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8/11/2019 Elucidating the Antimicrobial Mechanisms of Silver: Significance of Higher Oxidation States and Reduction Potential
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y =6.0638x+10195
R =0.9164
0
5000
10000
15000
20000
25000
30000
35000
0 500 1000 1500 2000 2500 3000
1/[Trp](M-1)
Time (seconds)
Elucidating the Antimicrobial Mech
Significance of Higher Oxidation St
CSC 2012, Calgary, Alberta
David Lischuk B.Sc, P. Chem; Carla Spina, Ph.D
Exciton Technologies Inc.
Abstract.
For centuries, silver has been used in medicinal and sanitation applications. As early as the 8th century, the
use of silver as a blood purifier and for bad breath was documented by the Muslim physician Avicenna1. The
biocidal property of silver and other metals has bee n described as an oligodynamic effect. From the Greek
(oligos = few, and dynamis = power), it is a term used to describe how low metal ion concentrations exert
powerful biocidal effects toward lower life forms, yet are non-toxic to humans and other higher life forms,
with the relative toxicity as described in the Horsfall Series:
Ag > Hg > Cu > Cd > Cr > Ni > Co > Zn > Fe > Ca
However, the exact biochemical mechanisms or actions and interactions of silver are still under debate, where
a hypothetical sub-division of the series may be: Ag3+> Ag2+> Ag+
To elucidate the impact of silver oxidation state and reduction potential, the biological chemistry of silver
compounds and select amino acids are explored as a model system. In particular: kinetic NMR and UV-Visible
spectrometry experiments will explore the biological oxidative effects of Ag7NO11, AgO, Ag2O, and AgNO3in
aqueous medium.
Introduction.
Silver has been a ubiquitous antimicrobial agent from early history to contemporary wound care. Since the
first silver wound care device brought to market in 19894 , there has been an surge of silver-based wound care
devices available, containing a variety of silver species as seen in Table 1.
Although it is well-accepted in the wound care community that silver must be present in an ionic state to
provide antimicrobial activity, with the vast array of medicinal silvers available and the risks of antimicrobial
resistance, a more thorough understanding of the mechanism of action of silver is needed6. Current literature
has identified three primary mechanisms: protein complexation and denaturation7; DNA complexation and/or
intercalation, impeding transcription8; and cellular respiration interference, either as an electron sink or via
disruption of membrane potentials9. This work examines the effect of medicinally relevant silver compounds,
of various reduction potentials and oxidation states, on amino acid oxidation as a model system for protein
degradation. The work presented here demonstrates the unique activity of various silver compounds and
supports further investigation into oxidative mechanisms towards antimicrobial activity.
Methods.
UV-Visible spectra were obtained on a Hewlett Packard 8452A UV-Visible spectrophotometer, using UV-rated
polymeric cuvettes (path length = 10 mm) unless otherwise stated. Tryptophan, glycine and D2O were
obtained from Sigma Aldrich, silver(I,III) oxide and silver(I) oxide were obtained from Alfa Aesar, and silver
nitrate was obtained from Anachemia; all chemicals were used without further purification. Ag7NO11was
prepared using published procedures5and stored protected from light 4 oC until needed. Water for
spectrophotometry was generated using a custom Reverse Osmosis and Deionizer system (Claysmore Spring
Water).
A UV-Vis calibration curve for Tryptophan (Trp) was generated using max
= 279 nm peak, with a linear range of
0.020.30 mM (Figure 1). Glycine (Gly) exhibited weak UV-Vis absorbance (Figure 2), therefore was studied
via 1H NMR spectroscopy, as detailed below. Preliminary UV-Vis degradation profiles for silver-Trp were carried
out in a syringe, filtering insoluble components with a 0.2 m syringe filter. A typical procedure went as
follows: 9.0 ml Trp solution (0.04 g/L, 0.20 mM) was loaded into a syringe with a clinically relevant
concentration of the identified silver compound (1 mg Ag equivalent/mL solution). The reaction mixture was
agitated for a specified time, then filtered through a 0.2 m syringe filter directly into a cuvette, acquiring a
UV-Vis spectrum (190-820 nm). Blank solutions were prepared using the equivalent volumes and
concentrations of silver without Trp; blank absorbance recorded at 279 nm . From each reaction syringe, 3
spectra at different time points were obtained for the TrpAg system up to 250 minutes. For those
preliminary systems demonstrating reactivity, subsequent kinetic studies were undertaken using a bulk
reaction sampling procedure. Briefly, 200 mL of stock Trp solution was prepared, to which 0.25 g Ag-OXS was
added under vigorous stirring. 2 ml samples were removed every 5 minutes for a period of 2 hours, filtered,
and spectrum obtained.
Proton NMR studies were performed at the NMR laboratory at the University of Alberta; an Agilent/Varian
Inova 400 MHz instrument was used for all spectra. A concentrated stock solution of Gly in D2O (100 g/L, 1.3
M) was prepared and sealed in a glass ampoule until needed. The concentrated stock solution was used to
ensure that spectra could be obtained rapidly, in order to obtain reliable kinetic data. A baseline spectrum was
obtained, and then 10 mg of either Ag7NO11or Ag2O was added directly to the tube, reacted for 30 minutes
until the first spectrum was obtained. Consecutive spectra were obtained at 30 minute intervals until 3 hours
had elapsed. The procedure was repeated for the other oxide, in fresh solution.
