Microchim Acta 158, 173180 (2007) DOI 10.

1007/s00604-006-0678-7 Printed in the Netherlands

Original Paper Evaluation of soybean seed protein extraction focusing on metalloprotein analysis
rcia F. Mesko2 , Diogo P. Moraes2 , Alessandra Sussulini1 , Jerusa S. Garcia1 , Ma 2 3 rez , and Marco A. Z. Arruda1; Erico M. M. Flores , Carlos A. Pe
1 2 3

Department of Analytical Chemistry, Institute of Chemistry, Universidade Estadual de Campinas (UNICAMP), PO Box 6154, 13084-971 Campinas, S~ ao Paulo, Brazil Department of Chemistry, Universidade Federal de Santa Maria (UFSM), 97105-900 Santa Maria, Rio Grande do Sul, Brazil ncrotron (LNLS), PO Box 6192, 13084-971 Campinas, S~ Laborat orio Nacional de Luz S ao Paulo, Brazil

Received May 16, 2006; accepted August 5, 2006; published online October 16, 2006 # Springer-Verlag 2006

Abstract. Two methods of protein extraction for soybean seeds were evaluated in terms of preservation of the metal ions bound to proteins after the extraction and separation procedures. The proteins were rstly separated according to their molar masses by polyacrylamide gel electrophoresis. Then, the protein bands were mapped by synchrotron radiation X-ray uorescence in order to establish which metal ions were present in each one. Finally, some mapped protein bands were decomposed by microwave-assisted combustion and Ca, Cu, K, Mg, Mn, and Zn were quantied by inductively coupled plasma mass spectrometry or inductively coupled plasma optical emission spectrometry. The extraction methods studied were Method A (based on the treatment of ground soybean seeds with hexane and their extraction with TrisHCl and  -mercaptoethanol) and Method B (based on the treatment of ground soybean seeds with petroleum ether and their extraction with TrisHCl, dithiothreitol, phenylmethanesulfonyl uoride, sodium dodecyl sulfate and potassium chloride). The best method was Method B, in which a 78% higher extraction efciency was obtained when compared to Method A. Additionally, the metal-protein in Author for correspondence. E-mail: zezzi@iqm.unicamp.br

teractions were more appropriately preserved when Method B was applied, where the most affected ions were those that are bound weakly to proteins, such as Ca, K, and Mg.
Key words: Soybean seed; protein extraction; metalloproteins; spectrometric techniques.

The diversity and complexity of samples as well as the different goals of an analysis generate the most challenging problems and, sometimes, the most creative solutions [1, 2]. These problems frequently push the development of new methods, strategies, equipment and interpretations, enabling interrelations between different knowledge areas. It is clear that the synergetic effect then produced is extremely salutary for the science involved in the sample preparation, as well commented by Pawliszyn: sample preparation is science, not art [3]. One of these challenges can be considered when focused biomolecules such as proteins and metalloproteins are analyzed. The complexity of proteins is extremely high and frequently they are instable, even under mild conditions [4]. Thus, gentle procedures are almost imperative for maintaining the integrity of the analytes. This fact can explain the great number of techniques, methods, strategies and

174

A. Sussulini et al.

reagents used for sample preparation when biomolecule determination=characterization is undertaken [5]. Another important point when metalloproteins analysis is considered is that the sample preparation step must be as effective as possible in order to allow the integrity not only of the protein but also of the metals bound to protein. In protein chemistry, the proteins to be analyzed must be extracted from the biological sample, freed from any substances which could interfere with the analytical technique and kept in solution during the whole separation process [6]. For extraction of proteins from biological samples, cell disruption must rst be made. In solid tissues, generally grinding [7] or mechanical homogenization [8] are used for disruption. The major interfering substances present in oleaginous seeds such as soybeans are lipids and they are commonly removed by a chemical delipidation process that is achieved by extraction of the biological material with organic solvents [6, 9]. The solubilization is usually carried out in a buffer containing surfactants, reducing agents and protease inhibitors, according to the sample to be analyzed [6]. Metalloprotein analyses are almost unexplored, specially for vegetal samples, although it is an important subject when dealing with metallomics, which consists in the identication of metal species present in a biological system as well as the elucidation of their biochemical and physiological functions [10]. Due to the complexity of proteins=metalloproteins and of the vegetal samples, sample preparation can be considered as an important and inestimable tool for promoting accurate results. However, most protein sample preparation protocols are utilized without any evaluation criteria for their efciencies. Additionally, to the best of our knowledge, these sample preparation protocols are considered only for protein analysis and not for metalloprotein analysis. Thus, this work aims to evaluate two different soybean seed protein extraction methods, whose differences are in the solvents used for the chemical delipidation of the sample (hexane and petroleum ether) and in the buffers that were employed in the aqueous extraction of the soybean seed proteins (one containing only TrisHCl buffer with  mercaptoethanol as reducing agent, called Method A, and another more complex method containing Tris HCl buffer, dithiothreitol as reducing agent, the protease inhibitor phenylmethanesulfonyl uoride, the surfactant sodium dodecyl sulfate and potassium chloride for adjusting the ionic strength of the medium,

