J. Photochem. Photobiol. B: Biol.

, 23 (1994) 43-48 43

Riboflavin-sensitized photoprocesses of tryptophan

E. Silva+, R. Ugarte, A. Andrade and A.M. Edwards

Facultad de Quimica, Pontificia Univemidad Catblica de Chile, Casilla 306, Santiago 22 (536) (Chile)

(Received August 24, 1993; accepted December 13, 1993)

Abstract

The photodegradation of tryptophan in oxygen saturated aqueous solution sensitized by riboflavin is accompanied
by the generation of the following reactive oxygen species: ‘Ou OH; H,O, and O;-. When parallel photodegradation
experiments are run with “C-riboflavin in one case and “C-tryptophan in the other and the irradiation products
are separated by Sephadex G-15 and C,,-HPLC, the generation of the following species is detected: aggregate
forms of riboflavin, indolic products associated to flavins, indolic products of molecular weight higher than
tryptophan, formylkynurenine, and other tryptophan photodecomposition products. The significance of the riboflavin
anion radical and tryptophan cation radical as intermediates in the photoproduct formation is discussed.

Key wordr: Reactive oxygen species; Photoproducts; Riboflavin; Tryptophan; Tryptophanyl radical; Flavin
radical

1. Introduction onstrated that irradiation of lysozyme with visible

Tryptophan (Trp), an essential amino acid, and
riboflavin (Rb), a vitamin, are natural constituents
of living organisms and are ingested by humans
in food. At the same time, however, the exposure
of these substances to visible light has been related
to hepatic dysfunction produced by parenteral
nutrition [l] and to toxic effects on mammalian
cells in culture medium [2]. An exceptional effi-
ciency for the photo-oxidation of Trp was found
when the vitamin Rb was used as sensitizer [3].
This process is characterized by quantum yields
higher than those observed with other sensitizers
such as methylene blue or rose bengal which involve
a type II (‘O,-mediated) photo-oxidation mech-
anism. Yoshimura and Ohno [4] found that the
semiquinone anion radical of lumiflavin and the
half-oxidized radical of indole were formed by the
reaction between triplet lumiflavin and indole. The
semiquinone anion radical of lumiflavin reacted
with oxygen to form superoxide. They also dem-
onstrated that the quantum yield of the lumiflavin
sensitized photo-oxygenation of dilute indole via
radical processes was much higher than that via
‘02 processes, though appreciable amounts of ‘02
were formed via a type II process. We have dem-
‘Author to whom correspondence should be addressed.
light in the presence of riboflavin and molecular
oxygen not only produces the photo-oxidation of
some amino acid residues of that enzyme, but also
generates a binding of this sensitizer to Trp residues
of this protein [S, 61. It was possible to isolate
and characterize spectrophotometrically at least
two types of photo-adducts (according to the degree
of modification of the flavin) obtained by anaerobic
irradiation of solutions of free tryptophan in the
presence of Rb [7]. It is noteworthy that the
riboflavin-lysozyme and riboflavin-tryptophan
binding have also been obtained in the dark [8,
91. In this paper the products generated during
aerobic irradiation of solutions containing Rb and
Trp are revised and subjected to further study.
2. Materials and methods
Solutions of Rb (3.5X10m5 M) and Trp
(3.5 x 10m4M) in 0.05 M phosphate buffer pH 7.0
(Trp/Rb solution) were irradiated for 1 h in a 1
cm light path cell thermostated at 37 “C. Oxygen
saturation condition was reached by oxygen bub-
bling (dissolved oxygen concentration at 37 “C is
1 X 10e3 M). Light from a 150 W slide projector
lamp, equipped with an interference filter, was