Acknowledgements.
The authorswouldlike tothank Dr.Michael Serpe for the use ofhisspectrophotometer,Mr.Avijeet Sarkerand Mr.Mark Mizolskifor helpfuldiscussions,and the University ofAlberta NMR laboratory for the performance ofthe NMRexperiments.
References.
1)Bhattacarya R, Mukherjee P.AdvancedDrugDelivery.Reviews. 2006; 60: 1289-1306. 2)Wadhera A,Fung M. DermatologyOnline Journal.2005; 11: 12 3) Fox, CL.ArchSurg .1968; 96(2): 184-188. 4)http://www.accessdata.fda.gov/scripts/cdrh/c
Trauma.1987; 27(3): 301-303.
7)Liau SL,et al. Lettersin AppliedMicrobiology. 1997; 25: 279-283. 8)Modak SM,Fox CL. BiochemistryPharmacology.1973; 22(2391): 404.9) Dibrov P,et al.AntimicrobialAgents inChemotherapy.2002; 46(8): 2668-2670. 10)Harriman,A. J PhysCh
ChemistryandPhysics: 88thEdition(Chemical Rubber Company),2007
This work was sponsored by Exciton Technologies Inc.
SilverSpeciesChemical
Compound
Oxidation
State
Solubilityin
water
(maximum
ppm Ag)
Generation ofclinically
relevant silverspecies
Metallic Silver Ag Ag0 0.0
Oxidation (byairor
biological fluids):
generatingsurfaceAg2O
Silver
Sulfadiazine
(SSD)
Ag(C10H9
N4O2S)Ag1+ 0.3
Dissolution: generating
Ag1+and the antibiotic
sulfadiazine
SilverChloride AgCl Ag1+ 1.4
Dissolution: generating
Ag1+and the Cl -counter-
ion
Silver(I)Oxide Ag2O Ag1+ 22.4
Dissolution: generating
Ag1+andOH-via reaction
with water
SilverSulfate Ag2SO4 Ag1+ 1638
Dissolution: generating
Ag1+andthe SO4-
counter-ion
SilverNitrate AgNO3 Ag1+ 775000
Dissolution: generating
Ag1+and the NO 3-
counter-ion
SilverSodium
Hydrogen
Zirconium
Phosphate
Ag0.1-0.5)Na(0.1-0.8)H(0.1-0.8)Zr2(PO)3
Ag1+ n/a
Dissociation/ion
exchange: generatingfree
Ag1+which canbe
replacedin thespeciesby
anequivalent cation from
the wound fluid
SilverOxysalts Ag7NO11Ag1+, Ag2+,
Ag3+n/a
Decomposition ofthe
solidmaterial: generating
Ag1+, Ag2+, Ag3+, aswell
asO 2, NO3-, and other
species
Table 1: Propertiesof Clinically Common Silver Species
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
255 355 455 555 655 755
Absorbance
(nm)
198 g/LGly
40 g/LGly
Figure 2: UV-Visspectraof glycine at 198 and 40 g/L.
0
0.5
1
1.5
2
2.5
3
180 280 380 480 580 680 780
Absorbance
(nm)
y =5.1254x+0.0204
R =0.9993
0
0.5
1
1.5
2
2.5
0 0.1 0.2 0.3 0.4 0.5
Absorbance
[Trp] (mM)
Figure 1: UV-Visspectraof tryptophanat aseries of applicable concentrations.Inset:Calibration curve, using the 279 nm peak.Note that the red point in the inset isnotincluded in the curve, indicating the limit of linearity.
0
0.2
0.4
0.6
0.8
1
1.2
190 290 390 490 590 690 790
Absorbance
(nm)
a
0
0.2
0.4
0.6
0.8
1
1.2
190 290 390 490 590 690 790
Absorbance
(nm)
b
0
0.2
0.4
0.6
0.8
1
1.2
190 290 390 490 590 690 790
Absorbance
(nm)
c
0
0.2
0.4
0.6
0.8
1
1.2
190 290 390 490 590 690 790
Absorbance
(nm)
d
Figure 3: Typical spectraof tryptophanupon contact with varioussilver species.Clockwise from upper-left: a)Ag-OXS; b)AgO; c) Ag2O; d)AgNO3.The initial spectrumin all casescomesfrom the stocktrp solution.
y =7.0452ln(x)+48.864
R =0.9788
0
10
20
30
40
50
60
70
80
90
100
0 50 100 150 200 250
%
[Trp]
Time (min)
Figure 4: %change in [Trp] over time, upon exposure to Ag-OXS
Figure 5: Linear fit of 1/[ Trp]vs.time, showing an approximately linear relation
Figure 6: Reference 1HNMR spectraof a)glycine stocksolution (1.3 M in D 2O); b)glycine and Ag2O; c)glycine and Ag7NO11
*Solubility in 0.1 M NaNO3,Clinical Chemistry, 199516
Information not available in published literatureSolubility not applicable asAg 7NO11slowly decomposesin solution (dataon file)