called Method B). They were tested and evaluated in terms of preservation of metal-protein bonds after extraction and separation procedures. After using the Bradford method [11] to determine total protein concentrations, polyacrylamide gel electrophoresis (SDSPAGE) was applied to separate the proteins, synchrotron radiation X-ray uorescence (SR-XRF) was used to identify the metal ions bound to proteins, and inductively coupled plasma mass spectrometry and inductively coupled plasma atomic emission spectrometry (ICP-MS and ICP OES, respectively) were carried out to quantify the investigated elements. Soybean samples were taken as examples due to their total protein content (41% m=m) [12], because they present some metalloproteins already catalogued in the protein data bank [13] and, nally, due to their nutritional and economic aspects [12, 14]. Experimental
Protein extraction In this investigation, two different extraction methods were used to extract the proteins from soybean seeds. In both methods, the seeds were frozen in liquid nitrogen and ground into a ne powder using a mortar and a pestle. The rst method (called Method A) was performed according to Mujoo et al. [15]. In this case, 1 g of the soybean powder was defatted twice with hexane (J. T. Baker, Phillipsburg, USA, www.mallbaker.com). Then, the proteins were extracted with 25 mL of a solution containing 0.03 mol L1 TrisHCl (Merck, Darmstadt, Germany, www.merck.de) pH 8.0 and 0.01 mol L1  mercaptoethanol (J.T. Baker) for 1 h, with vortexing every 10 min. Samples were then centrifuged at room temperature for 20 min at 11000 g in a model Bio-Spin-R ultracentrifuge (BioAgency, S~ ao Paulo, Brazil, www.bioagency.com.br) and the supernatant containing the soybean proteins was collected. The second method (called Method B) was adapted from the protocol described by Bellato et al. [16]. In this case, 1 g of the soybean powder was defatted three times with petroleum ether, b.p. 3560  C (J. T. Baker) for 15 min each. Then, the proteins were extracted with 10 mL of a solution containing 50 mmol L1 TrisHCl pH 8.8, 1.5 mmol L1 KCl (Merck), 10 mmol L1 dithiothreitol (DTT) (Pierce, Rockford, USA, www.piercenet.com), 1.0 mmol L1 phenylmethanesulfonyl uoride (PMSF) (Sigma, St. Louis, USA, www. sigmaaldrich.com) and 0.1% (m=v) sodium dodecyl sulfate (SDS) (Synth, Diadema, Brazil, www.synth.com.br). The samples were mixed for 10 min in an ice bath and insoluble materials were removed by centrifugation at 4  C for 5 min at 5000 g.

Determination of total protein concentration Total protein concentrations in all samples were determined according to the Bradford method, employing bovine serum albumin (Sigma) as a standard [11], in order to estimate the protein concentration after each extraction. For this purpose, the samples were appropriately diluted using 1.5 mol L1 TrisHCl (pH 8.8). The measurements were done in triplicate, at 595 nm, using a Micronal B582 spectrophotometer (S~ ao Paulo, Brazil, www.micronal.com.br).

