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olda Direct covalent attachment of C TPP to
Direct covalent attachment of C1TPP to the above peptides was accomplished under anhydrous conditions with carbodiimide-mediated coupling. Stock solutions of C1TPP, EDC, NHS, bactenecin, and indolicidin were prepared in absolute ethanol prior to mixing; as neither PME nor CeMe is highly soluble in absolute ethanol, stock solutions of these peptides were prepared in 4:1 (v/v) ethanol:acetonitrile. The composition of the reaction mixtures (molar equivalents) were as follows: 1 peptide: 1.1 C1TPP: 1.2 EDC: 1 NHS. After completion of the coupling reaction (≫2h), reaction mixtures were diluted with water and dialyzed (1000molecular weight cutoff) exhaustively against water and phosphate-buffered saline (PBS). Construct concentrations are estimated based on the initial concentration of AMP and the total final volume of the preparation. Metal variants were prepared through incubation of porphyrin-AMP constructs (25μM) with metal salts (vanadium (III) bromide; zinc chloride; cobalt (II) chloride; 50μM) in aqueous solution [8]. The solutions were thoroughly mixed and heated to 60°C for 3 h before storage at 4°C for at least 48h. Metal incorporation was evaluated based on changes in absorbance and fluorescence characteristics. Construct names are abbreviated to indicate the metal, porphyrin, and AMP used; for example, CoC1-Ind is the cobalt variant of C1TPP conjugated to the indolicidin peptide. The bacterial targets for binding studies, Escherichia coli (XL1 blue) and Bacillus cereus (ATCC 10987), were grown to mid-log in Luria (37°C) or tryptic soy broth (30°C), respectively, before harvesting by centrifugation at 1200×g for 10min (4°C). Cell pellets were washed twice with phosphate-buffered saline (PBS), pH7.4 and resuspended in 1/5 original volume of PBS. Cell numbers (in PBS) were then counted by flow cytometry (Accuri C6). Cell suspensions not used immediately were diluted with an equal volume of 60% glycerol in PBS before storage at −20°C. Prior to analysis, olda were diluted in PBS to the appropriate concentrations. A Tecan XSafire microtiter plate reader was used to measure the absorbance and fluorescence of the porphyrin-AMP constructs in the presence and absence of bacterial targets. Absorbance was measured from 360 to 800nm in steps of 2nm. Fluorescence emission spectra were collected from 500 to 800nm (2nm steps) using 415nm excitation while fluorescence excitation spectra were collected from 385 to 619nm (2nm steps) at 730nm emission. In both cases, a gain of 160 was applied with 50 flashes at 400Hz, and an integration time of 20μs was employed. All experiments were conducted in 15% DMSO in order to ensure a homogeneous solution; porphyrin-AMP constructs have low water solubility due to the hydrophobicity of the porphyrin utilized and inherent solubilities of the AMPs. Cell concentrations ranging from 107 to 103cells/mL were employed. Indicator concentrations were varied from 12 to 0.1μM. In all cases, difference spectra were calculated as the point-by-point subtraction of indicator only spectra from spectra collected for the indicator in the presence of the target. Fluorescence spectra for cell pellets utilized a total initial volume of 765μL with 8μM indicator and varying target cell concentrations in an Eppendorf tube (1.5mL). As above, all experiments were conducted in 15% DMSO. The fluorescence of the initial solution was measured before centrifuging at 7500rpm for 10min. Supernatant was then removed (665μL), and the remaining solution and pellet were mixed thoroughly to resuspend components. The fluorescence of both the resulting supernatant and the resuspended pellet were collected using the microtiter plate protocol described above. CD experiments were carried out using a Jasco J-815 circular dichroism spectrometer (Jasco Inc., Easton, MD). An estimated peptide concentration of ~10μM was used for each CD measurement. CD spectra were recorded in phosphate buffer (PB), pH7.0 in the presence or absence of 25mM sodium dodecylsulfate (SDS; critical micelle concentration of SDS ~8.0mM), as indicated. The wavelength scan was completed from 190 to 300nm in a thermally controlled (20°C) quartz cell having a 0.5cm path length. Each CD spectrum was the average of three scans collected at a scan rate of 50nm/min, using a data pitch of 1nm, digital integration time (D.I.T) of 8s, band width of 1nm, and a scan speed of 50nm per minute. The background spectra (i.e., PB buffer ±25mM SDS in the cuvette) were measured first, followed by that of the peptide solution. Subtraction of the background (solution) spectrum from that of the peptide solution yielded the spectrum of the peptide in the absence or presence of SDS.