Polycationic Peptides from Diatom Biosilica That Direct Silica Nanosphere Formation
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S. Mann J. Webb R. J. P. Williams Eds . Biomineralization: Chemical and Biochemical Perspectives (VCH Weinheim Germany 1989).
J. Pickett-Heaps A. M. M. Schmid L. A. Edgar in Progress in Phycological Research F. E. Round and D. J. Chapman Eds. (Biopress Bristol UK 1990) vol. 7 pp. 1–169.
B. E. Volcani in Silicon and Siliceous Structures in Biological Systems T. L. Simpson and B. E. Volcani Eds. (Springer New York 1981) pp. 157–200.
Silica precipitation assay. A solution of orthosilicic acid was freshly prepared by dissolving tetramethyl-orthosilicate ester in 1 mM HCl to a final concentration of 1 M. Proteins to be tested for silica precipitation activity were dissolved in 100 mM sodium phosphate–citrate (buffered to the appropriate pH) to a final volume of 10 μl. Subsequently 1 μl of the 1 M orthosilicic acid solution was added and samples were incubated for 5 min at ambient temperature. Samples were centrifuged for 5 min at 14000 g and pellets were washed three times with H 2 O to remove free silicic acid and phosphate. Washed pellets were resuspended in 10 μl of 1 M NaOH and silica was dissolved by incubation at 95°C for 30 min. Silicium concentrations were determined in these solutions by the β-silicomolybdate method (19).
Purification of silaffin-1A. HF extract from C. fusiformis cell walls was prepared as described (12) and fractionated on a size-exclusion column (Superdex-Peptide HR-10/30 Amersham Pharmacia Biotech). Chromatography was performed in buffer A [250 mM NaCl 20 mM tris-HCl (pH 7.5)] at a flow rate of 250 μl/min. Eluting material was recorded at 226 nm and fractions were analyzed by Tricine–SDS-PAGE (13) and Coomassie blue staining. Fractions containing pure silaffin-1A were pooled dialyzed against H 2 O lyophylized and dissolved in H 2 O to a final protein concentration of 10 to 30 μmol/ml.
N. Kröger R. Deutzmann M. Sumper data not shown.
R. K. Iler The Chemistry of Silica (Wiley New York 1979).
Protein concentration was determined by the bicinchoninic acid assay as described [
Peptide concentration was determined after acid hydrolysis by HPLC analysis of the PTC amino acid derivatives.
We thank G. Lehmann and E. Hochmuth for technical assistance; T. Maurer and H. R. Kalbitzer for NMR analysis; and F. Siedler and H. Sarioglou for MS/MS analysis. We are indebted to G. Wanner for field-emission SEM analysis and R. Wetherbee for help with TEM analysis. We greatfully acknowledge W. Tanner for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 521).