SpecrrochimicaAcm. Vol 43A. No 3.

pp 389-394
Printed in Great Britain

Spectroscopic

1987

0584&8539#87 $3 00 + 0 no
Ltd

(;: 1987
Pergamon Journals

study of molecular associations between flavins FAD

and RFN and some indole derivatives

ARMANDO CODORER,* ISIDRO S. MONZ~,

Departamento

de Quimica

Fisica, Facultad

de Quimicas,

FIEDAD MEDINA and FRANCISCO TOMAS

Universitat

de Valencia, Burjassot

(Received 20 Ma): 1986; in final form 29 July 1986; accrpted

(Valencia), Spain

30 JU/J 1986)

Abstract-The

formation
of complexes between two Aavins [flavine adenine dinucleotide
(FAD) and
(RFN)] and some indole derivatives has been studied in aqueous solution. The molecular
associations have been examined by means of electronic absorption spectra, since in each, a new chargetransfer like band has been located, and also by observing the variation of the fluorescence emission of FAD
or RFN on the solutions. The formation constants for the molecular complexes were determined from data of
absorption
using the Foster-Hammick-Wardley
method.
The quenching
fluorescence
phenomena
observed for the FAD and RFN were related to the concentration
of the indole derivatives and the
corresponding
quenching constants have been determined. Thermodynamic
parameters have been derived
from the values of association constants for the molecular complexes at several temperatures.
Clear evidence
is found for the influence on the stability of these complexes of the different substituent groups in the indole
derivatives and the molecular structure of both flavins.
riboflavin

INTRODUCTION
Riboflavin (RFN) is important
nutritionally
as the
vitamin Bz. Flavine adenine dinucleotide
(FAD) is a
coenzyme of flavoproteins found in the mitochondria
and it takes part in oxidative phosphorylation.
The flavins
are derivatives
of isoalloxazine
[benzo[g]pteridine-2,4(3H,lOH)-dione]
and, hence,
show a quinone like structure. In view of the well
established
electron acceptor character
of organic
quinones
such as chloranil, it would certainly be
expected that flavins function as electron acceptors [ 11.
Some flavins form complexes with some indole
derivatives and it has been found that charge transfer
forces play an important
role in the formation
of
flavin-indole
complexes [l-3]. Indeed ISENBERC and
SZENT-GYORGY [4],
PEREIRA and
TOLLIN
[5]?
WILSON [6], ISHIWATARIet al. [7], YAGI et al. [S], and
WILLIAMSON et ul. [9] have reported apparent association constants
of flavins with tryptophan,
methylindole and other organic compounds.
The object of the present investigation
is to determine the occurrence of complexes between either
FAD or RFN with a set of indole derivatives such as
tryptophan,
tryptamine and 5-hydroxytryptamine.
On mixing a solution of a donor D and an acceptor
A, one can assume the establishing of the following
equilibrium for the 1: 1 complex [lo, 111.
D + A<d%DA
Where K,,
determined
WARDLEY
HILDEBRAND

is the formation constant, which can be

by means of the FOSTER-HAMMICKmodification
[12]
of the
BENESIequation
[13-l, provided that a new

*To whom correspondence

should

be addressed.

band appears in the spectrum.

can be written as:
electronic

A
p=

CaI

-K,,.A+K,,.[A]o.~rc

This equation

(1)

where

A is the absorbance
at the maximum wavelength, A,,,,, , of the band of the complex, ETC is the
molar extinction
coefficient
of the complex
in
1. mol- .cm-, and [Alo and [D& denote the initial
molar
concentrations
of acceptor
and
donor
respectively.
On the other hand, the apparent constant K for the
quenching of the fluorescence can be derived from the
STERN-V LMER equation
[ 143.
+ = l+K.[D],
where I, and I represent the fluorescence intensities in
the absence and presence of the inhibitor, K is the
apparent constant and [D$, is the concentration
of the
donor (inhibitor).
We have carried out spectrophotometric
measurements of the absorption
complex band in aqueous
medium at several temperatures in phosphate buffer at
pH 6 in order to calculate the equilibrium constants of
the molecular complexes formed by FAD or RFN and
each of the indole derivatives, here considered. Some
thermodynamic
parameters have been computed from
the above results. On the other hand, the apparent
quenching
fluorescence
constants
of flavins when
forming these molecular complexes and related thermodynamic parameters have been also studied.
The aim of the present study is to get information
about these interactions, and try to shoti the influence
on the stability of the molecular complexes from some
factors, such as the different substituent groups carried
by the indole derivatives, or the nature of the flavin
considered.

