www.ietdl.org Published in IET Nanobiotechnology Received on 14th June 2013 Revised on 11th October 2013 Accepted on 17th October 2013 doi: 10.1049/iet-nbt.2013.0042
ISSN 1751-8741
Protein characterisation of Brosimum gaudichaudii Trécul latex and study of nanostructured latex film formation Eduardo F. Barbosa1,2, Victoria Monge-Fuentes2, Natiela B. Oliveira2, Rebecca Tavares2, Mary-Ann E. Xavier2, Marcelo Porto Bemquerer1, Luciano P. Silva1,2 1
Laboratory of Mass Spectrometry, Embrapa Genetic Resources and Biotechnology, Brasília, Brazil Postgraduate Program in Animal Biology, Institute of Biological Sciences, University of Brasília, UnB, Brasília, Brazil E-mail: [email protected] † These authors contributed equally to this work. 2
Abstract: Brosimum gaudichaudii Tréc. (Moraceae) is a common Brazilian Cerrado plant known by its pharmaceutical industry relevance. The authors investigated the latex protein components and potential biotechnological applications. Some protein fragments had their sequences elucidated, presenting similarities to jacalin and Kunitz-type trypsin inhibitors. Amino acid residue modifications were found, such as glutamine N-terminal residue cyclisation into pyroglutamic acid residue, and mass differences corresponding to hexoses and N-acetylhexosamine presence. The latex was used to produce a nanoscale structured film, which presented an increased attraction and reduced adhesion behaviours. The film presented high homogeneity, as observed by low nanoroughness values, probably because of its intrinsic components, such as the jacalin-like protein that has known agglutination properties. The immobilised Kunitz-type trypsin inhibitor presence in the latex film allow us to point out to applications related to this inhibition, as in active food packaging, since these peptidase inhibitors are able to inhibit pests and microorganism proliferation.
1
Introduction
Brosimum gaudichaudii Tréc. (Moraceae) occurs in the Atlantic and Amazon forests [1] and it is the only occurring species of the genus Brosimum in the Brazilian Cerrado [2]. This plant species has been largely employed by the pharmaceutical industry because of its considerable accumulation of furocoumarins derivatives [3] such as: xanthyletine, psoralen, bergapten (or 5-methoxypsoralen), luvangentine and the gaudichaudione, present in the bark of trunks and subterranean system. These furocoumarin derivatives have been isolated and used for the treatment of skin disorders such as vitiligo [4–7] because of their photosensitising and photochemotherapeutic properties [8, 9]. Even though some of the biomedical uses of Brosimum vegetal structures have been elucidated, there is no study related to plant secretions, such as latex extracted from B. gaudichaudii, much less concerning their protein content. Latex can be defined as a stable milky suspension or emulsion of polymer particles in an aqueous fluid, usually held under pressure in living plant cells known as lacticifers. This plant secretion exudes upon damage from specialised canals in about 10% of flowering plants and has no known metabolic function related to plant resource acquisition and allocation; however, it has been strongly implicated in defense against herbivorous insects [10, 11]. Latex from various plant species contain bioactive 222 & The Institution of Engineering and Technology 2014
compounds including alkaloids such as: morphine in Papaver spp. (Papaveraceae); cardiac glycosides in Asclepias spp. (Apocynaceae); terpenes such as the sesquiterpene lactone, lactucin from lettuce (Lactuca spp. Asteraceae); and digestive cysteine proteases in Carica genus (Caricaceae) and Ficus spp. (Moraceae) [12, 13]. Phenolic compounds and proteins have also been detected in latex from other species [14, 15]. The fact that latex is often highly rich in secondary metabolites, carbohydrates and enzymes suggests that it is not a waste product and can be considered a potential candidate for biotechnological applications [12]. Romaniuc Neto and Wanderley [16] emphasised the presence of abundant lacticifers in specimens belonging to Moraceae family. According to Lewinsohn [17], lacticifer plants are common in the Brazilian Cerrado vegetation, where the presence of latex provides resistance to herbivores via toxicity or anti-nutritional effects, and also through its stickiness, which can mire insect herbivores. Agrawal and Konno [12] pointed out that all plant parts can contain latex; however, the most commonly examined tissues of latex-bearing plants are stem and leaf tissues. In the case of B. gaudichaudii, latex is abundant in the bark of the subterranean system. In stems, latex is only abundant in the bark and pith of young stems and branches. Latex is scarce in both bark and pith of older stem parts and not present in B. gaudichaudii wood [18]. IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 222–229 doi: 10.1049/iet-nbt.2013.0042
www.ietdl.org Several studies have pointed out multiple applications attributed to plants latex, including activity as anti-tumour, anti-ulcer, anti-inflammatory agent [19]. Also, beer clarification [20], meat tenderising and preparation of protein hydrolysates [21] are useful in the food industry [22]. Another application of latex is related to its ability to form a coherent biofilm, which is one of the most important properties of latex [23]. Considering the potential biomedical, industrial and pharmaceutical applications and the lack of information about the molecular properties of latex extracted from B. gaudichaudii, the present study focused on purification, characterisation and sequencing of protein components from this plant latex. In addition, latex films were formed and analysed by atomic force microscopy (AFM) in order to elucidate some nanostructural and nanomechanical aspects of this biomaterial.
