Experiments evaluating protein size distribution pattern in EPS Si + and EPS Si 2 using sodium dodecyl sulfate – polyacrylamide gel electrophoresis

Laboratory studies were conducted to examine the sorption of selected radionuclides (234Th, 233Pa, 210Po, 210Pb, and 7Be) onto inorganic (pure silica and acid-cleaned diatom frustules) and organic (diatom cells with or without silica frustules) particles in natural seawater and the role of templating biomolecules and exopolymeric substances (EPS) extracted from the same species of diatom, Phaeodactylum tricornutum, in the sorption process. The range of partition coefficients (Kd, reported as logKd) of radionuclides between water and the different particle types was 4.78–6.69 for 234Th, 5.23–6.71 for 233Pa, 4.44– 5.86 for 210Pb, 4.47–4.92 for 210Po, and 4.93–7.23 for 7Be, similar to values reported for lab and field determinations. The sorption of all radionuclides was significantly enhanced in the presence of organic matter associated with particles, resulting in Kd one to two orders of magnitude higher than for inorganic particles only, with highest values for 7Be (logKd of 7.2). Results further indicate that EPS and frustule-embedded biomolecules in diatom cells are responsible for the sorption enhancement rather than the silica shell itself. By separating radiolabeled EPS via isoelectric focusing, we found that isoelectric points are radionuclide specific, suggesting that each radionuclide binds to specific biopolymeric functional groups, with the most efficient binding sites likely occurring in acid polysaccharides, iron hydroxides, and proteins. Further progress in evaluating the effects of diatom frustule–related biopolymers on binding, scavenging, and fractionation of radionuclides would require the application of molecular-level characterization techniques.


Dataset Description
Laboratory studies were conducted to examine the sorption of selected radionuclides (234Th, 233Pa, 210Po, 210Pb, and 7Be) onto inorganic (pure silica and acid-cleaned diatom frustules) and organic (diatom cells with or without silica frustules) particles in natural seawater and the role of templating biomolecules and exopolymeric substances (EPS) extracted from the same species of diatom, Phaeodactylum tricornutum, in the sorption process.
The range of partition coefficients (Kd, reported as logKd) of radionuclides between water and the different particle types was 4.  for 7Be, similar to values reported for lab and field determinations.The sorption of all radionuclides was significantly enhanced in the presence of organic matter associated with particles, resulting in Kd one to two orders of magnitude higher than for inorganic particles only, with highest values for 7Be (logKd of 7.2).Results further indicate that EPS and frustule-embedded biomolecules in diatom cells are responsible for the sorption enhancement rather than the silica shell itself.By separating radiolabeled EPS via isoelectric focusing, we found that isoelectric points are radionuclide specific, suggesting that each radionuclide binds to specific biopolymeric functional groups, with the most efficient binding sites likely occurring in acid polysaccharides, iron hydroxides, and proteins.Further progress in evaluating the effects of diatom frustule-related biopolymers on binding, scavenging, and fractionation of radionuclides would require the application of molecularlevel characterization techniques.

