Fig 1.
The chromatogram was acquired from a 200 μL standard solution of 5 μM mastoparan X to which 50 μL 5 mM POPC/POPG (3:1) LUV had been added. Both a peptide peak and a lipid peak are visible in the chromatogram.
Fig 2.
Peptide concentration standard curves.
(A, B, and C) Peptide peak area as a function of the peptide concentration of mastoparan X (A), melittin (B), and magainin 2 (C) standard solutions. The solid lines are the best least squares fits of Eq 1 to the data. (D, E, and F) Percentage of recovered peptide, as calculated by Eq 2, as a function of the peptide concentration of mastoparan X (D), melittin (E), and magainin 2 (F) standard solutions. The concentrations on the horizontal axes are, in all panels, the concentrations of the 200 μL standard solutions before 50 μL 5 mM POPC/POPG (3:1) LUV solutions were added to the standard solutions. The data are the average of three separate experiments. The error bars show the standard deviations. The error bars are not shown if they are smaller than the symbols. Linear concentration standard curves were obtained for all three peptides.
Fig 3.
Peptide loss during 1 h incubation of 220 μ L solutions in sample containers.
The percentage of recovered peptide was measured as a function of the peptide concentration for mastoparan X (A), melittin (B), and magainin 2 (C) solutions in borosilicate glass vials, polypropylene tubes, or Protein LoBind tubes. In all panels, the data are the average of two separate experiments. The error bars show the standard deviations. The error bars are not shown if they are smaller than the symbols. The data demonstrate that all three peptides tend to adsorb to the walls of the borosilicate glass vials and polypropylene tubes; at low peptide concentrations, only 10–20% of the expected peptide contents were recovered in these containers. In contrast, peptides do not absorb to Protein LoBind tubes to the same extent.
Fig 4.
Peptide loss during 1 h incubation of 2 mL 2 μ M solutions in sample containers.
The percentage of recovered peptide was measured for mastoparan X, melittin, and magainin 2 solutions in borosilicate glass vials, polypropylene tubes, Protein LoBind tubes, or quartz glass cuvettes. The data are the average of two separate experiments. The error bars show the standard deviations. The data demonstrate that peptide surface adsorption is not just a phenomenon that occurs at high surface area-to-solution volume ratios.
Fig 5.
Peptide loss during successive transfers of 250 μ L 5 μ M solutions between sample containers.
The solutions were successively transferred between borosilicate glass vials, polypropylene tubes, or Protein LoBind tubes. The percentage of recovered peptide was measured for mastoparan X (A), melittin (B), and magainin 2 (C) solutions as a function of the number of sample containers in which the solutions had been incubated. In all panels, the data are the average of two separate experiments. The error bars show the standard deviations. The error bars are not shown if they are smaller than the symbols. The data show that peptide is dramatically lost when peptide solutions are successively transferred between borosilicate glass vials or polypropylene tubes.
Fig 6.
Effect of NaCl concentration on peptide loss in 220 μ L 2 μ M solutions.
(A) Percentage of recovered peptide for 200 μL 2 μM standard solutions prepared in 10 mM HEPES buffers of varying NaCl concentration directly in limited volume inserts. (B, C, and D) Percentage of recovered peptide for 220 μL 2 μM mastoparan X (B), melittin (C), and magainin 2 (D) solutions incubated in 10 mM HEPES buffers of varying NaCl concentration for 1 h in borosilicate glass vials, polypropylene tubes, or Protein LoBind tubes. The data are the average of two separate experiments, except in (A) in which three experiments are averaged. The error bars show the standard deviations. The percentage of recovered peptide was not strongly influenced by the NaCl concentration.
Fig 7.
Adsorption and desorption kinetics of mastoparan X in 220 μ L 2 μ M solutions.
The kinetics were investigated in borosilicate glass vials and polypropylene tubes. (A) Adsorption kinetics in buffer. (B) Adsorption kinetics in 1 mM POPC/POPG (3:1) LUV solution. (C) Desorption kinetics. The desorption from the container walls was induced by 1 mM POPC/POPG (3:1) LUV. The data are the average of two separate experiments. The error bars show the standard deviations. Generally, adsorption and desorption are fast processes that take place within a few seconds.