(582ee) Structural and Activity Characterization of Irradiated Antimicrobial Peptide (WLBU2) for Use in Blood Processing and Biomedical Applications
AIChE Annual Meeting
2013
2013 AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Poster Session: Bioengineering
Wednesday, November 6, 2013 - 6:00pm to 8:00pm
Introduction:
Sepsis afflicts
approximately 750,000 individuals and kills 140,000 in North America annually,
costing an estimated $17 billion/year to treat sepsis.1 Current treatment involves
administration of IV fluids, antibiotics, vasopressors, and other medications,
which are suboptimal at best, resulting in prolonged hospital stays. Passing
blood through a sorbent device (hemoperfusion) to specifically remove targets
such as endotoxin and bacterial cells holds promise for rapid treatment of
acute sepsis. Clinical use of hemoperfusion in this context is based on
immobilized polymyxin B (PMB),2,3 but remains limited owing to
ineffective endotoxin removal, and serious complications (e.g., nephrotoxicity, neurotoxicity, monocyte stimulation, substantial protein loss)
associated with PMB and PMB-based devices.2,4-7 The synthetic, cationic
amphiphilic peptide (CAP) WLBU2 has greater
antimicrobial activity than PMB, works against a much broader spectrum of
Gram-positive and Gram-negative bacteria, and shows higher selectivity
for pathogenic entities over host cells.8,9 The long term
goal of work conducted in our laboratory is the covalent tethering of WLBU2 to
pendant polyethylene oxide chains for capture of LPS. One strategy for
covalently attaching these chains to surfaces involves the use of γ-irradiation.10,11 In this work the
effect of γ-irradiation on the structure and function of WLBU2 is
investigated.
Materials
and Methods:
WLBU2
Preparation
Lyophilized WLBU2
was purchased from Genscript (Piscataway, NJ). HPLC-grade water and deuterated
water (D2O) were used to make stock WLBU2 solutions at 5 mg/mL.
Solutions were aliquoted and diluted to 0.3 and 1.0 mg/mL and irradiated to 3
kGy by a 60Co source (OSU Radiation Center). Further dilutions and
sample preparations were performed using the irradiated samples with HPLC or
deuterated water.
Proton NMR
Spectroscopy
Proton nuclear magnetic
resonance (1H-NMR) spectra were obtained using a 400 MHz Robinson
NMR instrument with TopSpin 2.1 software at 25 °C using 1 mg/mL WLBU2 in D2O with
trimethylsilyl propanoic acid (TMSP) added as a spectrum reference. Each sample
was measured using 64 scans.
UV-Vis
Spectroscopy
Ultraviolet-visible
(UV-vis) absorbance measurement scans of peptide solutions were obtained
between 200 and 400 nm at 1 nm intervals, using a Genesys 6 UV-vis
spectrophotometer. Measurements were performed at 20 °C in a quartz cuvette.
CD
Spectroscopy
Circulary dichroism
(CD) measurements were taken with a Jasco J-815 CD Spectrometer at room
temperature between 180 and 280 nm with samples in water or HClO4
using 5 scans per experimental trial. Aliquots of WLBU2 and irradiated WLBU2
were diluted to 0.3 mg/mL in water and 0.2 mg/mL in 0.2 M or 0.5 M HClO4
to induce α-helix conformation. DichroWeb
was used to deconvolute and determine the percent helicity of the samples by
using the CONTIN or CDSSTR preset methods.
Radial Diffusion
Bacterial Inhibition Assay
The assay was
performed on E. coli (DH5α) and P. pentosaceus. Luria broth
(LB) and Lactobacilli MRS broth were used to grow E. coli and P.
pentosaceus, respectively, in suspended media at 37 °C overnight to
at least 4 x 108 CFU/mL. Bacteria were added to the under layer for
a final concentration of 4 x 107 CFU/mL and poured into plastic
petri dishes. Small wells were punched in the cooled bacteria-under layer gel
using a truncated P-1000 pipette tip. Peptide samples were added into the wells
and allowed to diffused for 2 hours. An over layer containing nutrients was
poured on top of the under layer and cooled to harden. Plates were incubated in
overnight at 37 °C. Image analysis was performed using ImageJ software. Trials
were performed in triplicates.
Results
and Discussion:
The deviation from standard WLBU2 spectra indicated a structural change upon
irradiation (Figure 1a). The NMR and UV-vis spectra
strongly suggest that irradiation oxidized or opened the tryptophan ring.
Although structure was altered, γ-WLBU2 activity against
Gram-positive and Gram-negative bacteria was not negatively affected (Figure 1b).
Figure
1:
(a) 1H-NMR result comparing WLBU2 (top, red) and γ-WLBU2
(bottom, blue). Large signal peaks at 0 and 4.8 represent the reference and H2O,
respectively. Arginine and valine signals were approximately the same from 0
to 6 ppm. Loss of the signal associated with tryptophan (7 to 7.6 ppm)
indicates a chemical change. (b) Mean diameter of WLBU2 kill zones against E.
coli and P. pentosaceus. A small effect of concentration was
observed. Deuterated water (D2O) had no effect on bacterial
inhibition, with or without WLBU2 peptide.
Conclusions: The data presented indicate
that while WLBU2 undergoes a chemical and structural change upon
γ-irradiation, this change does not negatively impact the activity of
WLBU2 against Gram-positive or Gram-negative bacteria. Further investigation
will be conducted to elucidate the exact structural changes of WLBU2 as well as
the extent of change in capture affinity of WLBU2 once tethered to pendant PEO chains.
References
6. Ikeda T. Hemoadsorption in critical care.
Therapeutic Apheresis 2002;6(3):189-192.