loll-1344/94/$07.00 Q 1994 Elsevier Sequoia. All rights reserved

SSDI 1011-1344(93)06984-B
44 E. Silva et al. / Riboflavin-sensitized photoprocesses of tryptophan
employed for irradiation at 450 nm. The energy
flow rate was 6.1 J mm2 set-‘. For radioactive
labeling, irradiations were carried out under similar
conditions, with previous addition of a D-[~-~~C]-
Rb (14C-Rb) solution or an L-[Methylene-‘4C]-Trp
(14C-Trp) solution in order to obtain a molar ratio
14C-Rb:Rb = 1:4.9, or 14C-Trp:Trp = 1:284. Radio-
activity was determined in a Beckman LS 5000
TD liquid scintillation counter. Gel filtration was
carried out on a 1.8 cm x42 cm Sephadex G-15
column equilibrated with distilled water.
High-performance liquid chromatography
(HPLC) was carried out with a reverse phase p-
Bondapack Cl8 column. The mobile phase was a
5 mM ammonium acetate/methanol 75/25 (v/v)
solution. Results were registered and evaluated
with a Waters 746 data module.
Absorption spectra were recorded on a Varian
Super Scan III spectrophotometer. Fluorescence
measurements were performed with a Perkin-
Elmer 650-10s fluorescence spectrometer. Oxygen
uptake measurements were carried out using a
YSI Model 5300 oxygen monitor.
Sephadex G-15 was obtained from Pharmacia,
Rb from Sigma Chemical Company and 14C-Rb
and 14C-Trp from Amersham International. All
other reagents were of analytical grade.
3. Results
Figure 1 shows the alteration in the absorption
spectra produced during irradiation of a Trp/Rb
solution under oxygen saturation. An increase is
observed of absorption in the region 31w40 nm,
which corresponds to the formation of products
during the photo-oxidation of Trp. The moditi-
cation of Trp was quantified by means of the
fluorescent characteristics of this amino acid. To
measure fluorescence, the sample was submitted
to a 50-fold dilution, in order to preclude the
filter effect due to the riboflavin present in the
medium, which can interfere both with the ex-
citation of Trp (A,= 280 nm) and with its flu-
orescent emission A,, = 355 nm). Figure 1 (inset)
shows the variation of Trp emission during the
photo-oxidation process. All these events are ac-
companied by oxygen uptake. With the aim to
determine the oxygen reactive species which par-
ticipate in the photoinduced events, different scav-
engers and quenchers were added to the system.
Results are summarized in Table 1. Thus, the
increased photoconversion of Trp in D,O relative
06-
Minutes
g O.L-
x
i
z
0.2 -
300 LOO 500 600
Nm
Fig. 1. Absorption spectra of a solution of tryptophan and riboflavin
both unirradiated and irradiated with monochromatic light
(h =450 nm) during 15,30,45 and 60 min under oxygen saturation
at pH 7.0 in a 0.05 M, phosphate buffer. Inset: Effect on the
fluorescence of tryptophan when irradiated in the presence of
riboflavin under the same conditions described above.
to H,O, together with the quenching effect of
w-, indicates the participation of ‘02 in Trp photo-
oxidation, while the decrease in the yield in the
presence of Fe(CN),3- suggests a role of electron
transfer processes.
The protective effect of benzoate points to par-
ticipation of OH and the lack of an effect of
catalase allows one to exclude H202 as a factor
in the photo-oxidation.
The presence of O,- was indirectly demon-
strated by the higher quantum yield detected after
irradiation in the presence of SOD, which catalyzes
the transformation of OZ.- to H202. This effect
was not observed when catalase and SOD were
simultaneously added to the system.
The irradiated sample was applied to a Sephadex
G-15 column. Figure 2 shows the elution pattern
of Rb (I) and of the Trp/Rb solution (II), both
irradiated for 60 min with monochromatic light
(450 mn) under oxygen saturation. The irradiated
Rb underwent a slight modification, characterized
by the presence of a new flavinic fraction which
elutes at a shorter retention time. This peak may
be assigned to an aggregate form of Rb [lo].
Six fractions are observed in the elution pattern
II, corresponding to the Trp/Rb solution irradiated
for 60 min; two of them correspond to unreacted
Trp and Rb (e and d respectively). This fact was
confirmed by their spectral properties and their
retention times on an HPLC Cl8 column.
To determine the origin - flavinic and/or indolic
- of the remaining fractions, Trp and Rb were
 E. Silva et al. / Riboflavin-sensitized photoprocesses of tryptophan 45

TABLE 1. Effect of various agents upon the quantum yields of the photoprocesses of tryptophan sensitized by riboflavin under
oxygen saturation

Compound added @ % Remarks

Control 0.029 100.0

NaNr (0.5 mM) 0.016 55.2 ‘Or quencher’
D2O 0.056 193.1 ‘02 enhancer
%Fe(CN), (0.1 mM) 0.014 48.3 Electron acceptor
Benzoate (5 mM) 0.021 72.4 Trap for ‘OH
Catalase (300 units) 0.029 100.0 H202 trap
Catalase (300 units denatured) 0.029 100.0
Superoxide dismutase (100 units) 0.033 113.8 O;- trap
Superoxide dismutase (100 units denatured) 0.029 100.0
Superoxide dismutasekatalase 0.029 100.0 H202 and 0; trap

‘Also ‘OH and R+ trap.

