An Action Spectrum of the Riboflavin Photosensitized Inactivation of Lambda Phage

Article (PDF Available)inPhotochemistry and Photobiology 81(2):474-80 · November 2004with 54 Reads
DOI: 10.1562/2004-08-25-RA-292 · Source: PubMed
Abstract
The Action Spectrum of riboflavin (RB) sensitized inactivation of lambda phage was determined between 266 and 575 nm. Below 304 nm, RB depresses the phage reduction by screening phage from radiation that it would otherwise absorb directly. Between 308 and 525 nm, RB sensitizes the inactivation of phage. Enhanced phage reduction is observed at 320 and 500 nm because of binding of RB to the phage and the shifting of the absorption curve of the phage-bound flavin relative to free flavin in phosphate-buffered saline. Enhanced inactivation at 320 and 500 nm and depressed phage inactivation between 360 and 410 nm is also influenced by the inner filter effect.
Photochemistry and Photobiology, 2005, 81: 474–480
An Action Spectrum of the Riboflavin-photosensitized Inactivation of
Lambda Phage
{
Christopher B. Martin
y1
, Erin Wilfong
1
, Patrick Ruane
2
, Raymond Goodrich
2
and Matthew Platz*
1
1
Department of Chemistry, The Ohio State University, Columbus, OH
2
Navigant Biotechnologies, Lakewood, CO
Received 25 August 2004; accepted 22 December 2004
ABSTRACT
The Action Spectrum of riboflavin (RB) sensitized inactivation
of lambda phage was determined between 266 and 575 nm.
Below 304 nm, RB depresses the phage reduction by screening
phage from radiation that it would otherwise absorb directly.
Between 308 and 525 nm, RB sensitizes the inactivation of
phage. Enhanced phage reduction is observed at 320 and 500
nm because of binding of RB to the phage and the shifting of
the absorption curve of the phage-bound flavin relative to free
flavin in phosphate-buffered saline. Enhanced inactivation at
320 and 500 nm and depressed phage inactivation between 360
and 410 nm is also influenced by the inner filter effect.
INTRODUCTION
Blood products are tested extensively for the presence of pathogens
before administration in the developed world. Nevertheless, there
exists a small, but finite, risk of transmission of infectious agents in
transfusion medicine (1). The risks of viral infection are because of
the ‘‘window period’’; the period of time between the infection of
a donor and the development of detectable levels of antibodies
(2,3). Nucleic acid testing was introduced for human immunode-
ficiency virus and hepatitis C virus in the United States in 1998 and
has shortened the window period and further decreased the
incidence of units of blood products containing pathogens (4,5).
This technology has greatly reduced the risk because the window
period has been decreased dramatically. Nevertheless, research
efforts to develop a methodology for the photochemical pathogen
reduction of blood products remains an active field. Psoralens (6),
methylene blue (7–9), porphyrins (10,11) and, more recently,
riboflavin (RB) (12) (Fig. 1) are under study as photosensitizers.
RB (vitamin B
2
) is a vitamin essential to the human diet. It is
present in aerobic organisms and is found in many foodstuffs such
as milk, beer, eggs, yeast and leafy vegetables. It is also the
precursor for flavin mononucleotide (FMN) and flavin adenine
dinucleotide, which are major coenzymes that participate in
a number of one-electron processes in the human body (13).
RB has absorption maxima at 220, 265, 375 and 446 nm in water
and is yellow-orange in color. When aqueous solutions containing
RB are exposed to sunlight, RB is converted into lumichrome (LC)
under neutral conditions and into lumiflavin (LF) in alkaline
solutions (13,14). LC is also a known metabolic breakdown
product of RB in the human body (Fig. 2) (15).
Flavin systems are known to be photochemically active, and
products of flavin photochemistry are known to react with a host of
biological molecules, often with clinical implications (13,16,17).
Neonates often have immature livers that cannot metabolize
bilirubin (BR), a metabolite of hemoglobin, into smaller water-
soluble molecules that can then be excreted (18). Jaundiced infants
are often treated by irradiation with visible light. Because of the
coincidental ultraviolet–visible (UV-VIS) absorption properties of
BR and RB, infants who have undergone phototherapy for BR also
experience in vivo RB photodegradation (19–21).