Acquisition Description
Diatom cultures, sample preparation, and EPS extraction P. tricornutum (UTEX 646) was selected for culturing in autoclaved f/2 and f/2-Si media (salinity of 26) at a temperature of 19 + 1 oC with a light cycling of 14 h : 10 h under a saturating irradiance of 100 umol quanta m-2 s-1.In order to deplete the diatom of Si supply, cultures were transferred into f/2-Si medium over at least six generations by harvesting cells (2694 g, 30 min) and resuspending them in fresh f/2-Si medium.Sterile polycarbonate bottles were also used to prevent Si supply from glassware.The growth status of P. tricornutum was monitored by changes in optical density at 750 nm.Cells, frustules, and EPS were collected when P. tricornutum reached the stationary phase.
Laboratory cultures of P. tricornutum were centrifuged (2694 x g, 30 min) and filtered (0.2 um) to collect the whole cells.The frustules were repeatedly treated by using a hydrogen peroxide (30%, room temperature) treatment until bubbles were no longer generated, followed by concentrated nitric acid (HNO3) digestion (85oC, 1 h) to remove organic matter adopted from Robinson et al. (2004).
The resulting organic carbon (C), nitrogen (N), and sulfur (S) contents of the cleaned frustules were measured using a Perkin Elmer CHNS 2400 analyzer to ensure the removal of organic materials using cysteine as a standard according to Guo and Santschi (1997).
EPS extraction was followed the procedures described in Xu et al. (2011b), which minimize cell rupture and molecular alterations and maximize extraction efficiency.EPS here is referring to those biopolymers that are attached on the diatom frustules.Hereafter, EPS Si+ and EPS Si2 denote the EPS extracted from diatoms cultured under Si-replete (f/2 medium) and Si-depleted (f/2-Si medium) conditions, respectively.Briefly, laboratory cultures were centrifuged (2694 x g, 30 min) and filtered (0.2 um) when diatoms reached stationary phase.
The diatom cells were soaked with 0.5 mol L-1 sodium chloride (NaCl) solution for 10 min and followed by centrifugation at 2000 x g for 15 min to remove the medium and weakly bound organic material on the cells.The pellet from previous step was resuspended in a new 100 mL 0.5 mol L-1 NaCl solution and stirred gently overnight at 4oC.The resuspended particle solution was ultracentrifuged at 12,000 x g (30 min, 4uC), and the supernatant was then filtered through a 0.2 um polycarbonate membrane.The filtrate was desalted and collected with a 1 kDa cutoff cross-flow ultrafiltration and diafiltration membrane and then freeze-dried for later use.

Characterization of exopolymeric substances
After partitioning EPS collected from lab cultures into aliquots for freeze-drying, subsamples were analyzed for individual components.Concentration of total carbohydrate (TCHO) concentration was determined by the TPTZ (2,4,6-tripyridyl-s-triazine) method using glucose as the standard, and uronic acids were measured by the meta-hydroxyphenyl method using glucuronic acid as the standard (Hung and Santschi 2001).Protein content was determined using a modified Lowry protein assay, using bovine serum albumin (BSA) as the standard (Pierce, Thermo Scientific).C, N, and S contents were determined as described above.Iron was measured using an atomic absorption spectrometer (Varian) after overnight digestion with 12 mol L-1 HNO3 at 85oC (Von Loon 1985).To evaluate the protein size distribution pattern in EPS Si+ and EPS Si2, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to Sambrook et al. (1989) using standard molecular weight markers (Dual Xtra Standards, Bio-Rad).
Fourier transform infrared spectroscopy (FTIR) was used to characterize samples using a Varian 3100 model interfaced with a single reflection horizontal attenuated total reflectance (ATR) accessory (PIKE Technologies).A diamond plate was used as the internal reflection element.A freeze-dried EPS sample was mounted at the surface of the diamond.
Absorbance spectra from 800 to 2000 cm21 were collected and integrated using Varian Resolution Pro 4.0 software.ATR-FTIR spectroscopy provides a noninvasive way to quickly gain information about the contents of major secondary structures of biopolymers (Xu et al. 2011b;Jiang et al. 2012).Major infrared (IR) peaks were assigned according to Xu et al. (2011b) and Jiang et al. (2012).Characteristic bands found in the IR spectra of proteins and polypeptides include the amide I (1652-1648 cm-1) and amide II (1550-1548 cm-1) band.

Related Publications
Alvarado Quiroz, N. G., Hung, C.-C., & Santschi, P. H. (2006).Binding of thorium(IV) to carboxylate, phosphate and sulfate functional groups from marine exopolymeric substances (EPS).Marine Chemistry, 100(3-4), 337-353.doi:10.1016/j.marchem.2005.10.023 The absorption associated with the amide I band leads to stretching vibrations of the C=O bond of the amide, and absorption associated with the amide II band leads primarily to bending vibrations of the N-H and C-N bond.The symmetric stretching peak due to deprotonated carboxyl groups is observed at 1400 cm-1 along with the CH2 bending mode at 1455 cm-1.In the 800-1200 cm-1 regions, responses from C-O, C-O-C, P-O-P, C-O-P, and ring vibrations of the main polysaccharide functional groups are present in polysaccharide mixtures.The peaks at 1241 and 1113 cm-1 correspond to P-O stretching in phosphate groups.Processing DescriptionBCO-DMO Processing Notes:-added conventional header with dataset name, PI name, version date -modified parameter names to conform with BCO-DMO naming conventions -combined the two submitted tables together on the pH column, indicating percent activity and percent composition in the parameter names.