(4

Fig. 2. Elution patterns on a Sephadex G-15 column (1.8 cm X 42
cm) of solutions of: riboflavin (I) and ttyptophan and riboflavin
(II), irradiated under the same conditions described in Fig. 1.
irradiated in two parallel solutions containing 14C-
Rb and 14C-Trp respectively. In each case the
irradiated sample was applied to the G-15 Seph-
adex column. Figure 3(a) shows the elution pattern
detected on the basis of the radioactivity initially
localized in the 14C-Trp. The presence of radio-
activity can be observed in several fractions of this
chromatogram, corresponding to peaks a, b, c, e,
and f (Fig. 2). Of these, peak e corresponds to
unreacted Trp. When 14C-Rb was used in the
parallel experiment, the elution profile shown in
Fig. 3(b) was obtained. In this figure, three fractions
present radioactivity, corresponding to peaks a, b,
and d of Fig. 2, where peak d corresponds to
unreacted Rb.
Fractions from filtration in Sephadex G-15 were
rechromatographed through the same column and,
to achieve a better resolution of the products,
each peak was applied to a reverse phase HPLC
Cl8 column (Fig. 4). Figure 4(a) shows the chro-
matogram obtained when applying fraction a from
Fig. 3 to the HPLC Cl8 column. Peak a corresponds
z
5
- 10000
-t” 8ooo
g 6OW
Loo0
2000
70 lb0
(b) mL
Fig. 3. Elution patterns on a Sephadex G-15 column (1.8 cm X 42
cm) of solutions of: riboflavin and “C-tryptophan (a), and tryp-
tophan and “C-riboflavin (b), irradiated under the same conditions
described in Fig. 1.
to the fraction of higher molecular weight and
presents components of flavinic and indolic origin.
From the analysis of this HPLC chromatogram,
together with the localization of the radioactive
label from either the indole or the flavin, we
conclude the following. The diagram shows an
initial zone (I) formed by at least five signals which
contains over 70% of the radioactivity from the
indolic component present in peak a from Fig. 3,
and approximately 43% of the radioactivity of the
flavinic component in that peak. Peak II shows a
1:l indole:flavin molar ratio. Peak III corresponds
to a modified form of Rb, and peak IV is formed
by a compound of indolic origin.
46 E. Silva et al. I Riboflavin-sensitized photoprocesses of hyprophan

VI

i
V

10 20
1
_ !k
20
I__
10
30
(a) (b) (4 (4Time (min)
Fig. 4. a, b, c and d correspond to HPLC chromatograms of peaks a, b, c and f shown in Fig. 3. HPLC conditions are given in
Section 2.

Figure 4(b) shows the chromatogram obtained

when applying peak b from Fig. 3 to the HPLC
Cl8 column. Peak b corresponds to a fraction of
intermediate molecular weight with respect to
peaks a and c and higher than that of the reactants.
Peak b is mainly due to indolic components which
resolve into two fractions, the larger of which has
93% of the radioactivity of the indolic component
present in peak b (peak VI). The smaller fraction
shows radioactivity of flavinic origin, but whose
magnitude is significantly lower than that of indolic
origin (peak V).
Figure 4(c) shows the chromatogram obtained
when applying peak c of Fig. 3 to the HPLC Cl8
column. The main and major fraction was of indolic
origin (peak VII). Both the absorption and fluor-
escence emission spectra of this compound agree
!Ll IO