Studies in Denmark have investigated long-term effects on
prematurely born individuals who have undergone BR photo-
therapy and simultaneous RB photodegradation. This study was
conducted over the time span of several decades and involved more
than 50 000 individuals. They concluded that there was no
increase, within statistical uncertainty, in the occurrence of cancer
in individuals receiving BR phototherapy (22). Thus, RB has
significant safety advantages relative to synthetic sensitizers. To
better understand the mechanism of action of RB, we have studied
the RB-sensitized photoinactivation of lambda phage as a function
of wavelength. In this study, we are pleased to report the action
spectrum (AS) of the inactivation of lambda phage in phosphate-
buffered saline (PBS).
MATERIALS AND METHODS
Luria Burtani media. Dehydrated Luria Burtani (LB) media (Sigma–
Aldrich Chemicals, St. Louis, MO; 25 g) is dissolved in 1000 mL of water,
and the pH is measured with a calibrated pH meter. The pH of the solution
is then adjusted to 7.4 60.1 by dropwise addition of concentrated NaOH.
The solution is sterile filtered through a 0.22 lm filter under reduced
pressure.
Growth media. Growth media is prepared by adding 10 mL of a 20%
maltose solution (20 g in 100 mL of water) to 1000 mL of LB media,
prepared as described earlier. This solution is then sterile filtered through
{Posted on the website on 28 December 2004
*To whom correspondence should be addressed: Department of Chemistry,
The Ohio State University, 100 West 18th Ave., Columbus, OH 43210,
USA. Fax: 614-292-5151; e-mail: platz@chemistry.ohio-state.edu
Current address: Department of Chemistry and Physics, Lamar University,
Beaumont, TX, USA.
Abbreviations: AS, action spectra (action spectrum); BR, Bilirubin; DEPC,
diethylpyrocarbonate; EDTA, ethylene diamine tetraacetate; FMN, flavin
mononucleotide; LB, Luria Burtani; LC, lumichrome; LF, lumiflavin;
PFU, plaque forming units; PBS, phosphate-buffered saline; RB,
riboflavin; RNase, ribonuclease; SM, storage media; TSA, Trypticase
soy albumin; UV-VIS, ultraviolet–visible.
Ó2005 American Society for Photobiology 0031-8655/05
474
a 0.22 lm filter under reduced pressure. Growth media is used in the
generation of Escherichia coli and the isolation of lambda phage.
LB (Top) agar. To 1000 mL of prepared LB media is added 7.0 g of
dehydrated agar (Bacto Laboratories Pty., Liverpool, Australia). The
resulting mixture is stirred magnetically and heated to a rapid boil to en-
sure that all the agar has dissolved. After the agar had dissolved, the
agar solution is then dispensed into 500 mL bottles in 400 mL portions
and sterilized by autoclaving. This is the agar that is referred to in the
plaque assay.
Storage media. To 950 mL of water is added 5.8 g of NaCl, 50 mL of 1 M
tris (hydroxymethyl) aminomethane hydrochloride buffer (OSU Reagent
Laboratories, Columbus, OH) and 1.0 g of anhydrous MgSO
4
. After all the
solids had dissolved, the pH was measured with a calibrated pH meter and
the pH was adjusted (as needed) to 7.3 60.1. The solution is sterile filtered
through a 0.22 lm filter under reduced pressure. This solution is used as the
medium in which the viral serial dilutions are performed.
Phosphate-buffered saline. To 1000 mL of water is added 8.5 g NaCl,
0.2 g KH
2
PO
4
and 2.9 g Na
2
HPO
4
. After all the solids had dissolved, the
pH was measured with a calibrated pH meter and the pH was adjusted
(as needed) to 7.3 60.1. This solution is then sterile filtered through a
0.22 lm filter under reduced pressure. This solution is used as the medium
in which the irradiation experiments were performed.
Stock RB solution. A saturated solution of RB was prepared by heating
a mixture of PBS and an excess of RB at 558C. The solution was then sterile
filtered (0.22 lm), and the optical density at 446 nm was recorded for
a sample that was diluted 10-fold. On the basis of the extinction coefficient
of aqueous RB (e
446 nm
511 400), the concentration of the diluted solution
was calculated (typically 750 lM) and the stock RB solution was then
diluted with sterile PBS to achieve a final RB concentration of 400 lM.
