Path: TopS3-DissertationsMathematics-FMIPA2008

DIFERENSIASI DAN DISTRIBUSI SPESI MAGNESIUM, KALSIUM, MANGAN, SENG, MOLIBDEN DAN KADMIUM DALAM CAIRAN FLOEM TANAMAN JARAK (Ricinus communis L.)

PhD Theses from JBPTITBPP / 2017-09-27 15:45:35
Oleh : NOOR FITRI (NIM 30506004), S3 - Mathematics and Natural Sciences
Dibuat : 2008, dengan 7 file

Keyword : Diferensiasi dan distribusi spesi (Mg, Ca, Mn, Zn, Mo, dan Cd), Floem, Ricinus communis L.

Spesiasi unsur (elemental speciation) merupakan salah satu topik utama dalam penelitian bidang kimia analitik, terutama untuk memenuhi kebutuhan informasi tentang perilaku dan karakter suatu unsur misalnya mobilitas, fungsi, ketersediaan, defisiensi dan toksisitasnya. Perilaku suatu unsur baik pada organisme maupun pada sistem ekologis tidak dapat diterangkan hanya dengan mengetahui jumlah total unsur tersebut dalam sampel yang bersesuaian melainkan juga ditentukan oleh bentuk spesi unsur tersebut.




Pada tanaman, sejumlah unsur dengan berbagai bentuk spesi kimia diperlukan untuk pertumbuhan, misalnya unsur Mg dan Ca sebagai unsur hara pokok, dan Mn, Zn, dan Mo sebagai unsur hara runut. Fungsi dan peranan unsur tersebut dalam tanaman telah diketahui dengan jelas, namun bagaimana spesiasi kimia unsur tersebut belum terungkapkan sepenuhnya. Berdasarkan latar belakang tersebut di atas, telah dilakukan spesiasi unsur Mg, Ca, Mn, Zn, Mo, dan Cd dalam cairan floem tanaman jarak (Ricinus communis L.), dengan target utama diferensiasi dan distribusi spesi kimia unsur hara pokok Mg dan Ca, spesi kimia unsur hara runut Mn, Zn, dan Mo serta spesi kimia logam berat (Cd) dalam sampel tersebut.




Penelitian ini difokuskan pada analisis spesiasi operasional, yaitu karakterisasi spesi yang tergantung pada metode analitik yang dipilih. Metode pemisahan utama dilakukan dengan menggunakan kromatografi eksklusi ukuran (size exclusion chromatography, SEC) sedangkan metode pemisahan pendukung adalah quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC PAGE). Pemisahan dua tahap (bidimensional) spesi Mo dilakukan dengan menggunakan kedua metode tersebut. Adapun pemisahan dua tahap spesi Ca, tahap pertama dengan SEC dan tahap kedua dengan elektroforesis kapiler (capillary electrophoresis, CE). Pendeteksian selektif unsur dilakukan dengan inductively coupled plasma quadrupole mass spectrometry (ICP QMS). Khusus untuk spesi Mo dilakukan pendeteksian selektif molekul dengan electrospray ionization –mass spectrometry (ESI MS). Dengan menggunakan metode SEC, spesi Mg, Ca, Mn, Zn, Mo, dan Cd terdistribusi berdasarkan perbedaan berat molekul relatifnya. Kolom pemisah yang digunakan adalah sephadex G-50 SF (700 mm x 24 mm) dan sephadex G-25 M (28 mm x 9 mm). Sebagai fasa gerak digunakan larutan 20 mM MES/1 mM NaN3 pH 8,0 dan larutan 20 mM NaCl/1 mM NaN3 pH 8,0. Kondisi operasional yang diaplikasikan pada kolom sephadex G-50 SF meliputi (i) laju alir 0,3 mL/min, (ii) volume fraksi 7,2 mL, jumlah fraksi 95 (fraksinasi berlangsung otomatis), (iii) pendeteksian UV pada panjang gelombang 254 nm, dan (iv) temperatur pemisahan 4 derajat C, sedangkan pada kolom sephadex G-25 M dilakukan pada kondisi: (i) laju alir 0,3 mL/min, (ii) volume fraksi 0,1-0,2 mL, (iii) jumlah fraksi 20-40 (manual), dan (iv) temperatur pemisahan pada temperatur ruang.




