Adsorption and desorption kinetics of mastoparan X in 220 μ L 2 μ M solutions.

<p>The kinetics were investigated in borosilicate glass vials and polypropylene tubes. (<i>A</i>) Adsorption kinetics in buffer. (<i>B</i>) Adsorption kinetics in 1 mM POPC/POPG (3:1) LUV solution. (<i>C</i>) 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.</p

Example of HPLC chromatogram.

<p>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.</p

Effect of NaCl concentration on peptide loss in 220 μ L 2 μ M solutions.

<p>(<i>A</i>) 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. (<i>B</i>, <i>C</i>, and <i>D</i>) Percentage of recovered peptide for 220 μL 2 μM mastoparan X (<i>B</i>), melittin (<i>C</i>), and magainin 2 (<i>D</i>) 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 (<i>A</i>) 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.</p

Adsorption of Cationic Peptides to Solid Surfaces of Glass and Plastic

<div><p>Cationic membrane-active peptides have been studied for years in the hope of developing them into novel types of therapeutics. In this article, we investigate an effect that might have significant experimental implications for investigators who wish to study these peptides, namely, that the peptides adsorb to solid surfaces of glass and plastic. Specifically, we use analytical HPLC to systematically quantify the adsorption of the three cationic membrane-active peptides mastoparan X, melittin, and magainin 2 to the walls of commonly used glass and plastic sample containers. Our results show that, at typical experimental peptide concentrations, 90% or more of the peptides might be lost from solution due to rapid adsorption to the walls of the sample containers. Thus, our results emphasize that investigators should always keep these adsorption effects in mind when designing and interpreting experiments on cationic membrane-active peptides. We conclude the article by discussing different strategies for reducing the experimental impact of these adsorption effects.</p></div

Peptide loss during 1 h incubation of 220 μ L solutions in sample containers.

<p>The percentage of recovered peptide was measured as a function of the peptide concentration for mastoparan X (<i>A</i>), melittin (<i>B</i>), and magainin 2 (<i>C</i>) 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.</p

Peptide concentration standard curves.

<p>(<i>A</i>, <i>B</i>, and <i>C</i>) Peptide peak area as a function of the peptide concentration of mastoparan X (<i>A</i>), melittin (<i>B</i>), and magainin 2 (<i>C</i>) standard solutions. The solid lines are the best least squares fits of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122419#pone.0122419.e001" target="_blank">Eq 1</a> to the data. (<i>D</i>, <i>E</i>, and <i>F</i>) Percentage of recovered peptide, as calculated by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122419#pone.0122419.e002" target="_blank">Eq 2</a>, as a function of the peptide concentration of mastoparan X (<i>D</i>), melittin (<i>E</i>), and magainin 2 (<i>F</i>) 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.</p

Peptide loss during successive transfers of 250 μ L 5 μ M solutions between sample containers.

<p>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 (<i>A</i>), melittin (<i>B</i>), and magainin 2 (<i>C</i>) 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.</p

Critical micelle concentration assessed via fluorescence spectroscopy.

<p>As an example, pyrene fluorescence excitation spectra are shown for a concentration sequence of Adec8 in (A). The formation of micelles is monitored via partitioning of pyrene into the hydrophobic cavity of the forming micelles. This induces the observed peak-shift of pyrene (A), which is quantified via the intensity ratio R = I₃₃₉/I₃₃₃ plotted in (B) as a function of peptide concentration. In the current work, the cmc is defined as the point where R deviates from the background signal.</p

Partitioning of MPX, Ala1 and Ala14 onto POPC∶POPG (3∶1) LUVs studied via ITC at 37°C.

<p>In panel (A), heat traces of 25 mM LUVs injected into 20 µM peptide (19×2 µL injections) are shown. Panel (B) shows the corresponding heat of reaction, Q, as a function of number of injections. The solid lines in (B) represent fits to the data. In panel C, the change in potency of Ala1 and Ala14 for neutral or anionic lipid membranes measured relative to MPX, is evaluated via the ratio <i>K_ins</i>/<i>K_ins(MPX)</i> and <i>K_eff/K_eff(MPX)</i> respectively. The membrane charge selectivity of the peptide (ability to select between neutral and anionic lipid membranes) is assessed via the partitioning coefficient ratio <i>K_eff/K_ins</i> shown in (C).</p

Structures of MPX and eight analogues.

<p>Ala1 and Ala14 have alanine substitution at position 1 or 14, and are analogues with reduced hydrophobicity. Leu8 has a leucine substitution in position 8 resulting in augmented hydrophobicity. Adec1, Adec8 and Adec14 have 2-amino-decanoic acid substitution in position 1, 8 or 14 respectively, and constitute the most hydrophobic analogues of MPX considered in this study. PAMPX and OAMPX are N^α-terminal propanoic and octanoic acid acyl analogues of MPX, respectively.</p