Aptamer solution preparation. The solutions used in the aptamer
experiments were prepared using autoclaved ribonuclease (RNase)–free
water containing 0.1% diethylpyrocarbonate (DEPC). To RNase-free water
(500 mL) was added 4.39 g NaCl, 0.82 g NaPO
4
, 0.19 g MgCl
2
and 15 mg
ethylene diamine tetraacetate (EDTA) to prepare RNase-free PBS. The
custom-synthesized aptamer (Dharmacon, Lafayette, CO) was used as
received (GGC GUG UAG GAU AUG CUU CGG CAG AAG GAC ACG
CC). The lyophilized sample (1.14 mg) was dissolved in 0.5 mL of RNase-
free PBS to make the 200 lL solution of aptamer. RNase-free PBS was
also used to prepare the solution of FMN.
Reconstitution of E. coli.A freeze-dried sample of E. coli c600 (ATCC,
Rockville, MD) was vortexed with 0.4 mL of LB media and then
transferred to an additional 5 mL of LB media. Using a flame-sterilized
inoculation loop, streak plates (Trypticase soy albumin [TSA] Becton
Dickinson, Franklin Lakes, NJ) were prepared and were placed in a 378C
gravity convection incubator to grow overnight. Again, using a flame-
sterilized inoculation loop, individual colonies of E. coli were isolated and
were used to inoculate several bottles containing 200 mL of sterile growth
media. These bottles were then shaken on an orbit mixer overnight in
a378C incubator. After 24 h, the previously clear amber solutions became
very milky with bacterial growth. Each bacterial suspension was divided
into 50 mL centrifuge tubes (40 mL) and was centrifuged at 1500 gfor
20 min. The supernatant was discarded, and the remaining pellet was
resuspended in 20 mL of 0.01 MMgSO
4
, which had been previously sterile
filtered (0.22 lm filter). The optical density of each of the resuspended
bacterial samples was approximately 2.2 at 600 nm. This solution is referred
to as the stock bacteria.
Reconstitution of lambda phage. A freeze-dried sample of lambda phage
(ATCC, Rockville, MD) was vortexed with 0.5 mL of LB media. This
initial solution of phage was diluted serially (10-fold) with storage media
(SM) to yield solutions ranging from 10
0
(undiluted) to 10
8
(1/10
8
of
original concentration). A portion of each of these phage solutions (100
lL) was mixed with an equal amount of E. coli and the mixture was
allowed to incubate for 20 min. Warm molten agar (558C, 4 mL) was then
briefly vortexed with each incubated mixture and poured on a TSA agar
plate (Becton Dickinson). After the agar overlay had hardened (10 min),
the plates were inverted and placed in the incubator (378C) overnight.
Plates that showed confluent lysis (complete plate coverage with clear
plaques) were retained, and 6 mL of growth media was added to each
plate. These plates were then placed on an orbit mixer in the incubator
overnight. After 24 h, the agar overlay was scraped off gently and
combined with the supernatant. The combined phage-containing mixture
was centrifuged at 1500 gfor 20 min. The supernatant was collected and
sterile filtered to yield an amber-colored liquid, which contained
approximately 8 logs of lambda phage (10
8
plaque forming units (PFU)/
mL) in growth media.
Generation of E. coli.Streak plates of E. coli on TSA were made either
from agar slants of the original reconstituted phage or of the most recently
used batch of bacteria. Using a flame-sterilized inoculation loop, individual
colonies of E. coli were isolated and were used to inoculate several bottles
containing 200 mL of sterile growth media. These bottles were then shaken
on an orbit mixer in a 378C incubator overnight. After 24 h, the previously
clear amber solutions became very milky with bacterial growth. Each
bacterial suspension was divided into 50 mL centrifuge tubes (40 mL) and
was centrifuged at 1500 gfor 20 min. The supernatant was poured off and
the remaining pellet was resuspended in 20 mL of previously sterile filtered
(0.22 lm filter) 0.01 MMgSO
4
. The optical density of each of the re-
suspended bacterial samples was approximately 2.2 at 600 nm. This solu-
tion is referred to as the stock bacteria.