Kalibrasi kolom sephadex G-50 SF dilakukan dengan menggunakan campuran standar protein thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), dan Vitamin B12 (1,35 kDa). Spesi yang terdeteksi setelah pemisahan dengan kolom sephadex G-50 SF dan kolom sephadex G-25 M berturut-turut diberi notasi subscript A dan subscript B. Subscript A1, A2, dan seterusnya menyatakan urutan spesi dari logam yang sama berdasarkan urutan berat molekul relatifnya. Metode QPNC PAGE diaplikasikan juga dalam penelitian ini untuk melihat profil elusi spesi unsur berdasarkan berbedaan m/z spesi. Kondisi operasional yang diterapkan meliputi (i) sistem buffer kontinyu 20 mM Tris-HCl/1 mM NaN3 pH 10, (ii) derajat polimerisasi poliakrilamid 4 %T / 2,67 % C, panjang gel 40 mm, fasa gerak 20 mM MES/1 mM NaN3 pH 8,0, dan (iii) temperatur analisis 4 derajat C. Sampel difraksinasi sebanyak 74 fraksi dengan volume setiap fraksi 5 mL (fraksinasi otomatis dengan pemograman). Pemisahan tahap dua spesi Ca dilakukan dengan menggunakan elektroforesis kapiler. Prinsip pemisahan CE adalah berdasarkan perbedaan mobilitas analit pada suatu medan listrik tertentu. Medan listrik diaplikasikan sepanjang kolom kapiler pada tegangan tinggi (15-30 kV) pada elektroda positif atau negatif. Pemisahan analit terjadi sebagai hasil pergerakan analit di bawah pengaruh fenomena elektroosmotik dan elektroforetik. Untuk mendapatkan metode pemisahan yang optimal dilakukan optimasi: komposisi dan konsentrasi buffer, pH, tegangan yang diaplikasikan, dan penggunaan surfaktan sebagai modifikator laju elektroosmotik. Pendeteksian selektif unsur dalam cairan floem tanaman jarak dan dalam fraksi SEC dan QPNC PAGE dilakukan dengan ICP QMS. Prinsip kerja metode ICP QMS adalah analit diionisasi dalam plasma menjadi muatan positif satu yang kemudian dicacah jumlahnya oleh spektrometri massa quadrupole. Kondisi operasional yang diaplikasikan antara lain (i) daya 880-1200 Watt, (ii) sistem detektor pulsa dan analog, (iii) laju gas argon pembawa 14 L/min, (iv) laju gas argon pengabut 0,9-0,98 L/min, (v) laju sampel 1,2 mL/min, (vi) pengolahan sinyal peak hopping, (vii) sweeps/reading 35-40/3-5, (viii) replikasi 5 kali, (ix) sample flush 45 detik, (x) read delay 15 detik, (xi) wash time 60 detik, dan (xii) standar internal 10 μg/L In dalam HNO3 1 % (b/b). Pengujian kemungkinan interaksi logam dengan protein/polipeptida diuji dengan menggunakan proteinase K. Cairan floem sebelum dan sesudah destruksi dengan proteinase K difraksinasi menggunakan kolom sephadex G-25 M kemudian secara selektif unsurnya dideteksi dengan ICP QMS. Konsentrasi protein dalam fraksi ditentukan berdasarkan metode Bradford. Profil elusi logam dalam fraksi sebelum dan sesudah destruksi proteinase dibandingkan untuk melihat kemungkinan adanya interaksi logam dengan protein/polipeptida. Hasil penelitian menunjukkan ada 6 kelompok spesi aktif UV berdasarkan profil serapan UV cairan floem pada kolom sephadex G-50 SF. Ke-enam kelompok aktif tersebut meliputi satu kelompok (kelompok A) terdeteksi pada daerah berat molekul tinggi (> 44 kDa) dan lima lainnya (kelompok B, C, D, E dan F) pada daerah berat molekul rendah. Sebagai hasil penelitian terdeteksi dua spesi karbon yaitu spesi CA1 dan CA2, 4 spesi fosfor, PA1, PA2, PA3, dan PA4 serta 11 spesi sulfur yang diberi notasi , SA1 hingga SA11.