Evaluation of soybean seed protein extraction focusing on metalloprotein analysis Separation of proteins by SDS-PAGE The samples obtained using both protein extraction methods were submitted to SDS-PAGE separation in order to establish the extraction efciencies. The separation was carried out with a vertical slab gel apparatus using a 185 135 1 mm gel plate. The SDS-PAGE was done using a separation gel composed of 12.5% (m=v) acrylamide (BioAgency) at pH 8.8 and 3.5% (m=v) stacking gel at pH 6.8, prepared according to Laemmli [17]. The samples were diluted in a solution containing 0.05 mol L1 TrisHCl (pH 6.8), 13.6% (m=v) glycerol (J.T. Baker), 2.7% (m=v) SDS and 5.4% (v=v)  -mercaptoethanol. Then, the diluted samples and the protein marker (MBI Fermentas, Hanover, USA, www.fermentas.com) were heated at 100  C for 5 min. For the electrophoretic separation, 25 mL of the diluted samples were applied in different lanes of the gel. The same volume of protein marker was applied in a separate lane of the gel, in order to allow the estimation of the molar masses of the separated proteins. The protein marker contains the proteins -galactosidade (116.0 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase (35.0 kDa), restriction endonuclease Bsp981 (25.0 kDa),  -lactoglobulin (18.4 kDa) and lysozyme (14.4 kDa). Electrophoresis was performed at 200 V and 30 mA for 9 h. As soon as the electrophoretic run was nished, the gel was stained with 1% (m=v) Coomassie brilliant blue (CBB) G-250 for 1 h. The excess of CBB G-250 was removed using a destaining solution, made of deionized water, methanol (J.T. Baker) and acetic acid (J.T. Baker) in a 6:3:1 (v=v) proportion, respectively. The gel was scanned and its image was analyzed by GelPro Analyzer version 3.1 software (Media Cybernetics, Maryland, USA, www.mediacy.com) for estimating protein molar masses.

175

quartz vessels. After placing the holders inside the quartz vessels, the system was closed and vessels were pressurized with oxygen (15 bar for 2 min). Then, the rotor with the vessels was inserted into the microwave cavity (Multiwave 3000, Anton Paar, Graz, Austria, www.anton-paar.com) and the program for microwave radiation started. The microwave energy program employed for the combustion procedure was as follows: (1) 5 min at 1400 W (combustion followed by a reux step) and (2) 20 min for cooling. The resulting solutions were diluted to 12 mL with deionized water. Finally, Ca, Cu, K, Mg, Mn and Zn were quantied using ICP-MS (Perkin-Elmer ELAN DRC II Axial Field Technology, Norwalk, USA, www.perkinelmer.com) and ICP OES (Perkin-Elmer Optima 4300 DV). The calibration curves ranged from 5 to 80 mg L1 for Cu, Mg, Mn, and Zn, which were determined by ICP-MS. Additionally, calibration curves for Ca and K (from 100 to 800 mg L1 ) were performed for ICP OES determinations.

Results and discussion Evaluation of protein extraction methods and separation by SDS-PAGE The initial evaluation of soybean seed protein extraction methods was made by comparison of the total protein concentrations determined by the Bradford method. For Method A, a protein concentration of 46 3 mg g1 (mg of protein per g of sample) was found and for Method B, the concentration was 209 41 mg g1 . The difference between the concentrations obtained was 78%. Considering the losses during the extraction processes, protein contents of 8 and 31% were found for Methods A and B, respectively. This last one is a value closer to that presented in the literature (41% of protein in terms of dry mass) [12]. According to these results, it can be noted that the extraction based on Method B, whose buffer contains the protease inhibitor PMSF and the surfactant SDS, and employs DTT as the reducing agent instead of  -mercaptoethanol, is much more efcient than the extraction performed by Method A, in terms of quantity of protein extracted. This can be explained due to the addition of PMSF that inhibits the action of the serine proteases, avoiding proteolysis (degradation) of the proteins with higher molar masses [21]. PMSF reacts with the activated serine of the catalytic center of serine proteases and prevents it from playing its catalytic role, so that irreversible inhibition of serine protease is obtained [6]. Another important fact is the addition of a surfactant to the buffer. It promotes the disruption of membranes, the solubilization of lipids, the delipidation and solubilization of proteins bound to the membranes or vesicles of the biological system [21], achieving the removal of the lipids (which interfere in the

Mapping of metal ions bound to proteins by SR-XRF The experiments using SR-XRF were carried out at the X-ray uorescence beam line of the Brazilian Synchrotron Light Source (LNLS) in Campinas, S~ ao Paulo (Brazil) [18]. A computer-controlled set of slits was used to collimate the white beam in order to deliver a 200 200 mm microbeam to the experimental station. An aluminum lter was placed in front of the microbeam, before the sample in order to reduce the intensity of the high-energy components of the spectrum. A HPGe energy dispersive detector was used to collect the uorescence as well as the scattered radiation coming from the samples. Before irradiation the protein bands were cut out from the gel, dried in an oven at 40  C to constant mass and then xed with sticky tape on the sample holder. The bands were irradiated for 100 s in a central point. This procedure was carried out in triplicate for each sample. The obtained spectra were processed with AXIL software [19] and were normalized to the incident intensity in order to correct for the variation of the incident photon ux on the sample during the collecting time. The analytical blank for SR-XRF analysis was the ovalbumin protein (45.0 kDa) and it was chosen after preliminary tests with the protein marker.