390

ARMANDOCODO~ER
EXPERIMENTAL

al.

and (vii) the buffer solutions do not interact with donor

and acceptor.
Thus, the spectra of FAD, RFN and each of indole
derivative have been carried out in aqueous and buffer
solutions and no bands have been found at the
wavelength of the complex formation (higher than
500 nm).
The Beers law for the complex DA is assumed
because at the dilute concentration range of work, the
absorbance values are obtained at the maximum
wavelength of the complex, and these well accomplish
the equation 1 (see Fig. la, b). Therefore, if A/[DI, is
plotted against A, a straight line with a slope of - K,,
and an y-intercept of K,, . 6X is obtained. Typical
examples of this are shown in Fig. 1 for (a) FAD-5hydroxytryptamine
and (b) RFN-5-hydroxytryptamine complexes at diverse temperatures. Similar plots
have been performed for the remaining complexes of
FAD and RFN with indole derivatives. The least
squares method was used and the values of KDAand
en: and their corresponding absolute error for 95%
confidence are gathered in Table 2.
Figure 2 shows the absorption spectra bands of
mixed solutions of FAD and varying concentration of
5-hydroxytryptamine in water at 5C. The negative
bands of the spectra are due to the fact that FAD
concentration in the sample cell is lower than in the
reference cell, because a quantity of FAD has been
involved in the complex formation in the sample cell. It
is clear the presence of two characteristic isosbestic
points. The corresponding
FOSTER-HAMMICK-

Materials

The indole derivatives listed in Table 1 were supplied by

Sigma Chemical Company. All of them were purified by
sublimation twice under reduced pressure. FAD and RFN
(Fluka AG purum grade) were used without further
purification.
All the solutions were made in phosphate buffer at pH 6
[lS] whose ionic strength was 0.2 M. It is assumed that, at
working concentration levels, the variations in ionic strength

are irrelevant.
Measurements

The details for the apparatus used to obtain the absorption

and emission spectra have been previously reported [lo].
Absorption spectra were recorded against a solution of the
electron acceptor (FAD or RFN) in the same solvent used to
make the solution of the complex. At each temperature
various measurements were performed with different donor
concentrations at a constant acceptor concentration.
The excitation wavelength for FAD and RFN were set at
445 nm and 470 nm respectively, in which no significant
excitation bands were found for any indole derivative. The
emission spectra of FAD and RFN in the presence of each
indole derivative were recorded from 450 nm to 700 nm. The
quenching fluorescence for FAD and RFN was carried out at
the maximum wavelength (530 nm).
The concentration range of donor in all cases was 10m4 to
10-r M. The concentration of acceptor was maintained
constant in all cases at a value of 1.10e4 M. The temperature
was varied in the 545C range.
RESULTS AND DISCUSSION
Absorption

et

spectroscopy

Equation (1) can be applied taking into account the

following assumptions: (i) only one complex with
defined stoichiometry can be formed from interactions
between D and A, (ii) the complex DA absorbs at a
wavelength where A and D are completely transparent,
(iii) in all cases [DJ, isz [Alo and [Alo has not necessarily to be constant, (iv) the complex is of the simple
1: 1 type, (v) the Beers law for the complex is obeyed,
(vi) a single value of the wavelength at which the
maximum appears is obtained when the ionic strength,
H + concentration and temperature remain constant,

WARDLEYplots and absorption spectra of the remaining complexes for FAD and RFN with the indole

derivatives listed in Table 1, are similar to Figs 1 and 2

and are not included.
The computed values of KDA at four different
temperatures (5, 15,25 or 27 and 45C) were used to
determine the thermodynamic parameters. Using the
Vant Hoff equation a plot of In KDA against l/T was
made to determine the change in enthalpy AH and
entropy AS. The values of AG at each temperature
are estimated by usual method. These results are listed

Table 1. Indole derivatives

Compound

L-Tryptophan
Tryptamine
5-Hydroxytryptamine

Formulae

Salt

R,

&

COOH
H
H

EH

HCl
Oxalate

Laboratory

Sigma
Sigma
Sigma

Associations

between

FAD and RFN and some indole derivatives

(a)

20 -

.
\

1l \

2
D

$15 ~

l
\.

\.,
10 -

\ .

\
.

\.,\
l

.\
%,

I
0 04

0 06

0 08

0 10

0 12

(b)

I!

I
0 10

0 05

015

Fig. 1. Plots of A/[D],

vs A for complexes: (a) FAD-S
hydroxytryptamine
(b) RFN-5-hydroxytryptamine
at various temperatures:
5C (O), 15C (A), 25C (+), 27C (0)
and 45C ( W).

in Table 2 with their corresponding

absolute errors.
It is assumed that all complexes are charge-transfer
type with stoichiometry
1: 1, since: (i) the straight lines
are found plotting equation 1, (ii) in all cases, two
isosbestic points are present in the absorption spectra,
(iii) the equilibrium constant decreases as the temperature increases, (iv) AH are less than 30 kJ mol- ; this
stoichiometry has been previously reported for similar
complexes between flavins and indole derivatives Cl].