2 2.1
Experimental section Latex source
Fresh latex sample was obtained from a B. gaudichaudii young tree located in Colinas do Sul, GO, Brazil (14° 03’06.82" S 48°09’23.10" W). Latex was obtained by clipping the petiole of some few leaves. A volume of 500 µl was collected in a plastic microtube containing trifluoroacetic acid (TFA) (0.1% by volume). Latex was stored at −20 °C prior to analysis. 2.2
Sample extraction
One hundred microlitres of latex sample were diluted in 900 µl of nanopure filtered water. Latex was then percolated for a period of 3 days. Sample was centrifuged at 8.500 × g for 45 min. 2.3
Protein quantification
Micro-Bradford assay was used to quantify the total amount of proteins in the latex sample. Latex aqueous extract was diluted in a factor of 10; thereafter, 100 μl from this dilution were added to 1 ml of Bradford reagent. Ten minutes after mixing, the absorbance was monitored using a Spectrophotometer SpectraMax® (Molecular Devices, Sunnyvale, CA) with a multiwell plate reader at a wavelength of 595 nm. The amount of protein was calculated using the standard curve made with bovine serum albumin. Experiment was performed in triplicate. 2.4 Reverse-phase high-performance liquid chromatography analysis Reverse-phase high-performance liquid chromatography (RP-HPLC) was performed with a Shimadzu LC-10VP (Shimadzu Corporation, Kyoto, Japan) equipped with a LC-10AD pump, controlled by a SCL-10A interface module and a Shimadzu SPD-10AV UV–Vis detector (Shimadzu Corporation, Kyoto, Japan). Analysis was performed on a semi-preparative NST C4300A C4 column (250 mm × 10.0 mm, 5 µm particle size). The mobile phase was 0.1% TFA and water (A) and acetonitrile and 0.1% TFA (B). Composition gradient was: 5% (B) → 95% (B), starting with 5% (B) for 5 min and then increasing 1% per minute until reaching 95% (B). This concentration was maintained for five additional minutes. Injection volume IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 222–229 doi: 10.1049/iet-nbt.2013.0042
was 300 µl and the samples were monitored at 216 and 280 nm. A 3 ml/min flow rate was used during analysis. HPLC grade solvents and Milli-Q (Millipore, Billerica, MA) water were used in the chromatographic studies. All chromatographic experiments were performed at room temperature. 2.5
Mass spectrometry analysis
All chromatographic fractions were further submitted to exact molecular mass determination by using an UltraFlex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, MA). Chromatographic fractions were resuspended in 10 μl of nanopure water, mixed in an α-cyano-4-hydroxycinnamic acid matrix solution (1:3, v:v), spotted onto a MTP AnchorChip var/384 MALDI target plate (Bruker Daltonics) and dried at room temperature for 2 h. Average and monoisotopic molecular masses were obtained in linear (m/z 4000–20 000 mass range) and reflector positive ion modes (m/z 600–5940 mass range), respectively, with external calibration, using the Protein and Peptide Calibration Standards I for mass spectrometry (m/z 1000–3000 and up to 6000 mass range, Bruker Daltonics). Ions were generated by a Smart Beam laser and were accelerated at 20 kV with optimised parameters. MS/MS experiments were performed with the LIFT® cell voltage parameters set at 19 kV for a final acceleration of 29 kV (reflector voltage) and pressure in the LIFT® cell around 4 × 10−7 mbar. The precursor ion was selected by timed ion selector and in some situations post lift metastable suppressor was used. MS/MS spectra were acquired from around 2000 laser shots by adjusting the laser intensity above the threshold for generation of molecular ions for each sample. FlexAnalysis 2.4 and 3.0 softwares (Bruker Daltonics) were used to interpret mass spectra. In addition, PepSeq software (Waters-Micromass, Milford, MA, USA) was used for the peptide sequencing and peaks labelling after spectra were exported in txt format. 2.6