Quantication of metal ions bound to proteins using ICP-MS or ICP OES The same bands used for mapping the metal ions were also used for their quantication. Thus, after mapping the metal ions, the bands containing these ions were decomposed by microwave-assisted sample combustion in closed vessels, as proposed by Flores et al. [20]. For that, the samples (13 mg) were put on a quartz holder containing lter paper impregnated with 35 mL of a 6 mol L1 NH4NO3 solution, which acts as combustion igniter. After that, 6 mL of a 4 mol L1 HNO3 solution (absorber solution) were added to the

176

A. Sussulini et al.

solubilization of proteins) from the medium. As 21% (in terms of dry mass) of a soybean seed is oil content [11], it is necessary to carry out a chemical delipidation on the sample prior to the resolubilization of proteins in the presence of surfactants. This was accomplished by employing organic solvents. In Method A, the solvent employed was hexane (a shortchain hydrocarbon) and in Method B, the solvent employed was petroleum ether (a mixture of liquid hydrocarbons). Both solvents are nonpolar; however, as petroleum ether has a variety of hydrocarbons of different chain lengths in its composition, it has more possibilities of interacting with the different lipids present in the sample than does hexane. Therefore, the delipidation is improved when petroleum ether is used, allowing a more efcient actuation of the surfactant on the protein extraction. The use of DTT as reducing agent instead of  mercaptoethanol is preferable because DTT breaks the disulde bonds of the proteins more efciently and as a consequence it can be employed at lower concentrations. Breaking the disulde bonds of proteins occurs by a process of equilibrium dislocation where the reducing agent (in excess in the system) is oxidized while the proteins are reduced to the thiol form [6]. Method B also has two additional favorable factors for the extraction of proteins: rst, the sample is maintained in an ice bath during the extraction with the buffer, which prevents proteins denaturation [22] and, second, the buffer contains KCl. It improves protein solubility and maintains the ionic strength of the medium constant, minimizing counterion effects [23]. After establishing the total protein concentration, protein separation was carried out by SDS-PAGE, which was performed in order to compare the extraction methods in a qualitative way. The molar mass prole from soybean seed proteins is shown in Fig. 1. The protein band molar masses ranged from approximately 12 to 107 kDa. Comparing lanes (c) and (d), where the same dilution factor (1:5) of the protein extract was used, it is possible to note that Method B showed higher protein band intensities than Method A, as expected due to the higher concentration of proteins available in this case. A comparison between lanes (b) and (f) in Fig. 1, which have similar concentrations of proteins, shows that the proteins extracted by the different methods are expressed in the same way, in terms of molar mass. Then, it is possible to state, at this point, that the main difference between

Fig. 1. SDS-PAGE electrophoresis of soybean seed samples. Lanes: (a) protein marker; extraction method A (b) 10 and (c) 5 mg of protein; extraction Method B (d ) 41, (e) 23 and ( f ) 10 mg of protein

the methods studied is the extraction efciency. Nevertheless, this statement is not so important when dealing with metallomics studies because the main focus is not the quantity of proteins obtained from the extraction, but also the quantity of metal ions that are bound to proteins preserved during the extraction process. Hence, the identication and quantication of the metal ions bound to the separated proteins was performed. Mapping of metal ions bound to proteins by SR-XRF In order to verify which metal ions are bound to the proteins separated by SDS-PAGE, SR-XRF spectra were obtained. During the experiments, a signicant background contribution appears in the SR-XRF spectra due to an increase of the elastically (Rayleigh) as well as inelastically (Compton) scattering of the incoming photon beam in the gel matrix, mainly composed by low-Z elements [2426]. The analytical blank was taken from the ovalbumin protein band (45.0 kDa), not only because it does not contain metal ions in its structure, as veried in the protein data bank [13], but also because it has passed through the same processes of staining and destaining that the other protein bands in the gel underwent. The

Evaluation of soybean seed protein extraction focusing on metalloprotein analysis