Diferensiasi dan distribusi spesi Mg, Ca, Mn, Zn, Mo, dan Cd juga dapat dipelajari berdasarkan profil elusinya setelah pemisahan SEC. Pada kolom sephadex G-50 SF terdeteksi minimal masing-masing 3 spesi Mg (MgA1, MgA2 dan MgA3), Ca (CaA1, CaA2 dan CaA3), Mn (MnA1, MnA2 dan MnA3), dan Zn (ZnA1, ZnA2 dan ZnA3), serta 2 spesi Mo (MoA1 dan MoA2) dan 5 spesi Cd (CdA1 -CdA5). Spesi pertama dari semua logam terdeteksi pada volum mati (> 44 kDa) yang bersesuaian dengan serapan aktif UV cairan floem.

Kelimpahan relatif spesi pertama berkisar 0,01 % (spesi Mg) hingga 10,37 % (spesi Cd) dibandingkan spesi lainnya untuk masing-masing logam. Spesi utama logam dengan kelimpahan relatif yang tertinggi terdeteksi pada daerah berat molekul rendah (< 1350 Da). Spesi utama logam-logam tersebut berkorelasi positif dengan serapan aktif UV cairan floem. Adapun hasil pemisahan SEC pada kolom sephadex G-25 M terdeteksi 3 spesi Mg (MgB1, MgB2 dan MgB3) dan Zn (ZnB1, ZnB2 dan ZnB3), dua spesi Ca (CaB1 dan CaB2), Mn (MnB1 dan MnB2), dan Mo (MoB1 dan MoB2), serta satu spesi Cd (CdB1).




Perbedaan pengkulturan tanaman jarak tidak berpengaruh terhadap diferensiasi spesi logam, namun berpengaruh terhadap jumlah total spesi. Pada media aeroponik, jumlah total spesi MnA2, spesi MoA2, dan semua spesi CdA lebih besar dibandingkan pada media pot. Hal ini menunjukkan pada media aeroponik, spesi biologis aktif (Bioavailability) logam Mn, Mo, dan Cd terserap lebih optimal dibandingkan dalam media pot.




Hasil pemisahan dengan QPNC PAGE menunjukkan keberhasilan penelitian mendeteksi spesi Mn dan Mo dengan sangat baik. Spesi Mo terdeteksi pada daerah elusi 35-80 mL sedangkan spesi Mn pada daerah elusi 90-170 mL. Pemisahan spesi Mg, Ca, Zn, dan Cd dengan QPNC PAGE kurang optimal yang teramati dengan profil elusi spesi bergradasi dan tingginya batas kuantifikasi pendeteksian selektif unsur Mg, Ca, Zn, dan Cd.




Hasil pengujian kemungkinan spesi logam berinteraksi dengan protein/polipeptida menunjukkan bahwa konsentrasi total spesi MgB1, spesi CaB1, spesi MnB1, MoB1, dan CdB1 berkurang secara signifikan sedangkan konsentrasi total spesi dengan berat molekul rendah dari Mg, Ca, Mn, Mo dan Cd bertambah setelah destruksi dengan proteinase K. Berkurangnya konsentrasi total spesi tersebut merupakan indikasi kemungkinan interaksi spesi dengan protein/polipeptida. Berbeda dengan spesi logam lainnya, tidak ada perbedaan profil elusi spesi Zn sebelum dan sesudah uji proteinase K. Hal ini menunjukkan kecil kemungkinan Zn berikatan dengan protein/polipeptida.

