evaluation of soybean seed protein extraction focusing on metalloprotein analysis

Microchim Acta 158, 173–180 (2007)

DOI 10.1007/s00604-006-0678-7

Printed in the Netherlands

Original Paper

Evaluation of soybean seed protein extraction focusingon metalloprotein analysis

Alessandra Sussulini1, Jerusa S. Garcia1, Marcia F. Mesko2, Diogo P. Moraes2,

Erico M. M. Flores2, Carlos A. Perez3, and Marco A. Z. Arruda1;�

1 Department of Analytical Chemistry, Institute of Chemistry, Universidade Estadual de Campinas (UNICAMP),

PO Box 6154, 13084-971 Campinas, S~aao Paulo, Brazil2 Department of Chemistry, Universidade Federal de Santa Maria (UFSM), 97105-900 Santa Maria, Rio Grande do Sul, Brazil3 Laborat�oorio Nacional de Luz Sıncrotron (LNLS), PO Box 6192, 13084-971 Campinas, S~aao 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 soy-

bean seeds were evaluated in terms of preservation of

the metal ions bound to proteins after the extraction and

separation procedures. The proteins were firstly sepa-

rated according to their molar masses by polyacryl-

amide gel electrophoresis. Then, the protein bands were

mapped by synchrotron radiation X-ray fluorescence in

order to establish which metal ions were present in each

one. Finally, some mapped protein bands were decom-

posed by microwave-assisted combustion and Ca, Cu,

K, Mg, Mn, and Zn were quantified by inductively

coupled plasma mass spectrometry or inductively cou-

pled plasma optical emission spectrometry. The extrac-

tion methods studied were Method A (based on the

treatment of ground soybean seeds with hexane and

their extraction with Tris–HCl and �-mercaptoethanol)

and Method B (based on the treatment of ground soy-

bean seeds with petroleum ether and their extraction

with Tris–HCl, dithiothreitol, phenylmethanesulfonyl

fluoride, sodium dodecyl sulfate and potassium chlo-

ride). The best method was Method B, in which a 78%

higher extraction efficiency was obtained when com-

pared to Method A. Additionally, the metal-protein in-

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 chal-

lenging 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 syner-

getic effect then produced is extremely salutary for

the science involved in the sample preparation, as well

commented by Pawliszyn: ‘‘sample preparation is sci-

ence, not art’’ [3]. One of these challenges can be con-

sidered 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� Author for correspondence. E-mail: [email protected]

reagents used for sample preparation when biomole-

cule determination=characterization is undertaken [5].

Another important point when metalloproteins analy-

sis 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 pro-

teins from biological samples, cell disruption must

first be made. In solid tissues, generally grinding [7]

or mechanical homogenization [8] are used for disrup-

tion. The major interfering substances present in olea-

ginous seeds such as soybeans are lipids and they are

commonly removed by a chemical delipidation pro-

cess that is achieved by extraction of the biological

material with organic solvents [6, 9]. The solubili-

zation 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, spe-

cially for vegetal samples, although it is an important

subject when dealing with metallomics, which con-

sists in the identification 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 consid-

ered as an important and inestimable tool for promot-

ing accurate results. However, most protein sample

preparation protocols are utilized without any evalua-

tion criteria for their efficiencies. Additionally, to the

best of our knowledge, these sample preparation pro-

tocols 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 em-

ployed in the aqueous extraction of the soybean seed

proteins (one containing only Tris–HCl buffer with �-

mercaptoethanol as reducing agent, called Method A,

and another more complex method containing Tris–

HCl buffer, dithiothreitol as reducing agent, the pro-

tease inhibitor phenylmethanesulfonyl fluoride, the

surfactant sodium dodecyl sulfate and potassium chlo-

ride 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 con-

centrations, polyacrylamide gel electrophoresis (SDS-

PAGE) was applied to separate the proteins, synchrotron

radiation X-ray fluorescence (SR-XRF) was used to

identify the metal ions bound to proteins, and induc-

tively coupled plasma mass spectrometry and induc-

tively 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, finally, 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 fine powder using a