AFM of latex film
2.6.1 Film production: Ten microlitres aliquots of the latex solution were deposited onto freshly cleaved mica surface and dried on a hot plate at 55°C overnight. Controls were composed by just cleaved mica surface. 2.6.2 AFM analysis: Film samples were analysed with a commercial AFM instrument SPM-9600 (Shimadzu, Kyoto, Japan) operating in dynamic phase mode. Scanner used has a travel of 125 µm in XY-directions and 7 µm in Z-direction. Images were obtained in air, at room temperature (23°C), and at ∼30–40% relative humidity. Scanned areas were perfect squares measuring 5 µm × 5 µm with scan rate of 1 Hz. Trace and retrace procedures were performed in order to prove that the samples were not modified during scanning steps. All AFM images contained 512 × 512 data points and were processed by SPM-9600 off-line software (Shimadzu, Kyoto, Japan). The processing consisted in an automatic X-line, Y-line and plane fit levelling of the surface. After processing, surface analysis was performed and the nanoroughness parameters such as: arithmetic mean roughness (Ra), maximum height (Rz), 10-point mean roughness (Rzjis), root-mean-square roughness (Rq), average height (Rp) and average depth (Rv) were obtained. Statistical analyses [one-way analysis of variance (ANOVA) 223
& The Institution of Engineering and Technology 2014
www.ietdl.org and Tukey test] were performed by Origin Pro 8 software (OriginLab, Northampton, MA, USA). 2.7
Force spectroscopy of latex film
To analyse the film surface interaction forces, the same previously described commercial AFM instrument was used in contact mode was used. A pyramidal silicon nitride tip (curvature radius
ISSN 1751-8741
Protein characterisation of Brosimum gaudichaudii Trécul latex and study of nanostructured latex film formation Eduardo F. Barbosa1,2, Victoria Monge-Fuentes2, Natiela B. Oliveira2, Rebecca Tavares2, Mary-Ann E. Xavier2, Marcelo Porto Bemquerer1, Luciano P. Silva1,2 1
Laboratory of Mass Spectrometry, Embrapa Genetic Resources and Biotechnology, Brasília, Brazil Postgraduate Program in Animal Biology, Institute of Biological Sciences, University of Brasília, UnB, Brasília, Brazil E-mail: [email protected] † These authors contributed equally to this work. 2
Abstract: Brosimum gaudichaudii Tréc. (Moraceae) is a common Brazilian Cerrado plant known by its pharmaceutical industry relevance. The authors investigated the latex protein components and potential biotechnological applications. Some protein fragments had their sequences elucidated, presenting similarities to jacalin and Kunitz-type trypsin inhibitors. Amino acid residue modifications were found, such as glutamine N-terminal residue cyclisation into pyroglutamic acid residue, and mass differences corresponding to hexoses and N-acetylhexosamine presence. The latex was used to produce a nanoscale structured film, which presented an increased attraction and reduced adhesion behaviours. The film presented high homogeneity, as observed by low nanoroughness values, probably because of its intrinsic components, such as the jacalin-like protein that has known agglutination properties. The immobilised Kunitz-type trypsin inhibitor presence in the latex film allow us to point out to applications related to this inhibition, as in active food packaging, since these peptidase inhibitors are able to inhibit pests and microorganism proliferation.