Generation of lambda phage. An existing solution of phage was diluted
serially (10-fold) with SM to yield solutions ranging from 10
0
(undiluted) to
10
8
(1/10
8
of original concentration). A portion of each of these phage
solutions (100 lL) was mixed with an equal amount of E. coli and the mix-
ture was allowed to incubate for 20 min. Warm molten agar (558C, 4 mL)
was then briefly vortexed with each incubated mixture and poured on a TSA
agar plate. After the agar overlay had hardened (10 min), the plates were
inverted and placed in the incubator (378C) overnight. Plates that showed
confluent lysis (complete plate coverage with clear plaques) were retained,
and 6 mL of growth media was added to the plates. These plates were then
placed on an orbit mixer in the incubator overnight. After 24 h, the agar
overlay was scraped off gently and combined with the supernatant. The
combined phage containing mixture was centrifuged at 1500 gfor 20 min.
The supernatant was collected and sterile filtered to yield an amber-
colored liquid, which contained approximately 8 logs of lambda phage
(10
8
PFU/mL) in growth media.
AS (phage) sample preparation. All samples were handled using
standard aseptic techniques. In a 3.0 mL quartz cuvette was added 100 lL
of a solution of lambda phage (;8 logs), PBS and the required volume of
400 lMRB in PBS to achieve a total solution volume of 2.5 mL with the
desired RB concentration.
Plaque assay. Bacteriophage (lambda) solution (100 lL) to be analyzed
was diluted serially (10-fold) in SM to achieve solutions ranging from 10
0
to 10
8
in concentration. Each of these dilutions (100 lL) was mixed with
100 lL of bacteria (E. coli) in a 15 mL centrifuge tube and was incubated at
room temperature for 20–30 min. To each tube was added 3–4 mL of TOP
agar (40–558C), and this vortexed mixture was poured evenly over a TSA
agar plate (Becton Dickenson) and allowed to harden. When the agar had
solidified sufficiently, the plates were inverted (to prevent pooling of
condensation on agar) and allowed to incubate for 24 h. Plates that
Figure 2. The structure of LF and LC.
Figure 1. The structure of RB.
Photochemistry and Photobiology, 2005, 81 475
contained greater than 300 plaques (clear regions) were labeled as TNTC,
too numerous to count. Viral titers were determined by using the following
formula in which the number of PFU was between 30 and 300.
Number of PFU ¼Number of plaques counted 310ðdilutionþ1Þ
For example, a plate containing 42 plaques on the plate labeled 10
5
would
be considered to have:
42 3105þ1¼4:23107PFU=mL:
Dye laser system. With the exception of the data at 266 and 355 nm, all
samples were irradiated using a Lambda Physik ScanMate UV dye laser
pumped by a Lambda Physik LPX-100 XeCl excimer laser (Lambda
Physik, Gottingen, Germany). Wavelengths between 288 and 320 nm were
produced using rhodamine 6G, rhodamine B and rhodamine 101 as dye
solvents and the doubling unit. Energies in this wavelength range were
between 0.15 and 1.2 mJ/pulse at 10–50 Hz. Wavelengths between 330
and 550 nm were produced using the fundamental laser wavelength of
the dyes p-terphenyl, DMQ, QUI, PBBO, coumarin 47 or coumarin 120.
Energies in this second wavelength range were between 0.5 and 15
mJ/pulse at 10–50 Hz.
YAG laser system. Irradiations at 266 and 355 nm were performed using
a Spectra Physics Quanta-Ray YAG laser system (Spectra Physics, Irvine,
CA) operating at 10 Hz with energies averaging 10 mJ/pulse.
RESULTS
Action spectrum
The AS of the inactivation of lambda phage in PBS with 0, 50, 100,
150 and 200 lMRB was determined between 266 and 575 nm.
Each irradiated sample contains 2.5 mL of solution in a 3.0 mL
quartz cuvette. Each data point was performed in triplicate, and the
reported value reflects the average of these three values. In the case
where one value fell clearly outside the area of the other two
values, it was omitted. This occurred in only 13% of the data sets
throughout the AS. The AS (Fig. 3) can be divided into two distinct
regions on the basis of the energy required for inactivation and the
effect of the sensitizer, RB.