Pembuktian keberadaan dan kestabilan spesi MoA2 dilakukan dengan SEC dan QPNC PAGE. Cairan floem pertama dipisahkan dengan SEC kolom sephadex G-50 SF. Fraksi SEC dengan konsentrasi Mo tertinggi (Fraksi 43/volume elusi 309,6 mL) dipisahkan lebih lanjut dengan QPNC PAGE. Strategi kedua, pemisahan tahap pertama dilakukan dengan QPNC PAGE kemudian fraksi QPNC PAGE dengan konsentrasi Mo tertinggi (fraksi 10/volume elusi 50 mL) dipisahkan lebih lanjut dengan SEC kolom sephadex G-50 SF. Hasil penelitian membuktikan keberadaan dan kestabilan spesi Mo yang terdeteksi secara kuantitatif dengan menggunakan kedua metode tersebut berhasil dipisahkan lebih lanjut dengan terdeteksinya 13 spesi aktif UV dalam waktu kurang dari 10 menit.

Diharapkan kontribusi ilmiah penelitian yang dilakukan yaitu tentang diferensiasi dan distribusi spesi Mg, Ca, Mn, Zn, Mo, dan Cd dapat bermanfaat untuk mengetahui mekanisme proses transformasi dan transport spesi tersebut dalam cairan floem tanaman jarak.

Deskripsi Alternatif :

Elemental speciation is the main topic of research on analytical chemistry, particularly to attain some information related to behaviors and characteristics of elements, for instance; their mobility, function, occurrence, deficiency and toxicity. The elemental behaviors both in organism and ecological system are not only elucidated by determining a total amount of elements in suitable samples, but also by determining the pattern of elemental species. In turn, this will help in better understanding of the properties and influence of the element.




Some elements with various patterns of chemical species are needed for plant growth and development, for examples; Mg and Ca frequently act as the major nutrients, whereas Mn, Zn and Mo are mostly needed to be micronutrients. The functions and roles of those elements in the plants are already well known, however, what and how the elemental species are not yet fully understood. On those reasons, the elemental speciation of Mg, Ca, Mn, Zn, Mo and Cd in phloem sap of castor bean (Ricinus communis L.) was studied. The elemental speciation was primarily emphasized on the differentiation and distribution of chemical species of major nutrients Mg and Ca, micronutrients Mn, Zn and Mo, as well as chemical species of heavy metal Cd in the phloem sap samples.




This work was focused on the operational speciation analysis, defined as species characterization, depending on the selected analytical methods. The main separation method employed size exclusion chromatography (SEC), whereas the supporting separation method was performed using quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC PAGE). Bidimensional separation of Mo species was done by applying of both methods above. Additionally, bidimensional separation of Ca species in the first stage was used SEC, whereas in the second stage, the separation was conducted using capillary electrophoresis (CE). Selective detection of elements was performed by inductively coupled plasma quadrupole mass spectrometry (ICP-QMS). In particular, molybdenum (Mo) was selectively detected by electrospray ionization-mass spectrometry (ESI-MS).




The distribution of Mg, Ca, Mn, Zn, Mo, and Cd species according to the differences of their relative molecular weights were identified by using SEC method. Two SEC columns were applied, namely Sephadex G-50 SF (700 mm x 24 mm) and sephadex G-25 M (28 mm x 9 mm). In accordance with 20 mM MES/1 mM NaN3 pH 8,0 and 20 mM NaCl/1 mM NaN3 pH 8,0 solutions were used as the mobile phase, respectively. The operational parameters of separation applied for the sephadex G-50 SF column were (i) eluent flow of 0.3 mL/min, (ii) fraction volume of 7.2 mL with the total of fraction of 95 (automatic running fractionation), (iii) UV detection wavelenght of 254 nm, and (iv) temperature of separation system at 4 degrees C, whereas the operational conditions of separation applied for the sephadex G-25 M column were (i) eluent flow of 0.3 mL/min, (ii) fraction volume ranging from 0.1 to 0.2 mL, (iii) total of fraction of 20-40 (manual running fractionation), and (iv) separation system at room temperature. In order to standardize the molecular weight range of the chromatographic separation, a mixture protein standard consisting of thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and Vitamin B12 (1,35 kDa) were employed on sephadex G-50 SF column. After separation using sephadex G-50 SF and sephadex G-25 M column, the detected species are consecutively written in notations of subscript A and subscript B. Subscript A1, A2, etc, denotes the subsequent species of the same metal on the basis of their subsequent relative molecular weight.




Quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC PAGE) method was also utilized in this study. The application of this method was aimed to obtain the species elution profiles on the basis of the m/z differences of the species. The experimental operational conditions were: (i) continuous buffer system of 20 mM Tris-HCl/1 mM NaN3 pH 10, (ii) polyacrylamide polymerization degree of 4 %T / 2,67 % C, with a length of gel of 40 mm, and mobile phase of 20 mM MES/1 mM NaN3 pH 8,0, and (iii) temperature of analysis of 4 degrees C. The samples were fractionated on 74 fractions with each fraction volume of 5 mL (automatic fractionation by programming).




The further separation of Ca species was carried out by means of capillary electrophoresis (CE). This separation is based on the distinction of their analyte mobility for a given electric field. The electric field was applied along capillary column at a high voltage (15-30 kV) on both positive and negative electrodes. The separation of analytes was a result of analyte movement under influences of electroosmosis and electrophoresis phenomena. To attain an optimal separation method, the composition and concentration of buffer, pH, used voltage and the usage of surfactant as a modifier of electroosmosis flow were optimized.




Selective elements in phloem sap of castor bean and in fractions of SEC and QPNC PAGE were detected by using inductively coupled plasma quadrupole mass spectrometry (ICP-QMS). In this method, analytes were ionized in plasma to be a cation (M+), and its count was calculated by quadrupole mass spectrometry (QMS). The operational conditions applied in the analysis consist of (i) power of 880-1200 Watt, (ii) pulse and analog detector system, (iii) argon gas flow of 14 L/min, (iv) nebulizer argon gas flow of 0,9-0,98 L/min, (v) sample flow of 1,2 mL/min, (vi) peak hopping signal process, (vii) sweeps/reading of 35-40/3-5, (viii) replication of 5 times, (ix) sample flush of 45 seconds, (x) read delay of 15 seconds, (xi) wash time of 60 seconds, and (xii) internal standard of 10 μg/L In in HNO3 1 % (b/b).




The interaction probability of metals and protein/polypeptide was tested by using proteinase K. Prior to and after destruction using proteinase K, phloem sap was fractionated by means of sephadex G-25 M column and the elements were selectively detected by ICP-QMS. The protein content of the fractions was determined by Bradford method. The metal elution profiles of fractions resulted before and after destruction using proteinase K are compared and interpreted if any probability of interaction between metals and protein/polypeptide.




On the basis of UV absorption profiles of phloem sap on sephadex G-50 SF column, 6 (six) groups of UV active species were identified. The six groups of species consisting of 1 (one) group (group A) was detected at a high molecular weight area (> 44 kDa), whereas the other 5 (five) groups (group B, C, D, E and F) were detected at a low molecular weight area.




After SEC fractionation, elution profiles of C (carbon), P (phospor) and S (sulphur) species show a differentiation of C, P and S compouds in phloem sap. On sephadex G-25 M column, two C species (CB1 and CB2), four P species (PB1, PB2, PB3, and PB4), seven S species (SB1, SB2, SB3, SB4, SB5, SB6, and SB7). Fractions in an elution area ranging from 0,5 to 1,5 mL are collected and further separated on sephadex G-50 SF column by using a similar mobile phase (20 mM NaCl/ 1 mM NaN3 pH 8,0). As a result, two C species (CA1 and CA2), four P species (PA1, PA2, PA3, and PA4) and eleven S species annotated as SA1 to SA11 were well detected.




Differentiation and distribution of Mg, Ca, Mn, Zn, Mo, and Cd species in phloem sap of castor bean were also investigated on the basis of their elution profiles after SEC separation. On the sephadex G-50 SF column, three Mg species (MgA1, MgA2 and MgA3), three Ca species (CaA1, CaA2 and CaA3), three species of Mn (MnA1, MnA2, and MnA3), three Zn species (ZnA1, ZnA2 and ZnA3), as well as two Mo species (MoA1 and MoA2) and five Cd species (CdA1 -CdA5) were identified. The first species of all metals were observed at a void volume (> 44 kDa), which coincides with UV active absorption of phloem sap. The relative abundance of the first species varies from 0.01 % (Mg species) to 10.37 % (Cd species) compared to other species for each metals. The major species of metals with a highest relative abundance were detected at low molecular weight area (< 1350 Da). The major species are positively correlated to UV active absorption of phloem sap.