1
Introduction
Brosimum gaudichaudii Tréc. (Moraceae) occurs in the Atlantic and Amazon forests [1] and it is the only occurring species of the genus Brosimum in the Brazilian Cerrado [2]. This plant species has been largely employed by the pharmaceutical industry because of its considerable accumulation of furocoumarins derivatives [3] such as: xanthyletine, psoralen, bergapten (or 5-methoxypsoralen), luvangentine and the gaudichaudione, present in the bark of trunks and subterranean system. These furocoumarin derivatives have been isolated and used for the treatment of skin disorders such as vitiligo [4–7] because of their photosensitising and photochemotherapeutic properties [8, 9]. Even though some of the biomedical uses of Brosimum vegetal structures have been elucidated, there is no study related to plant secretions, such as latex extracted from B. gaudichaudii, much less concerning their protein content. Latex can be defined as a stable milky suspension or emulsion of polymer particles in an aqueous fluid, usually held under pressure in living plant cells known as lacticifers. This plant secretion exudes upon damage from specialised canals in about 10% of flowering plants and has no known metabolic function related to plant resource acquisition and allocation; however, it has been strongly implicated in defense against herbivorous insects [10, 11]. Latex from various plant species contain bioactive 222 & The Institution of Engineering and Technology 2014
compounds including alkaloids such as: morphine in Papaver spp. (Papaveraceae); cardiac glycosides in Asclepias spp. (Apocynaceae); terpenes such as the sesquiterpene lactone, lactucin from lettuce (Lactuca spp. Asteraceae); and digestive cysteine proteases in Carica genus (Caricaceae) and Ficus spp. (Moraceae) [12, 13]. Phenolic compounds and proteins have also been detected in latex from other species [14, 15]. The fact that latex is often highly rich in secondary metabolites, carbohydrates and enzymes suggests that it is not a waste product and can be considered a potential candidate for biotechnological applications [12]. Romaniuc Neto and Wanderley [16] emphasised the presence of abundant lacticifers in specimens belonging to Moraceae family. According to Lewinsohn [17], lacticifer plants are common in the Brazilian Cerrado vegetation, where the presence of latex provides resistance to herbivores via toxicity or anti-nutritional effects, and also through its stickiness, which can mire insect herbivores. Agrawal and Konno [12] pointed out that all plant parts can contain latex; however, the most commonly examined tissues of latex-bearing plants are stem and leaf tissues. In the case of B. gaudichaudii, latex is abundant in the bark of the subterranean system. In stems, latex is only abundant in the bark and pith of young stems and branches. Latex is scarce in both bark and pith of older stem parts and not present in B. gaudichaudii wood [18]. IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 222–229 doi: 10.1049/iet-nbt.2013.0042
www.ietdl.org Several studies have pointed out multiple applications attributed to plants latex, including activity as anti-tumour, anti-ulcer, anti-inflammatory agent [19]. Also, beer clarification [20], meat tenderising and preparation of protein hydrolysates [21] are useful in the food industry [22]. Another application of latex is related to its ability to form a coherent biofilm, which is one of the most important properties of latex [23]. Considering the potential biomedical, industrial and pharmaceutical applications and the lack of information about the molecular properties of latex extracted from B. gaudichaudii, the present study focused on purification, characterisation and sequencing of protein components from this plant latex. In addition, latex films were formed and analysed by atomic force microscopy (AFM) in order to elucidate some nanostructural and nanomechanical aspects of this biomaterial.
