Characterization of particulate proteins in Pacific surface waters

Abstract

We investigated molecular characteristics of particulate proteins in Pacific surface waters using two-dimensional electrophoresis (2DE). Most proteinaceous materials estimated by dye-binding methods were characterized by the 2DE unresolved acidic materials with a broad range of molecular mass and the 2DE unresolved low molecular mass materials with a broad range of isoelectric point. The 2DE unresolved acidic and low molecular mass materials were considered to comprise peptides conjugated with acidic saccharides and degradation products (peptides) of proteins, respectively, which indicates that almost all proteins in living organisms failed to survive in detrital particulate organic matter (POM). Nevertheless, 23 discrete proteins were distinguished by the 2DE. Electrophoretic patterns of the discrete proteins indicated that they were a component of detrital POM. Three discrete proteins were subjected to N terminal amino acid sequence analysis. Two proteins out of three could not be determined because their N termini were blocked, and one protein was determined from the N terminus to the ninth amino acid residue. A homology search revealed that the N terminal amino acid sequence of the protein agreed completely with that of 70 kDa heat shock protein (HSP70) derived from photosynthetic organisms. HSP70 is a major member of the molecular chaperones that protect or repair proteins from damage under conditions of environmental stress. The occurrence of HSP70 in this study demonstrated that phytoplankton were able to induce the molecular chaperone(s). Clarification of factor(s) controlling induction of chaperones will enable us to assess the actual environmental stress on phytoplankton at the biomolecular level.The particulate organic matter (POM) in surface water is a complex mixture of living biomass and nonliving detritus. The contribution of living biomass to the bulk POM is generally 1 order of magnitude lower than that of detrital organic matter (e.g., Volkman and Tanoue 2002). The particulate combined amino acids (PCAA) are the largest identified fraction in surface POM (e.g., Wakeham et al. 1997), and dye-binding colorimetric methods demonstrated that proteinaceous materials were a major component of POM (Setchell 1981;Long and Azam 1996). Phytoplankton is a primary producer of organic matter in the sea, and most combined amino acids in phytoplankton are in the form of proteins. However, heterotrophic processing may convert cellular proteins of phytoplankton to other forms before its incorporation into detrital pool. At present the chemical nature of the detrital combined amino acids and proteinaceous materials is not well documented, and the processes by which organic matter produced by phytoplankton is transferred to detrital POM are not clear. The characterization of proteinaceous materials in POM at the molecular level is 1 Corresponding author (tanoue@ihas.nagoya-u.ac.jp). AcknowledgmentsWe thank the captains and crews of the RV Hakuho-maru KH93-4, KH99-3, RV Tansei-maru KT00-13, and TS Seisui-ma...

Proteomics in environmental and technical microbiology

“…In another study, filtered particulate proteins from Pacific surface water were subsequently extracted with lysis buffer, further concentrated by precipitation trichloroacetic acid, and separated by 2DE. Despite a low number of spots in the 2D gels and high background staining, the HSP 70 was detected by Edman sequencing . A slightly different protocol was applied to microbes filtered from Chesapeake Bay.…”

Protein expression from zooplankton communities in a metal contaminated NW mediterranean coastal ecosystem

“…Proteins for 2-DE were differently extracted, according to a modified method of Saijo and Tanoue (2004), more intensive than that used for 1-DE. Indeed, a fraction of 0.1 g of zooplankton powder was homogenized with 1 mL of a sample solution consisting of urea (7 M), thiourea (2 M), 3-[3-(cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS, 1% w/v), polyethyleneglycol p-teroctylphenylether (Triton X-100, 3% v/v), dithiothreitol (DTT, 1% w/v), carrier ampholytes (0.2% v/v), BPB (0.002% v/v).…”

Evaluation of tangential flow ultrafiltration procedures to assess trace metals bound to marine dissolved organic matter

“…This technique offers the advantage of high pre-concentration factors and the possibility of using large sample volumes. UF procedures usually require membranes with a nominal molecular weight cut-off (MWCO) of 10 kDa, which guarantees the isolation of marine DOM of high molecular weight (marine proteins included) [10][11][12][13][14][15][16][17][18][19][20]. Nevertheless, marine DOM can be lost by adsorption onto the UF membranes [14,15], and this problem is especially important when dealing with marine proteins due to their highly adsorptive nature.…”

“…Nevertheless, marine DOM can be lost by adsorption onto the UF membranes [14,15], and this problem is especially important when dealing with marine proteins due to their highly adsorptive nature. Different reagents such as sodium azide and sodium dodecylsulphate (SDS) are commonly added to the filtered seawater before UF to prevent protein adsorption onto the ultrafiltration membrane [15][16][17][18][19][20]. On other occasions, UF procedures based on membranes with nominal MWCO of b 10 kDa have been proposed, and 1 kDa MWCO membranes have been used in several studies [11,[21][22][23][24][25].…”