The result of SEC separation on sephadex G-25 M column indicates the presence of three Mg species (MgB1, MgB2 and MgB3), three Zn species (ZnB1, ZnB2 and ZnB3), two Ca species (CaB1 and CaB2), two Mn species (MnB1 and MnB2), two Mo species (MoB1 and MoB2), and one Cd species (CdB1). The culture difference of castor bean did not influence the differentiation of metal species, but influencing the total amount of species. In an aeroponic culture, the total amounts of MnA2 and MoA2 species as well as all CdA species were greater than those in a soil culture. This suggests that Mn, Mo, and Cd metals in the aeroponic culture were optimally absorbed compared those in the soil culture.




The results of separation using QPNC PAGE indicate that Mn and Mon species are successfully detected. Molybdenum species were detected at elution area of 35-80 mL, whereas Mn species were identified at elution area ranging from 90 to 170 mL. The separation of Mg, Ca, Zn, and Cd species by using QPNC PAGE was not really working optimally, which is shown by a gradational species elution profiles and a high detection of quantification of Mg, Ca, Zn and Cd.




Study on interaction probability between metal species and protein/polypeptide indicates that the total concentration of MgB1, spesi CaB1, spesi MnB1, MoB1, and CdB1 species decreases significantly, whereas the total concentration for low molecular weight species of Mg, Ca, Mn, Mo and Cd increases after destructed by proteinase K. The decrease of MgB1, spesi CaB1, spesi MnB1, MoB1, and CdB1 species suggests that these species may interact with protein/polypeptide. In contrary, there are no differences of Zn elution profiles before and after destructed by proteinase K. This suggests that Zn may not bind with protein/polypeptide.




The occurrence and stability of MoA2 species were proven by SEC dan QPNC PAGE. Phloem sap was initially separated by SEC on sephadex G-50 SF column. The SEC fraction containg a highest concentration of Mo (43/elution volume of 309,6 mL) was further separated using QPNC PAGE. Alternatively, the first stage of separation was done by QPNC PAGE and the QPNC PAGE fraction containing a highest concentration of Mo (10 fractions/elution volume of 50 mL) was furthermore separated using SEC on sephadex G-50 SF column. As a result, by means of SEC and QPNC PAGE methods, the occurrence and stability of Mo species were well detected quantitatively. This is supported by ESI-MS detection results, which indicates that the relative molecular weight of MoA2 was less than 1000 Da.




In addition, further separation of CaB2 species using capillary electrophoresis (CE) was also performed. The optimal conditions of the separation method was obtained at (i) composition and concentration of background electrolit (BGE) of 10 mM Na2HPO4/ 10 mM NaH2PO4/0,5 mM CTAB, (ii) pH of 8.0, (iii) voltage of -20 kV, (iv) wavelenght of detection/reference of 214/500 nm, and (v) optimal temperature at 14degrees C. As a result, CaB2 species was successfully separated by identifying a total of 13 UV active species in less than 10 minutes.




Differentiation and distribution of Mg, Ca, Mn, Zn, Mo, and Cd species in phloem sap of castor bean were successfully carried out by means of SEC, QPNC PAGE, and CE separation methods as well as modified ICP QMS technique for selective detection of elements. The advantages of the modified separation and detection methods are their ability in differentiating and detecting minor species at a minimal relative abundance of 0.01 % (MgA1) and a lowest mass in pikogram orde (50 pg for CdA1 species). The study on differentiation and distribution of Mg, Ca, Mn, Zn, Mo, and Cd species could be a significant scientific contribution and undoubtedly benefited for a better understanding of mechanism of transformation and transport processes of those species in phloem sap of castor bean.

Copyrights : Copyright Â(c) 2001 by ITB Central Library. Verbatim copying and distribution of this entire article is permitted by author in any medium, provided this notice is preserved.

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