with those of the N-formylkynurenine, which is a
classical product of Trp photo-oxidation [ll].
Figure 4(d) shows the chromatogram obtained
when applying peak f of Fig. 3 to the HPLC
column. Peak f corresponds to a fraction of mo-
lecular weight somewhat lower than that of the
reactants and it resolves into three components
which present only the indolic type radioactivity
(peaks VIII, IX and X).
When the irradiated sample was applied directly
to the HPLC column, the chromatogram shown
in Fig. 5(b) was obtained. The last three peaks
(arrows) do not appear in the chromatograms
shown in Fig. 4. These fractions elute after Rb,
indicating that they are more apolar and probably
(b)
10 20 30
Time (min)
Fig. 5. Chromatograms on a reverse phase Crs column of a
solution of tryptophan and riboflavin both unirradiated (a) and
irradiated (b) under the same conditions described in Fig. 1.
HPLC conditions are given in Section 2.
have been insolubilized during the isolation pro-
cesses. To obtain information on the nature of
these compounds, two irradiations were performed
in the presence of 14C-Trp and 14C-Rb, respectively,
and the irradiated samples were directly applied
to the HPLC C,, column. The last three fractions
were collected and it was verified that they pos-
sessed 14.3% and 15.5% of the total radioactive
label from the r4C-Trp and r4C-Rb respectively.
 E. Silva et al. / Rilmjlavin-sensitized photoprocesses of hyptophan
Labeling from the two reactants was simultaneously
found in all these fractions.
4. Discussion
The great number of different products obtained
during the aerobic irradiation of a Trp solution
in the presence of Rb led us to reconsider the
mechanism of the photo-processes involved.
The photo-oxidation of indolic compounds in
the presence of Rb has been widely documented
[12-171. Yoshimura and Ohno [4] have shown
kinetically that Type I photoconversion of indole
sensitized by lumiflavin is more efficient than the
competing Type II process. They have also dem-
onstrated experimentally the presence of the half-
reduced radical of lumiflavin and the half-oxidized
radical of indole. These reports are in accordance
with our results which show a drastic decrease of
the photoinduced events when K,Fe(CN), (a
known acceptor of electrons) was added to the
system. Accordingly, we postulated the transfer of
one electron from the Trp indolic ring (IndH) to
Rb in its triplet state (Scheme 1). Although the
reduction potential of riboflavin is -0.3 V at pH
7.0 [18], the redox potential of the triplet flavin
can be considered to be shifted to 1.7 V [4], which
is higher than the redox potential of IndI-P/IndH
(1.5 V) [19].
The presence of both ‘OH and ‘02 (Table 1)
accounts for the appearance of formylkynurenine
in peak VII of Fig. 4, and also probably for other
products of low molecular weight obtained from
indole, which appear in peaks VIII, IX, and X
RbH’ + Ind’ + Products
3Rb
T
IndH F Rb’- + IndH’+
19
Rb + O,‘-
1 toward O2 and also that indole is two orders of
H202
1 We conclude that the products obtained during
*OH m P Oxidation
products
Rb + ‘0, Inw ) Oxidation
products
Scheme 1. (IndH represents tryptophan)
41
of the same figure. The presence of aggregate
forms of Rb found in peak III of Fig. 4 could be
explained by the action of light on this sensitizer
(20-221. Radical species of Rb may be obtained
from the charge transfer complex produced be-
tween triplet Rb and the amino acid (Scheme 1).
Scheme 1 also shows the presence of In&, which
can lead to reaction products comprised of two
or more Trp units. These processes can be rep-
resented as follows
Ind’ + Ind’ -+ Ind-Ind (I)
Ind’+ IndH - Ind-IndH’ (2)
The radical dimer shown in eqn. (2) could give
rise to a trimer if it reacted with another Ind’, or
else it could interact with other IndH molecules,
generating derivatives of higher molecular weight.
Humphries and Dryhurst [23] have reported the
generation of 5-hydroxytryptophan dimers and tri-
mers from radical species of this amino acid de-
rivative. In this study, the In& was obtained both
electrochemically and by the action of a peroxidase.
In opposition to dimerization reactions by radi-
cal-radical coupling processes, they proposed that
a radical-substrate reaction occurs in which a non-
radical indole derivative is attacked by a radical
one (process (2)). This kind of process can account
for the appearance of indolic products of higher
molecular weight than Trp (peaks IV and VI, Fig.
4). The indolic products associated to flavins (zone
I, peaks II and V of Fig. 4 and the last three
peaks (arrows) of Fig. 5(b)) can be explained as
having originated from radical reactions, which
would involve the reaction of an indolic radical
with the corresponding flavin radical
(Ind),’ + RbH - (Ind), _ ,-Ind-RbH (3)
where ‘n” can be equal to or greater than one.
The new products obtained in this work (eqns.
(l-3)) can be explained as a consequence of the
predominance of a Type I mechanism. Yoshimura
and Ohno [23] have reported that triplet lumiflavin
is three times more reactive toward indole than
magnitude more reactive toward triplet lumiflavin
than toward ‘OZ.
the aerobic irradiation of the Trp/Rb solution are:
(i) aggregate forms of Rb; (ii) indolic products
associated to flavins; (iii) indolic products of higher
molecular weight than Trp; (iv) N-formylkynu-
renine and other products of photodecomposition
of Trp of lower molecular weight.
48 E. Silva et al. I Riboflavin-sensitized photoprocesses of tryptophan
Acknowledgments
The authors thank Mrs. I. Aguirre for assistance
in preparing the manuscript and Miss Viviana
Ulloa for excellent typing. Special thanks are also
given to Dr. Eduardo Lissi (Universidad de
Santiago, Chile) for critical reading of the man-
uscript. This work was supported by Fondo Na-
cional de Investigation Cientffica y Tecnologica
(FONDECYT Grant 1930571).
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