mortar and a pestle. The first method (called Method A) was per-

formed according to Mujoo et al. [15]. In this case, 1 g of the soy-

bean powder was defatted twicewith hexane (J. T. Baker, Phillipsburg,

USA, www.mallbaker.com). Then, the proteins were extracted with

25 mL of a solution containing 0.03 mol L�1 Tris–HCl (Merck,

Darmstadt, Germany, www.merck.de) pH 8.0 and 0.01 mol L�1 �-

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~aao

Paulo, Brazil, www.bioagency.com.br) and the supernatant con-

taining the soybean proteins was collected. The second meth-

od (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. 35–60 �C

(J. T. Baker) for 15 min each. Then, the proteins were extracted

with 10 mL of a solution containing 50 mmol L�1 Tris–HCl pH

8.8, 1.5 mmol L�1 KCl (Merck), 10 mmol L�1 dithiothreitol (DTT)

(Pierce, Rockford, USA, www.piercenet.com), 1.0 mmol L�1 phenyl-

methanesulfonyl fluoride (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 5000g.

Determination of total protein concentration

Total protein concentrations in all samples were determined accord-

ing to the Bradford method, employing bovine serum albumin

(Sigma) as a standard [11], in order to estimate the protein concen-

tration after each extraction. For this purpose, the samples were

appropriately diluted using 1.5 mol L�1 Tris–HCl (pH 8.8). The

measurements were done in triplicate, at 595 nm, using a Micronal

B582 spectrophotometer (S~aao Paulo, Brazil, www.micronal.com.br).

174 A. Sussulini et al.

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 extrac-

tion efficiencies. 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) acryl-

amide (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 L�1 Tris–HCl (pH 6.8), 13.6% (m=v)

glycerol (J.T. Baker), 2.7% (m=v) SDS and 5.4% (v=v) �-mercap-

toethanol. 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, 25mL 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 sep-

arated proteins. The protein marker contains the proteins �-galacto-

sidade (116.0 kDa), bovine serum albumin (66.2 kDa), ovalbumin

(45.0 kDa), lactate dehydrogenase (35.0 kDa), restriction endonu-

clease 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 finished, 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 Analyz-

er version 3.1 software (Media Cybernetics, Maryland, USA,

www.mediacy.com) for estimating protein molar masses.

Mapping of metal ions bound to proteins by SR-XRF

The experiments using SR-XRF were carried out at the X-ray fluores-

cence beam line of the Brazilian Synchrotron Light Source (LNLS)

in Campinas, S~aao Paulo (Brazil) [18]. A computer-controlled set of

slits was used to collimate the white beam in order to deliver a

200�200mm microbeam to the experimental station. An aluminum

filter 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 fluorescence 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 fixed 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 soft-

ware [19] and were normalized to the incident intensity in order to

correct for the variation of the incident photon flux on the sample

during the collecting time. The analytical blank for SR-XRF analy-

sis was the ovalbumin protein (45.0 kDa) and it was chosen after

preliminary tests with the protein marker.

Quantification 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 quantification. Thus, after mapping the metal ions, the bands

containing these ions were decomposed by microwave-assisted sam-

ple combustion in closed vessels, as proposed by Flores et al. [20].