177

Fig. 2. Electropherogram of 10 mg of soybean seed proteins, extracted by Method B, with bands marked (115) for metal ion identication and quantication

extraction methods were not taken into consideration for analysis of blank because it is a specic protein band from the molar mass marker, applied in the electrophoresis gel in the same way for both cases. Thus, the amounts of metal ions found in the ovalbumin band were subtracted from the values obtained for the metal ions present in the sample proteins. Figure 2 shows an electropherogram where the protein bands analyzed are marked. The metal ions detected in the protein bands by SRXRF were Ca, Co, Cr, Cu, Fe, K, Mn, Ni and Zn. Because the SR-XRF beam line has an Al lter, elements such as Na and Mg were not detected by this technique. Figure 3 shows spectra in which the extraction methods are compared for the same molar mass protein band. Comparing Fig. 3(a) and (b), it is possible to observe that when the protein is extracted by Method B (Fig. 3b), the number of counts of the metal ions is higher and more species are detected than when the protein is extracted by Method A (Fig. 3a).

Fig. 3. SR-XRF spectra, with background correction, for 31.5 kDa protein band (number 10 in Fig. 2): (a) extracted by Method A and (b) Method B

This tendency was observed with the other bands evaluated. Such observations can be explained as the metal ions bind to proteins in different ways, and those which have a non-specic binding are easily lost during the extraction or during the electrophoretic procedure [24]. Although the electrophorectic separation system is quite denaturant, which can cause some loss of metal ions during the process, several authors [24, 2731] have already used this same strategy. These losses can not be signicant, since only a small part of these metal ions would be strongly bound to the proteins. Quantication of metal ions bound to proteins by ICP OES or ICP-MS In the following analyses, some metal ions that act as macronutrients (Ca, K, Mg) and as micronutrients

178
Table 1. Metal ion concentrations (mg g1 ); n 3 Band= extraction method 3=A 3=B 5=A 5=B 6=A 6=B 7=A 7=B 8=A 8=B 10=A 10=B 12=A 12=B Metal ion Ca(II)a 334 72 356 80 1915 37 1421 77 1960 170 Cu(II)b 5.4 0.9 10 1 8.4 0.9 13 2 27 5 4.5 0.7 Ka 484 58 692 96 676 90 626 81 595 84 521 30 498 35 638 72 712 95 926 62 786 89 Mg(II)b 314 56 393 35 534 44 Mn(II)b 38 7 41 7 39 6 33 3 40 8 49 9 60 3 63 3 60 2

A. Sussulini et al.

Zn(II)b 68 2 57 5 70 9 87 9

Detected but below LOQ; adetermined by ICP OES; bdetermined by ICP-MS.

(Cu, Mn, Zn) of plants [32] were quantied. Ca and K were quantied by ICP OES and Cu, Mg, Mn and Zn were quantied by ICP-MS. The selection of the protein bands to have their metal ions quantied was made based on the results obtained by SR-XRF and on the protein data bank [13]. Preferentially, bands were chosen that present a great number of proteins containing metal ions in their structure. The results related to metal ion concentrations are shown in Table 1. The bands 3, 58, 10 and 12 correspond to those in Fig. 2. The analytical blank for quantitative analyses was the same as used in the SR-XRF experiments (the ovalbumin protein band). Calcium, potassium and magnesium showed the highest concentration levels among the metal ions evalu-

ated. This fact demonstrates the importance of these elements in metabolic processes. Potassium and magnesium are activators (or cofactors) of several different enzymes [33]. The inuence of the protein extraction method on metal ion binding preservation was also investigated. In general, according to the results shown in Table 1, it is possible to note that the proteins extracted by Method B presents a higher (or the same) concentration of metal species when compared to those extracted by Method A. The results from bands 7 and 8 exemplify such behavior. Ca, Mn, Mg and Zn were the metal ions that showed a concentration signicantly higher when the proteins were extracted by Method B.