Action spectra: 266–304 nm
It is well known that UV radiation with wavelengths of less than or
equal to 304 nm is lethal to cells (23,24). Thus, the AS for the
inactivation of lambda phage in PBS from 266 to 304 nm required
only a small amount of incident energy, 0.1 J/mL, to achieve a
maximum phage reduction of 2.5 logs in this region. As the AS
approaches 304 nm, the reduction of phage drops off sharply as
the absorption curve of DNA approaches zero.
Phage inactivation is dominated by direct light absorption by the
phage from 266 to 304 nm. Not only does the presence of RB fail
to increase the extent of phage inactivation but also the presence of
RB reduces the amount of phage inactivation by acting as a
screening agent by decreasing the amount of light that is absorbed
by the phage. This screening effect is proportional to the concentra-
tion of RB in solution. Because the competitive absorption of
RB dominates any sensitization of the phage, it is concluded
that direct absorption of light by the phage is much more efficient
at phage inactivation compared with sensitized inactivation in
this region.
Action spectra: 308–575 nm
To achieve the same magnitude of log reduction that was observed
between 266 and 304 nm, the energy required for the experiments
between 308 and 575 nm was increased 50-fold from 0.1 to 5.0
J/mL. There is no inactivation of lambda with light 330 nm in
the absence of RB.
The AS do not correlate perfectly with the absorption curve of
either RB or LC in PBS over the entire wavelength regimen. There
appears to be essentially an identical amount of viral inactivation
at 355 and 500 nm, although the optical densities (of solutions
containing the same concentration of RB) differ by a factor of five
at these wavelengths. The phage reduction obtained at 320 and
500 nm is greater than that expected based on the absorption
spectrum of RB in PBS at these wavelengths. The effect is shown
clearly in Fig. 4, which plots the difference in lambda reduction
achieved in the presence of RB minus that realized in its absence.
There is another deviation of lambda inactivation from the
absorption spectrum of RB in PBS. The inactivation obtained at
375 nm, where RB has an absorption maximum, is lower than
expected.
DISCUSSION
We hypothesize that RB is largely free in PBS solution and only
a small amount of flavin (not detectable by absorption spectros-
copy) is bound to phage. We speculate that the absorption spectrum
Figure 3. The AS of the RB-sensitized inactivation of lambda phage
in PBS.
Figure 4. AS containing either no RB or the average inactivation because
of 50–200 lMRB. The net effect from the addition of RB (sensitized–
unsensitized) on the AS and the absorption curve of RB is also shown.
476 Christopher B. Martin et al.
of bound RB will differ from that of free RB. We hypothesize that
photolysis of the RB–phage complex is orders of magnitude more
efficient at inactivating lambda phage than photolysis of free RB.
This can be responsible for differences between the AS and the
absorption spectrum of free RB because regions of enhanced ab-
sorption of bound flavin will appear as regions of enhanced patho-
gen reduction.
The aptamer sequence (GGC GUG UAG GAU AUG CUU
CGG CAG AAG GAC ACG CC) is known (25) to have a high
affinity for FMN, was custom synthesized, purified and deprotected
and was used as received. The aptamer was prepared in RNAse-
free PBS (autocloaved with 0.1% DEPC) containing 0.1 mM
EDTA and 4 mMMg
þ2
. The magnesium is present to allow proper
folding of the aptamer and consequent binding of the flavin ring of
FMN. Difference spectra were obtained with both 100 and 200 lM
RNA aptamer and FMN. Only the data for the 200 lMcon-
centrations are shown in Fig. 5.