2 2.1
Experimental section Latex source
Fresh latex sample was obtained from a B. gaudichaudii young tree located in Colinas do Sul, GO, Brazil (14° 03’06.82" S 48°09’23.10" W). Latex was obtained by clipping the petiole of some few leaves. A volume of 500 µl was collected in a plastic microtube containing trifluoroacetic acid (TFA) (0.1% by volume). Latex was stored at −20 °C prior to analysis. 2.2
Sample extraction
One hundred microlitres of latex sample were diluted in 900 µl of nanopure filtered water. Latex was then percolated for a period of 3 days. Sample was centrifuged at 8.500 × g for 45 min. 2.3
Protein quantification
Micro-Bradford assay was used to quantify the total amount of proteins in the latex sample. Latex aqueous extract was diluted in a factor of 10; thereafter, 100 μl from this dilution were added to 1 ml of Bradford reagent. Ten minutes after mixing, the absorbance was monitored using a Spectrophotometer SpectraMax® (Molecular Devices, Sunnyvale, CA) with a multiwell plate reader at a wavelength of 595 nm. The amount of protein was calculated using the standard curve made with bovine serum albumin. Experiment was performed in triplicate. 2.4 Reverse-phase high-performance liquid chromatography analysis Reverse-phase high-performance liquid chromatography (RP-HPLC) was performed with a Shimadzu LC-10VP (Shimadzu Corporation, Kyoto, Japan) equipped with a LC-10AD pump, controlled by a SCL-10A interface module and a Shimadzu SPD-10AV UV–Vis detector (Shimadzu Corporation, Kyoto, Japan). Analysis was performed on a semi-preparative NST C4300A C4 column (250 mm × 10.0 mm, 5 µm particle size). The mobile phase was 0.1% TFA and water (A) and acetonitrile and 0.1% TFA (B). Composition gradient was: 5% (B) → 95% (B), starting with 5% (B) for 5 min and then increasing 1% per minute until reaching 95% (B). This concentration was maintained for five additional minutes. Injection volume IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 222–229 doi: 10.1049/iet-nbt.2013.0042
was 300 µl and the samples were monitored at 216 and 280 nm. A 3 ml/min flow rate was used during analysis. HPLC grade solvents and Milli-Q (Millipore, Billerica, MA) water were used in the chromatographic studies. All chromatographic experiments were performed at room temperature. 2.5
Mass spectrometry analysis
All chromatographic fractions were further submitted to exact molecular mass determination by using an UltraFlex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, MA). Chromatographic fractions were resuspended in 10 μl of nanopure water, mixed in an α-cyano-4-hydroxycinnamic acid matrix solution (1:3, v:v), spotted onto a MTP AnchorChip var/384 MALDI target plate (Bruker Daltonics) and dried at room temperature for 2 h. Average and monoisotopic molecular masses were obtained in linear (m/z 4000–20 000 mass range) and reflector positive ion modes (m/z 600–5940 mass range), respectively, with external calibration, using the Protein and Peptide Calibration Standards I for mass spectrometry (m/z 1000–3000 and up to 6000 mass range, Bruker Daltonics). Ions were generated by a Smart Beam laser and were accelerated at 20 kV with optimised parameters. MS/MS experiments were performed with the LIFT® cell voltage parameters set at 19 kV for a final acceleration of 29 kV (reflector voltage) and pressure in the LIFT® cell around 4 × 10−7 mbar. The precursor ion was selected by timed ion selector and in some situations post lift metastable suppressor was used. MS/MS spectra were acquired from around 2000 laser shots by adjusting the laser intensity above the threshold for generation of molecular ions for each sample. FlexAnalysis 2.4 and 3.0 softwares (Bruker Daltonics) were used to interpret mass spectra. In addition, PepSeq software (Waters-Micromass, Milford, MA, USA) was used for the peptide sequencing and peaks labelling after spectra were exported in txt format. 2.6
AFM of latex film
2.6.1 Film production: Ten microlitres aliquots of the latex solution were deposited onto freshly cleaved mica surface and dried on a hot plate at 55°C overnight. Controls were composed by just cleaved mica surface. 2.6.2 AFM analysis: Film samples were analysed with a commercial AFM instrument SPM-9600 (Shimadzu, Kyoto, Japan) operating in dynamic phase mode. Scanner used has a travel of 125 µm in XY-directions and 7 µm in Z-direction. Images were obtained in air, at room temperature (23°C), and at ∼30–40% relative humidity. Scanned areas were perfect squares measuring 5 µm × 5 µm with scan rate of 1 Hz. Trace and retrace procedures were performed in order to prove that the samples were not modified during scanning steps. All AFM images contained 512 × 512 data points and were processed by SPM-9600 off-line software (Shimadzu, Kyoto, Japan). The processing consisted in an automatic X-line, Y-line and plane fit levelling of the surface. After processing, surface analysis was performed and the nanoroughness parameters such as: arithmetic mean roughness (Ra), maximum height (Rz), 10-point mean roughness (Rzjis), root-mean-square roughness (Rq), average height (Rp) and average depth (Rv) were obtained. Statistical analyses [one-way analysis of variance (ANOVA) 223
& The Institution of Engineering and Technology 2014
www.ietdl.org and Tukey test] were performed by Origin Pro 8 software (OriginLab, Northampton, MA, USA). 2.7
Force spectroscopy of latex film
To analyse the film surface interaction forces, the same previously described commercial AFM instrument was used in contact mode was used. A pyramidal silicon nitride tip (curvature radius