Table 2. Figures of merits of comparable methods for determination of metalloproteins Sample Human liver cytosol Extraction method buffer containing HEPES (pH 7.4) and sucrose; glass bead homogenization buffer containing SDS and derivatization with iodoacetic acid buffer containing TrisHCl (pH 6.8), SDS, glycerol and -mercaptoethanol; grinding homogenization Method A or B Metal ions analyzed (technique of determination) Cu, Fe and Zn (SR-XRF) Results only qualitative results. Detection of six Zn-, four Fe- and one Cu-containing protein bands in the sample LOD: 50 ng mL1 per protein band LOQ (mg L1 ): Ca 48.1, Cu 6.02, Fe 12.3, K 40.0 and Zn 4.64 (SR-TXRF); Ca 193 and Mg 3.32 (FAAS); Na 397 (FAES) LOQ (mg g1 ): Ca 305 and K 204 (ICP OES); Cu 3.8, Mg 302, Mn 33 and Zn 55 (ICP-MS) Ref. [24]

Yeast

Se (ETV-ICP-MS)

[29]

Embriogenic callus

Ca, Cu, Fe, K and Zn (SR-TXRF); Ca and Mg (FAAS); Na (FAES) Ca and K (ICP OES); Cu, Mg, Mn and Zn (ICP-MS)

[31]

Soybean seeds

present work

Evaluation of soybean seed protein extraction focusing on metalloprotein analysis

179

Usually, monovalent ions such as K are weakly bound to the protein structure, owing to the fact that only van der Waals forces are involved [7]. For this reason, the metal-protein interactions can be easily broken during sample handling and no conclusive result can be obtained for such metals. On the other hand, metal ions such as Ca and Mg interact moderately with proteins, although these interactions can also be lost, depending on the extraction procedure employed. Furthermore, transition metal ions such as Cu, Mn and Zn have the strongest coordination with proteins, through electrostatic forces [7, 33]. Due to this fact, in the majority of the protein bands evaluated, signicant differences related to copper and manganese levels were not observed. It is important to emphasize that the quantitative data agree with the results obtained by SR-XRF, which allows conrmation that a better preservation of the binding of these species with proteins occurs when they are extracted by Method B. This method, in addiction to extracting and solubilizing a larger quantity of proteins, is more appropriate to preserve metal-protein homeostasis. The features of the proposed method in comparison to others used for metalloproteins analysis in different samples are summarized in Table 2. In all cases shown, the proteins were separated by SDS-PAGE. Conclusions This work pointed out the necessity of a careful evaluation of sample preparation procedures, due to their intrinsic differences, when protein or metalloprotein analysis is desired. The extraction medium is decisive to preserve each metal species in the protein structure. In this way, Method B (whose buffer contained Tris HCl, KCl, DTT, PMSF and SDS) presented the best performance, not only for total protein extraction (78% higher when compared to Method A) but also for metal-protein binding preservation, corroborated by the identication and quantication of the metal species bound to proteins. This is particularly important when metallomic studies are concerned.
Acknowledgments. The authors would like to thank the Fundac ~ ao de Amparo  a Pesquisa do Estado de S~ ao Paulo and the Conselho co e Tecnol Nacional de Desenvolvimento Cient ogico for nancial support (grant numbers 05=54892-3 and 475474=2004-0, respectively) and for fellowships to A.S. (grant number 04=11960-6) and M.A.Z.A. This work has been supported by the Brazilian Synchrotron Light Source (LNLS) under proposal D09B-XRF-4206=05. The authors also thank Prof. Carol H. Collins for language assistance.