The negative values in the difference spectrum (ca 370 and
445 nm) are because of the lower molar absorbtivity of the bound
flavin compared with the free flavin. It is obvious that FMN bound
to the RNA aptamer has increased absorbance at 500 nm. This
is consistent with increased inactivation of lambda by a small
quantity of RB bound to phage nucleic acid. The difference spec-
trum observed in the presence of aptamer does not explain the in-
creased efficiency of inactivation of lambda phage between 320
and 340 nm and the lower than expected amount of inactivation
observed between 350 and 400 nm. However, the effect of solvent
polarity on the absorption spectrum of RB mirrors the deviation of
the AS of RB in PBS. In 50% dioxane–water (Fig. 6), there is
enhanced absorption between 320 and 350 nm and decreased
absorption between 360 and 410 nm (26). Thus, it seems likely
that the enhanced pathogen reduction observed at 320 nm in the
AS is because of the binding of RB to regions of the phage
resembling 50% dioxane–water. This is consistent with, but not
limited to, the polarity observed for the major groove region of
DNA.
Fluorescence excitation spectroscopy
To investigate the relationship between the AS (photochemistry)
and the (fluorescence) excitation scan, a series of excitation scans
were performed over the concentration range in which the AS was
obtained (Fig. 7).
The excitation scan with 10 lMRB most resembles the UV-VIS
absorption spectrum of RB. However, as the concentration of RB
increases, the excitation scan gradually changes. The excitation
scan contains three maxima for 75 lMRB, and the excitation scan
obtained with 100 lMRB bears no resemblance to the absorption
spectrum of RB. The shape of the absorption spectra of RB at
comparable concentrations obtained in the excitation scans does
not change over the concentration range. No deviations from
Beer’s law were observed arguing against the formation of ground-
state aggregates under these experimental conditions. Similar results
were also obtained in formamide, a solvent that forms hydrogen
bonds to RB and dissociates hydrogen-bonded aggregates. We also
obtained concentration-dependent fluorescence excitation spectra
with FMN in PBS. This is significant because unlike RB, the equi-
librium constant for FMN aggregation is known. The aggregation of
FMN K
eq
5265 638 mol
1
is quite minimal at micromolar FMN
concentrations (27), yet the distorted fluorescence excitation spectra
are still observed. We can also rule out the formation of an exciplex
because the fluorescence spectrum does not change between 10 and
100 lMRB.
Under certain conditions of RB concentration, the fluorescence
excitation spectra much more closely resemble the AS than does
the absorption spectrum of RB. Thus, the origin of the fluorescence
excitation effect will likely have implications for understanding
the AS as well.
The fluorescence excitation spectra are explained readily as
a consequence of the inner filter effect (28). As the optical density
of the sample increases, less incident light will penetrate to the
center of the cuvette. Fewer RB molecules in the center of the
cuvette will be excited and emit light that reaches the detector.
This effect tends to elevate the intensity of the chromophore at the
sides of its absorption maxima, relative to the wavelength of
maximum absorption. Thus, the fluorescence spectrum broadens
and distinct maxima present in the absorption spectra are
obscured.
The same principles apply to the AS. Incident light at wave-
lengths of the RB absorption maxima will have limited penetration
of the sample and inactivate phage in a relatively small volume.
Incident light off of the RB absorption maxima will penetrate more
deeply into the cuvette and inactivate phage over a larger volume.
Therefore, the total pathogen inactivation obtained on the sides of
the RB absorption maxima can roughly equal the logs of reduction
of lambda at the absorption maxima. To test this interpretation, we
studied the photoconversion of RB to LC in the samples used to
generate the AS.
Figure 6. UV-VIS absorption shift of RB in mixtures of water and dioxane
or dimethyl sulfoxide compared with RB in water.
Figure 5. Difference spectrum of 200 lMFMN and 200 lMaptamer com-
pared with 200 lMFMN. [path length 50.1 cm].
Photochemistry and Photobiology, 2005, 81 477
Photochemical conversion of RB to LC
The absorption spectrum of each sample used in the AS was
recorded before and after exposure to laser radiation. If the
assumption is made that the absorption at 374 and 446 nm is only
because of RB and LC, then the absorbance at those wavelengths
can be expressed and the concentrations of RB and LC before
and after photolysis can be evaluated.
A374 nm ¼½RBe374 nm ðRBÞþ½LCe374 nm ðLCÞ
A446 nm ¼½RBe446 nm ðRBÞþ½LCe446 nm ðLCÞ
The amount of RB that was photobleached during the irradiation
used to generate the AS is compared with the (scaled) excitation
scan of 100 lMRB. It can be seen in Figs. 8 and 9 that the two
spectra, generated in two completely independent manners, are in
remarkable agreement.