For that, the samples (1–3 mg) were put on a quartz holder contain-

ing filter paper impregnated with 35mL of a 6 mol L�1 NH4NO3

solution, which acts as combustion igniter. After that, 6 mL of a

4 mol L�1 HNO3 solution (absorber solution) were added to the

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 combus-

tion procedure was as follows: (1) 5 min at 1400 W (combustion

followed by a reflux step) and (2) 20 min for cooling. The result-

ing solutions were diluted to 12 mL with deionized water. Final-

ly, Ca, Cu, K, Mg, Mn and Zn were quantified 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 80mg L�1 for

Cu, Mg, Mn, and Zn, which were determined by ICP-MS. Addi-

tionally, calibration curves for Ca and K (from 100 to 800mg L�1)

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 extrac-

tion 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 g�1 (mg of protein per g of sample) was

found and for Method B, the concentration was 209�41 mg g�1. 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 lit-

erature (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 efficient than the

extraction performed by Method A, in terms of quan-

tity 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 surfac-

tant to the buffer. It promotes the disruption of mem-

branes, the solubilization of lipids, the delipidation

and solubilization of proteins bound to the mem-

branes or vesicles of the biological system [21], achiev-

ing the removal of the lipids (which interfere in the

Evaluation of soybean seed protein extraction focusing on metalloprotein analysis 175

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 resolubiliza-

tion of proteins in the presence of surfactants. This

was accomplished by employing organic solvents. In

Method A, the solvent employed was hexane (a short-

chain 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 efficient actuation of the sur-

factant on the protein extraction.

The use of DTT as reducing agent instead of �-

mercaptoethanol is preferable because DTT breaks

the disulfide bonds of the proteins more efficiently

and as a consequence it can be employed at lower

concentrations. Breaking the disulfide bonds of pro-

teins 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: first, the sample is main-

tained 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 medi-

um 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 extrac-

tion methods in a qualitative way. The molar mass

profile from soybean seed proteins is shown in Fig. 1.

The protein band molar masses ranged from approxi-

mately 12 to 107 kDa. Comparing lanes (c) and (d),

where the same dilution factor (1:5) of the protein ex-

tract 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 pro-

teins available in this case. A comparison between

lanes (b) and (f) in Fig. 1, which have similar concen-

trations 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

the methods studied is the extraction efficiency. Nev-

ertheless, this statement is not so important when

dealing with metallomics studies because the main fo-

cus 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 pro-

cess. Hence, the identification and quantification 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 significant

background contribution appears in the SR-XRF spec-

tra due to an increase of the elastically (Rayleigh) as

well as inelastically (Compton) scattering of the in-

coming photon beam in the gel matrix, mainly com-

posed by low-Z elements [24–26].

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 verified 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

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) 10mg

of protein


extraction methods were not taken into consideration

for analysis of blank because it is a specific protein

band from the molar mass marker, applied in the elec-

trophoresis 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 SR-

XRF were Ca, Co, Cr, Cu, Fe, K, Mn, Ni and Zn.

Because the SR-XRF beam line has an Al filter, ele-

ments such as Na and Mg were not detected by this

technique. Figure 3 shows spectra in which the extrac-

tion methods are compared for the same molar mass

protein band. Comparing Fig. 3(a) and (b), it is pos-

sible 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).

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-specific binding are easily lost

during the extraction or during the electrophoretic

procedure [24]. Although the electrophorectic separa-

tion system is quite denaturant, which can cause some

loss of metal ions during the process, several authors

[24, 27–31] have already used this same strategy.

These losses can not be significant, since only a small

part of these metal ions would be strongly bound to

the proteins.

Quantification 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

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

Fig. 2. Electropherogram of 10mg of soybean seed proteins, ex-

tracted by Method B, with bands marked (1–15) for metal ion

identification and quantification


(Cu, Mn, Zn) of plants [32] were quantified. Ca and K

were quantified by ICP OES and Cu, Mg, Mn and Zn

were quantified by ICP-MS. The selection of the pro-

tein bands to have their metal ions quantified 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, 5–8, 10 and 12 cor-

respond 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 high-

est concentration levels among the metal ions evalu-

ated. This fact demonstrates the importance of these

elements in metabolic processes. Potassium and mag-

nesium are activators (or cofactors) of several differ-

ent enzymes [33].

The influence 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) concentra-

tion of metal species when compared to those ex-

tracted 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 signifi-

cantly higher when the proteins were extracted by

Method B.