References
[1] Arruda M A Z (ed) (2006) Trends in sample preparation. Nova Science, New York (in press) rcel M (2006) Analytical chemistry in [2] Simonet B M, Valca modern society: what can we expect. Microchim Acta 153: 1 [3] Pawliszyn J (2005) Personal communication, during the 7th International Symposium on Advances in Extraction Technologies, Campinas [4] Voet D, Voet J G (2004) Biochemistry. John Wiley & Sons, London, p 127 [5] Garcia J S, Magalh~ aes C S, Arruda M A Z (2006) Trends in metal-binding and metalloprotein analysis. Talanta 69: 1 [6] Rabilloud T (1996) Solubilization of proteins for electrophoretic analyses. Electrophoresis 17: 813 [7] Posch A, van den Berg B M, Burg H C J, G org A (1995) Genetic variability of carrot seed proteins analyzed by oneand two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 16: 1312 [8] Geigenheimer P (1990) Preparation of extracts from plants. Methods Enzymol 182: 174 [9] van Renswoude J, Kempf C (1984) Purication of integral membrane proteins. Methods Enzymol 104: 329 [10] Haraguchi H (2004) Metallomics as integrated biometal science. J Anal At Spectrom 19: 5 [11] Bradford M M (1976) Rapid and sensitive method for quantitation of microgram quantities of proteins utilizing principle of protein-dye binding. Anal Biochem 72: 48 [12] Yaklich R W (2001)  -conglycinin and glycinin in highprotein soybean seeds. J Agric Food Chem 49: 729 [13] ExPASy Proteomics Server (2006) http:==ca.expasy.org=, accessed on January 13th [14] Achouri A, Boye J I, Belanger D (2005) Soybean isoavones: efcacy of extraction conditions and effect of food type on extractability. Food Res Inter 38: 1199 [15] Mujoo R, Trinh D T, Ng P K W (2003) Characterization of storage proteins in different soybean varieties and their relationship to tofu yield and texture. Food Chem 82: 265 [16] Bellato C M, Garcia A K M, Mestrinelli F, Tsai S M, Machado M A, Meinhardt L W (2004) The induction of differentially expressed proteins of Xyllela fastidiosa with citrus extract. Braz J Microbiol 35: 235 [17] Laemmli U K (1970) Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227: 680 rez C A, Radtke M, Sa nchez H J, Tolentino H, [18] Pe Neuenshwander R T, Barg W, Rubio M, Bueno M I S, Raimundo I M, Rohwedder J J R (1999) Synchrotron radiation x-ray uorescence at the LNLS: beamline instrumentation and experiments. X-Ray Spectrom 28: 320 [19] Vekemans B, Janssens K, Vincze L, Adams F, van Espen P (1994) Analysis of X-ray spectra by iterative least squares (AXIL): new developments. X-Ray Spectrom 23: 278 [20] Flores E M M, Barin J S, Paniz J N G, Medeiros J A, Knapp G (2004) Microwave-assisted sample combustion: a technique for sample preparation in trace element determination. Anal Chem 76: 3525 [21] Shaw M M, Riederer B M (2003) Sample preparation for twodimensional gel electrophoresis. Proteomics 3: 1408 [22] G org A, Weiss W, Dunn M J (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4: 3665 [23] Melvin M (1987) Electrophoresis. John Wiley & Sons, London, p 51 [24] Gao Y, Chen C, Zhang P, Chai Z, He W, Huang Y (2003) Detection of metalloproteins in human liver cytosol by syn-

180

Evaluation of soybean seed protein extraction focusing on metalloprotein analysis chrotron radiation X-ray uorescence after sodium dodecyl sulfate polyacrylamide gel electrophoresis. Anal Chim Acta 485: 131 van Gysel M, Lemberge P, van Espen P (2003) Description of Compton peaks in energy-dispersive X-ray uorescence spectra. X-Ray Spectrom 32: 139 Vincze L, Vekemans B, Janssens K, Adams F (1999) Modeling of photon scattering at high X-ray energies: experiment versus simulation. J Anal Atom Spectrom 14: 529 cs K (1987) Sz okefalvi-Nagy Z, Demeter I, Bagyinka C, Kova PIXE analysis of proteins separated by polyacrylamide gel electrophoresis. Nucl Instrum Methods Phys Res, Sect B 22: 156 cs K, Sz okefalvi-Nagy Z, Demeter I, Bagyinka C, Kova Quynh L H (1990) Location and quantication of metal ions in enzymes combining polyacrylamide gel electrophoresis and particle-induced X-ray emission. Biol Trace Elem Res 26: 93 ry C C, Chassaigne H, Verbeeck L, Cornelis R, Vanhaecke [29] Che F, Moens L (2002) Detection and quantication of selenium in proteins by means of gel electrophoresis and electrothermal vaporization ICP-MS. J Anal At Spectrom 17: 576 [30] Chen C, Zhao J, Zhang P, Chai Z (2002) Speciation and subcellular location of Se-containing proteins in human liver studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and hydride generation-atomic uorescence-spectrometric detection. Anal Bioanal Chem 372: 426 guez A P M, Pe rez C A, Arruda [31] Verbi F M, Arruda S C C, Rodr M A Z (2005) Metal-binding proteins scanning and determination by combining gel electrophoresis, synchrotron radiation X-ray uorescence and atomic spectrometry. J Biochem Biophys Methods 62: 97 [32] Fox T C, Guerinot M L (1998) Molecular biology of cation transport in plants. Annu Rev Plant Physiol 49: 669 [33] Williams R J P, Silva J J R F (2000) The distributions of elements in cells. Coord Chem Rev 200202: 247

[25]

[26]

[27]

[28]