391

392

ARMANDOCBDOGER

350

400

450

etal.

Temperature 5C

500

550

600

650

x(nm)

Fig. 2. Absorption spectra of FABS-hydroxytryptamine

solutions. Concentration of FAD: 1.00
x 10e4 M; concentration of 5-hydroxytryptamine (M): (1) 8.87 x 10m3, (2) 7.09 x 10m3, (3) 5.32 x W3,
(4) 4.26 x lo-, (5) 3.55 x 10-3, (6) 2.84 x 1O-3.

For the flavins studied and at a definite temperature,

RFN leads to greater stability of the complexes than
FAD, (see Table 2). This fact is possibly due to the
internal differences in both flavin structures. It is wellknown [16], that FAD undergoes intramolecular
associations in which the adenine residue (not present
in RFN) reacts with the isoalloxazine structure.
Moreover, simultaneously with the formation of
charge-transfer complex adducts in the presence of
donors, FAD has the chance to form hydrogen bond
oligomers [ 171. Thus, an increase of temperature will
lead to a minor repercussion on the complexes
FAD-indole derivatives than the RFN-indole derivatives studied, as it is actually found (see Table 2).

On the other hand, comparing the results obtained

for the different donors, it can be seen that the
complexes with L-tryptophan and tryptamine have
similar stabilities and behaviours with FAD and RFN.
On the contrary, Shydroxytryptamine
leads to a
major stability and different behaviour with the two
flavins studied. This fact suggest that the presence of
polar groups, as hydroxy or methoxy [lo] substituents
on the indole nucleus, results in the increase of the
charge-transfer-complex activity while polar groups in
the amine residue (-CH2-CH(R2kNH2)
do not show
any considerable influence. This is consistent with the
idea [6,18] that the delocalized n-electronic structures
play an important role on the charge-transfer-complex

U I)

Temperature 5C

Fig. 3. Quenching of FAD fluorescence caused by added 5-hydroxytryptamine. Concentration of FAD: 0.96
x 1O-4 M; concentration of 5-hydroxytryptamine (M): (2) 0.67 x 10e3, (3) 1.01 x lo-, (4) 1.67 x 10A3,
(5) 2.70 x 10-3, (6) 3.38 x 10-3, (7) 4.06 x lo-, (8) 5.07 x 10-3, (9) 6.76 x 10-3, (10) 8.45 x lo-.

Associations between FAD and RFN and some indole derivatives

interaction. Moreover, in the case of 5-hydroxytryptamine with FAD and RFN acceptors, the chance to
form hydrogen bonds with FAD is mainly supported
by the fact that at 45C the K,,
for FAD5hydroxytryptamine
becomes greater than the K,, for
RFN-5-hydroxytryptamine.
These further assessments
are supported
by the
lowest value of AS which involves less freedom
degrees of the FADS-hydroxytryptamine
complex.
Fluorescence spectroscopy
The progressive changes of the fluorescence spectra
of FAD caused by adding 5-hydroxytryptamine
are
shown in Fig. 3. From the values of the fluorescence
intensities and applying the STERN-V LMER relation
good straight lines have been obtained as it can be seen
in Fig. 4, and from them, the apparent constant, K, has
been derived at each temperature.
Similar representations
as those given in Figs 3 and
4, have been obtained for each of the other complexes
of FAD and RFN with tryptophan, tryptamine and 5hydroxytryptamine,
and the corresponding
values of
K derived from them are included in Table 3.
In all cases an increase of temperature
leads to a
decrease of the slope of the STERN-V LMER relation.
This fact could be attributed
to the typical static
quenching due to the complex formation. However, it is
well known [19] that FAD molecules quench themselves by dynamic inhibition. So, the K values derived
from the STERN-V LMER relation are higher than
those found with absorption
spectroscopy
method.

[D], x lo3

for the FAD-SFig. 4. Plots of lo/l

vs [D].
hydroxytryptamine
complex at various temperatures:
5C (a), 15C (A), 25C (+) and 45C (m).

393

394

ARMAND~COIXIAER et al.

This fact reflects the presence of several concurrent

the
quenching
fluorescence
mechanisms
on
phenomena.
The complexes formed with RFN are slightly more
stable than those formed with FAD as it can be seen in
Table 3, in good agreement with the trend observed by
the absorption spectroscopy method.
However, it is noted that the stabilities of the 5hydroxytryptamine complexes with both RFN and
FAD are greater than those observed for the other
indole derivatives, emphasizing, once more, that the
presence of polar groups in the indole nucleus, as, e.g.
hydroxyl group, plays an important role on the
stability of the complex.
All the thermodynamic parameters have been obtained by usual methods and are similar to those found
through the absorption method.
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