, & Li, 2006). Partial aromatic amino acid substitution, caused by hydrogen abstraction, results in a decrease in the fluorescence emission. The fluorescence intensity is very informative in itself. The magnitude of the intensity, however, cannot serve as a probe for the folded state. Such oxidative damage distorts the hydrophobic surface and the hydrophobic core of proteins, favouring denaturation and aggregation. Thus, the quantum yield and wavelength of the maximum fluorescence emission were not changed. Shifts in the fluorescence spectrum were detected, and the intensity of fluorescence decreased, even though the solvent polarity surrounding the tryptophan residue remained unaffected. A survey of the literature showed that lectins bind to their ligands most commonly through hydrogen bonds (some mediated by water) and hydrophobic interactions with the CRD (Sharon & Lis, 2004). The hydrophobic face of sugar rings interacts with aromatic surfaces; this is a common feature of complexes between

Fluorescence intensity (A. U.)

60 50 40 30

a

20 10 0 C

0.02 0.05 0.07 0.1

Control

0.05

0.1

0.2

0.4

0.6

0.8

0.4 0.8 Dose (kGy)

1

3

3

6

12.5

12.5

25

35

35

520 500

Fluorescence intensity (A.U.)

Relative integral intensity (%)

70

c

205 185

35 kGy Control

165 145 125 105 85

3 kGy

65 45 25 400 205 185

Control

145 125 105 85

3 kGy

65 45 25

Fluorescence intensity (A.U.)

Wavelength (nm)

400

460 440 420 400

C 0.02 0.05 0.05 0.07 0.1 0.1 Control

0.2 0.4 0.4

0.6

0.8 0.8

Dose (kGy)

1

33

6

12.5 12.5

25

35 35

600

d

35 kGy

165

480

b

450 500 550 Wavelengt (nm)

450 500 550 Wavelenght (nrn)

600

205

e

185 165

35 kGy

Control

145 125 105 85 65

3 kGy

45 25 400

450 500 550 Wavelengt (nm)

600

Fig. 4. SejaBL fluorescence after treatment with bis-ANS: (a) bis-ANS fluorescence. The relative intensity (%) of lectin at (N) 1 h, (s) 24 h and (h) 7 days after irradiation. (b) Mass centre; error in the fluorescence determination from the relative peak area of the integrated intensities to different doses is approximately ± 1%, which is less than the size of the symbols. (c–e) Fluorescence of lectin at 1 h, 24 h and 7 days after irradiation, respectively. Lectin excitation (360 nm) and emission (400–600 nm); ( ) nonirradiated and lectin irradiated with (- - -) 0.02–0.075 kGy, (—) 0.02–6 kGy and (. . .) 12.5–35 kGy.

A.F.M. Vaz et al. / Food Chemistry 124 (2011) 1289–1295

interchain hydrophobic surfaces, which are transiently solvent-exposed in the folding intermediates (Safar, Roller, Gadjusek, & Gibbs, 1994). Gamma rays are penetrative, and in theory, they can hit the protein anywhere along the polypeptide chain, even after food processing and packaging. The finding of misfolded states in SejaBL sheds some light on the mechanisms responsible for protein radiolysis, which can reduce unwanted or intolerant immunological effects exerted by lectins. Currently, little is known about how processing may alter allergens, and hence there is a need to systematically investigate the relationship between food protein allergenicity and the effect of physical processing (irradiation) on protein modification, including protein denaturation and aggregation. In addition to potentially increasing the allergenicity of food proteins, such modifications may also reduce allergenicity. This paper reveals structural changes and a loss of activity of lectins after irradiation, which might reduce the allergenicity by compromising the function of the CRD. However, further experiments are required to investigate the allergenicity of irradiated lectins. In conclusion, changes in the structure and activity of an irradiated food allergen revealed a novel application of gamma radiation for food processing. We hypothesise that transitory interactions between hydrophobic surfaces that persist in misfolded states are responsible for the initial collapse of the polypeptide chain after irradiation. Thus, protein aggregations arise, principally driven by hydrophobic interactions, formed from the association of fragmented polypeptides and unfolding intermediates that seek alternatively stable but poorly defined conformations after high doses of radiation. Furthermore, the total activity of lectin was not inhibited using radiation at relatively low doses that are usually effective against microorganisms and insects. We believe that this unusual phenomenon observed for SejaBL may also be ubiquitously applicable to other irradiated lectins, such as cereal lectin, to reduce the incidence of food intolerance. Acknowledgements This research was financially supported by research grants and fellowships from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), as well as the Ministério da Ciência e Tecnologia (Brazilian) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We are grateful to the Departamento de Energia Nuclear from the Universidade Federal de Pernambuco (UFPE) for access to their facility and for assistance. References Bhattacharyya, A., Mandal, A. K., Banerjee, R., & Roy, S. (2000). Dynamics of compact denatured states of glutaminyl-tRNA synthetase probed by bis-ANS binding kinetics. Biophysical Chemistry, 87, 201–212. Charlier, M., Eon, S., Sèche, E., Bouffard, S., Culard, F., & Spotheim-Maurizot, M. (2002). Radiolysis of lac repressor by c-rays and heavy ions: A two-hit model for protein inactivation. Biophysical Journal, 82, 2373–2382. Correia, M. T. S., & Coelho, L. C. B. B. (1995). Purification of a glucose/mannose specific lectin, isoform 1, from seeds of Cratylia mollis Mart. (Camaratu bean). Applied Biochemistry and Biotechnology, 55, 261–273. Davis, M. D., Parniak, M. A., Kaufman, S., & Kempner, E. S. (1997). Structure–function relationships of phenylalanine hydroxylase revealed by radiation target analysis. Archives of Biochemistry and Biophysics, 94, 491–495. Farkas, J. (1998). Irradiation as a method for decontaminating food. International Journal of Food Microbiology, 44, 189–204.