This suggests that the inner filter effect is the major factor
influencing the photochemistry of bulk (but not necessarily free)
RB in PBS under the conditions used in the AS.
The AS and the RB (scaled) excitation scan do not overlay
perfectly, thus examination of the difference between these two
plots should reveal any difference between the photochemistry of
RB and any added effect because of flavin bound to phage.
An examination of the increase in the log reduction of lambda
beyond the predicted inactivation based on the excitation scan
(and photobleaching) of RB reveals two interesting features.
There is significantly more phage inactivation at 350 nm and
shorter wavelengths than would be predicted by examining the
excitation spectrum of RB. This additional amount of inactivation
is attributed to direct absorption by the phage for irradiation
wavelengths of less than 320 nm. When the irradiation
Figure 7. Fluorescence excitation scans
of (a) 10, (b) 25, (c) 50, (d) 75 and (e)
100 lMRB in PBS compared with the
UV-VIS absorption of (f) 50 lMRB
in PBS.
Figure 8. Comparison of the calculated amount of RB photobleached in
the AS and the 100 lMRB fluorescence excitation scan, front face (FF)
fluorescence detection (scaled).
478 Christopher B. Martin et al.
wavelength is between 320 and 350 nm, the inactivation is
clearly due to the addition of RB because there is no inactivation
in the absence of the sensitizer. Because the inactivation in this
range is also beyond that predicted for the bulk photochemistry of
RB, it is reasonable to conclude that the inactivation originates
from the direct absorption of a phage–RB complex in an
environment resembling 50% dioxane–water.
As mentioned previously, there is less inactivation obtained
between 350 and 400 nm than from 400 to 500 nm as expected
based on the absorption spectrum of RB in PBS. The formation
of LC (which absorbs in this region) is probably responsible for
the difference between these two wavelength regimens. As RB is
photobleached, the optical density decreases in the region beyond
where the photoproduct, LC, absorbs (k.400 nm). As the
absorbance decreases, the inner filter effect will also decrease
and photochemistry can proceed more deeply in the cuvette.
Conversely, the photobleaching of RB does not lead to
a reduction in the optical density of the solution from 350 to
400 nm because the conversion of RB leads to the formation of
LC, which therefore maintains the optical density in this region
resulting in decreased photochemistry because of the inner filter
effect.
The inner filter effect on the AS is not the result of inefficient
mixing. The sample does not mix on the 17 ns timescale of the
laser pulse. When samples are irradiated at a wavelength where the
optical density of the solution is high, very few photons penetrate
past the first millimeter of the cuvette. Therefore, the statistical
probability that a photon is absorbed by an appropriate chro-
mophore decreases greatly beyond the first millimeter in the
cuvette. In this situation, the viral inactivation is limited by the
decreased penetration of the irradiating photons caused by the inner
filter effect. Most of the phage killed is within the first millimeter of
the cuvette. When the irradiating wavelength is at a point where the
solution is more optically thin, the photons can pass much deeper
into the solution and therefore excite a larger volume of sample
containing RB and phage particles as the light penetrates beyond
the first millimeter of the cuvette. Therefore, the total kill from
a laser pulse at a wavelength of low RB absorption can equal the
level of inactivation at the absorption maximum. The result is that
the distinct maxima observed in the absorption spectrum are
blurred and broadened in the AS, much as is observed in the
fluorescence excitation scan.
CONCLUSIONS
We have studied the RB-sensitized inactivation of lambda phage
in PBS as a model pathogen for the reduction of blood-borne
pathogens in the blood supply. The AS does not follow the
absorption spectrum of aqueous RB exactly. The shape of the AS
and the fluorescence excitation spectrum is influenced by the inner
filter effect. The high optical density of the solution limits the depth
of penetration of the incident laser and therefore limits the amount
of fluorescence and phage inactivation. The shape of the AS also
reflects the binding of minuscule amounts of RB to lambda phage.
Binding of RB to lambda shifts the absorption envelope of the
phage-bound flavin relative to that of free flavin in PBS. Photolysis
of the phage–flavin complex is more effective at phage reduction
than photolysis of free RB and results in enhanced phage reduction
at 320 and 500 nm.
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