Table 1. Metal ion concentrations (mg g�1); n¼ 3

Band=extraction

Metal ion

method Ca(II)a Cu(II)b Ka Mg(II)b Mn(II)b Zn(II)b

3=A – 5.4 � 0.9 484 � 58 – – –

3=B – – 692 � 96 – 38 � 7 –

5=A 334 � 72 – 676 � 90 – 41 � 7 –

5=B – – 626 � 81 – 39 � 6 –

6=A – 10 � 1 595 � 84 – – –

6=B – 8.4 � 0.9 521 � 30 – – –

7=A – 13 � 2 498 � 35 – – –

7=B 356 � 80 27 � 5 638 � 72 – 33 � 3 –

8=A – – 712 � 95 – 40 � 8 –

8=B 1915 � 37 – – 314 � 56 49 � 9 68 � 2

10=A 1421 � 77 4.5 � 0.7 – 393 � 35 – 57 � 5

10=B 1960 � 170 – – 534 � 44 60 � 3 70 � 9

12=A – – 926 � 62 – 63 � 3 –

12=B – – 786 � 89 – 60 � 2 87 � 9

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

Table 2. Figures of merits of comparable methods for determination of metalloproteins

Sample Extraction method Metal ions analyzed

(technique of determination)

Results Ref.

Human

liver cytosol

buffer containing HEPES

(pH 7.4) and sucrose; glass

bead homogenization

Cu, Fe and Zn (SR-XRF) only qualitative results.

Detection of six Zn-,

four Fe- and one Cu-containing

protein bands in the sample

[24]

Yeast buffer containing SDS and

derivatization with iodoacetic

acid

Se (ETV-ICP-MS) LOD: 50 ng mL�1 per protein band [29]

Embriogenic

callus

buffer containing Tris–HCl

(pH 6.8), SDS, glycerol and

�-mercaptoethanol; grinding

homogenization

Ca, Cu, Fe, K and Zn

(SR-TXRF); Ca and Mg

(FAAS); Na (FAES)

LOQ (mg L�1): 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)

[31]

Soybean

seeds

Method A or B Ca and K (ICP OES);

Cu, Mg, Mn and Zn

(ICP-MS)

LOQ (mg g�1): Ca 305 and K 204

(ICP OES); Cu 3.8, Mg 302,

Mn 33 and Zn 55 (ICP-MS)

present work


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 re-

sult can be obtained for such metals. On the other

hand, metal ions such as Ca and Mg interact moder-

ately with proteins, although these interactions can also

be lost, depending on the extraction procedure em-

ployed. Furthermore, transition metal ions such as Cu,

Mn and Zn have the strongest coordination with pro-

teins, through electrostatic forces [7, 33]. Due to this

fact, in the majority of the protein bands evaluated,

significant differences related to copper and manga-

nese levels were not observed.

It is important to emphasize that the quantitative data

agree with the results obtained by SR-XRF, which

allows confirmation 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 pro-

teins, 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 eval-

uation 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 identification and quantification of the metal

species bound to proteins. This is particularly impor-

tant when metallomic studies are concerned.

Acknowledgments. The authors would like to thank the Fundac�~aao de

Amparo �aa Pesquisa do Estado de S~aao Paulo and the Conselho

Nacional de Desenvolvimento Cientıfico e Tecnol�oogico for financial

support (grant numbers 05=54892-3 and 475474=2004-0, respec-

tively) and for fellowships to A.S. (grant number 04=11960-6) and

M.A.Z.A. This work has been supported by the Brazilian Synchro-

tron Light Source (LNLS) under proposal D09B-XRF-4206=05. The

authors also thank Prof. Carol H. Collins for language assistance.

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180 Evaluation of soybean seed protein extraction focusing on metalloprotein analysis

evaluation of soybean seed protein extraction focusing on metalloprotein analysis

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