1295

Farris, F. J., Weber, G., Chiang, C. C., & Paul, I. C. (1978). Preparation, crystalline structure, and spectral properties of the fluorescent probe 4,4-bis-1phenylamino-8-naph-thalenesulfonate. Journal of the American Chemical Society, 100, 4469–4475. Ferrand, M., Dianoux, A. J., Petry, W., & Zaccai, G. (1993). Thermal motions and function of bacteriorhodopsin in purple membranes: Effects of temperature and hydration studied by neutron scattering. Proceedings of the National Academy of Sciences, 90, 9668–9672. Franzini, E., Sellak, H., Hakim, J., & Pasquier, C. (1993). Oxidative damage to lysozyme by the hydroxyl radical: Comparative effects of scavengers. Biochimica et Biophysica Acta, 1203, 11–17. Frauenfelder, H., Sligar, S. G., & Wolynes, P. G. (1991). The energy landscapes and motions of proteins. Science, 254, 1598–1603. Garrison, W. M. (1987). Reaction mechanisms in the radiolysis of peptides, polypeptides, and proteins. Chemical Reviews, 87, 381–398. Gupta, A., & Sandhu, R. S. (1997). In vivo binding of mannose specific lectin from garlic to intestinal epithelium. Nutrition research, 17, 703–711. Horowitz, P., Prasad, V., & Luduena, R. F. (1984). Bis (1.8anilinonaphthalenesulfonate): A novel and potent inhibitor of microtubule assembly. Journal of Biological Chemistry, 259, 14647–14650. Kathir, K. M., Kumar, T. K. S., Rajalingam, D., & Yu, C. (2005). Time-dependent changes in the denatured state(s) influence the folding mechanism of an all bsheet protein. Journal of Biological Chemistry, 280, 29682–29688. Kempner, E. S. (2001). Effects of high-energy electrons and gamma rays directly on protein molecules. Journal of Pharmaceutical Sciences, 90, 1637–1646. Kume, T., Furuta, M., Todoriki, S., Uenoyama, N., & Kobayashi, Y. (2009). Status of food irradiation in the world. Radiation Physics and Chemistry, 78, 222–226. Laemmli, U. K. (1970). Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265. Madsen, C. (1997). Prevalence of food allergy/intolerance in Europe. Environmental Toxicology and Pharmacology, 4, 163–167. Oliveira, C. L. P., Hoz, L., Silva, J. C., Torriani, I. L., & Netto, F. M. (2006). Effects of gamma radiation on b-lactoglobulin: Oligomerization and aggregation. Biopolymers, 85, 284–294. Pastukhov, A. V., & Ropson, I. J. (2003). Fluorescent dyes as probes to study lipid-binding proteins. Proteins: Structure, Function, and Genetics, 53, 607– 615. Peumans, W. J., & Van Damme, E. J. M. (1996). Prevalence, biological activity and genetic manipulation of lectins in food. Trends in Food Science and Technology, 7, 132–138. Puchala, M., & Schuessler, H. (1995). Oxygen effect in the radiolysis of proteins. IV. Myoglobin. International Journal of Peptide and Protein Research, 46, 326–332. Riley, P. A. (1994). Free radicals in biology: Oxidative stress and the effects of ionizing radiation. International Journal of Radiation Biology, 65, 27–33. Rini, J. (1995). Lectin structure. Annual Review of Biophysics and Biomolecular Structure, 24, 551–577. Rosen, C. G., & Weber, G. (1969). Dimer formation from 1-anilino-8naphthalenesulfonate catalyzed by bovine serum albumin. A new fluorescent molecule with exceptional binding properties. Biochemistry, 8, 3915–3920. Safar, J., Roller, P. P., Gadjusek, D. C., & Gibbs, C. J. (1994). Scrapie amyloid (prion) protein has the conformational characteristics of an aggregated molten globule folding intermediate. Biochemistry, 33, 8375–8383. Sathe, S. K., Teuber, S. S., & Roux, K. H. (2005). Effects of food processing on the stability of food allergens. Biotechnology Advances, 23, 423–429. Sharon, N., & Lis, H. (2004). History of lectins: From hemagglutinins to biological recognition molecules. Glycobiology, 14, 53–62. Shi, W. Q., Dong, J. C., Zhao, Y. F., & Li, W. Y. (2006). Hydroxylation of 3-nitrotyrosine and its derivatives by gamma irradiation. Radiation Research, 166, 639–645. Taylor, M. E., & Drickamer, K. (1991). Carbohydrate-recognition domains as tools for rapid purification of recombinant eukaryotic proteins. Biochemical Journal, 274, 575–580. Tsonev, L. I., & Hirsh, A. G. (2000). Fluorescence ratio intrinsic basis states analysis: A novel approach to monitor and analyze protein unfolding by fluorescence. Journal of Biochemical and Biophysical Methods, 45, 1–21. Vaz, A. F. M., Costa, R. M. P. B., Melo, A. M. M. A., Oliva, M. L. V., Santana, L. A., SilvaLucca, R. A., et al. (2010). Biocontrol of Fusarium species by a novel lectin with low ecotoxicity isolated from Sebastiania jacobinensis. Food Chemistry, 119, 1507–1513. Xu, G., & Chance, M. R. (2005). Radiolytic modification of sulfur-containing amino acid residues in model peptides: Fundamental studies for protein footprinting. Analytical Chemistry, 77, 2437–2449.