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Spectrochimica Acta, Vol. 42A, No. 7, pp. 765 769, 1986.

0584-8539/86 S3.00 +0.00


Pergamon Journals Ltd.

Printed in Great Britain.

Spectroscopic study of intermolecular complexes between FAD and


some fl-carboline derivatives
ARMANDO CODOI~IER,* ISIDRO S. MONZO, FRANCISCOTOMAS and ROSA VALERO
Departamento de Quimica Fisica, Facultad de Ciencias Quimicas, Universidad de Valencia, Burjassot,
Valencia, Spain

(Received 12 September 1985; infinal form 7 January 1986; accepted 13 January 1986)
Abstract--The formation of molecularcomplexes between flavineadenine dinucleotide (FAD)and some flcarboline derivatives [antidepressant drugs that have a pronounced inhibition of monoamine oxidase
(MAO)] has been studied by using electronic absorption and fluorescence spectroscopic methods.
Thermodynamicparameters havebeen determined from the valuesof associationconstants for the molecular
complexes at various temperatures.The influenceof substituents in the fl-carbolinemoleculeon the stability
of the complexes formed was also investigated.

INTRODUCTION
It is well known that MAO acts on cerebral deposits of
amines producing an oxidative deamination of these
compounds [1-6], The inhibitors of MAO block this
enzyme hindering this oxidative domination and thus
lead to an increase in the concentration of cerebral
amines [7, 8]. This mechanism of inhibition therefore
leads to increasing cerebral activity and makes the
inhibitors of MAO antidepressant drugs [9, 113].Some
derivatives of fl-carboline (9H-3,4b-pyrido indole)
have been described as MAO inhibitors [11-20].
FAD (flavine adenine dinucleotide) is a prosthetic
group of MAO [21-26]. We have selected this compound to study its interaction with fl-carboline derivatives and to characterize the molecular complexes
formed between FAD and each of these fl-carboline
derivatives.
On mixing a solution of a donor D and an acceptor
A, the following equilibrium is established for the 1 : 1
complex
D + A " KDA DA
where

KDAis the

[DA]
KDA -- [D] [A]

formation constant, written as


[DA]
([D]o - IDA])([A]o - IDA])

0)
where [DA], [A]0 and [D]o denote the molar concentration of the complex and the initial molar concentrations of the acceptor and donor, respectively.
The FOSTER-HAMMICK--WARDLEY[27] modification of the BENESI-HILDEBRANDequation [28] can
be used to calculate the equilibrium constant for the
FAD-fl-carboline complexes, This equation can be
written as
A

[D]o

KDA" a + KDA" [A]o" eTC

(2)

*Author to whom correspondence should be addressed.

where A is the absorbance at the maximum wavelength


of the band of the complex, 2~x, exc is the molar
extinction coefficient of the complex in 1-mol- 1. cm- 1
and other quantities are the same as in Eqn. (1).
On the other hand, the apparent constant K for the
quenching of the fluorescence is deduced from the
STERN-VOLMER equation [29]
I = 1 + K. [D]0
I

where Io and I are the fluorescence intensities in the


absence and presence of the inhibitor, K the apparent
constant and [D]0 the concentration of the donor
(inhibitor).
In this paper we report the spectroscopic and
thermodynamic properties of the molecular complexes
obtained by mixing FAD with some fl-carboline
derivatives in aqueous solutions. The aims of the
present study are to get information about this interaction and to point out the influence of the substituents
of fl-carboline on the stability of the molecular complexes studied. A comparison is made between the
association constants obtained by means of electronic
absorption spectroscopy and the values of the thermodynamic parameters of the molecular complexes.
The apparent constants for the quenching of the
fluorescence of FAD by the formation of these molecular complexes and its thermodynamic parameters have
been also studied. The values of constants and thermodynamic parameters obtained by the two experimental
techniques utilized are compared.

EXPERIMENTAL

Materials
The fl-carbolinederivativeslisted in Table 1 were supplied
by Fluka AG and Sigma Chemical Company. All of them
were purifed by sublimation twice under reduced pressure.
FAD (Fluka AG purum grade) was used without further
purification.
All the solutions were made in phosphate buffer at pH 6
except the ones correspondingto harmane that were prepared
at pH 5.5 using water as solvent.
765

SA(AJ~2:~-A

(3)

766

ARMANDOCODO~ERet o2.

x.

Table 1. p-Carboline derivatives


Compound

Formula

Salt

Laboratory

RI~~~/N
H
R,
Norimrmane
Harmane
Harmol
Harmine

18

R2
16

Rz

H
H
OH
OCH3

H
CHa
CH3
CHs

20

HCI
HC1
HCI
HC1

Sigma
Sigma
Sigma
Fluka

14

12

Measurements
The electronicabsorption spectra were recorded on a Cary
219 spectrophotometer with a double beam system and a
temperature-regulated cell holder adapted for 1 crn cells.
Circulating water maintained the holder temperature constant to an accuracy of +0.1C. The temperature was
monitored with a thermocoupleattached to the referencecell.
Matched stoppered quartz cellsof I emgathlength wereused.
Spectra were recorded against a solution of the electron
acceptor (FAD)in the same solvent used to make the solution
of the complex. At each temperature six measurementswere
made with different donor concentrations keeping the acceptor concentration constant.
The fluorescence emission of FAD and the fl-carboline
derivatives was recorded with a MPF 44A Perkin-Elmer
spectrofluorimeter. For all the compounds studied the
fluorescenceexcitation spectra correspond to the absorption
band localizedin the visibleregion of FAD. The quenchingof
the fluorescence of FAD with increase in electron donor
concentration was recorded.
The concentration range of donors (~-carbolines) employed in all cases was 10-2-10 -4 M. The concentration of
aeceptor was constant and of the order of I x 10-4 M. The
temperature range studied was 5-45C.

RESULTS
Absorption spectroscopy
Equation (2) 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 [D]o ~> [A]o and [A]o has not necessarily to be constant, (iv) the complex is of the simple
1 : 1 type, and (vi) Beer's law is obeyed.
If A/[D]o is plotted against A, a straight line with
slope --KDA and y-intercept KDA'STC can be obtained. To minimize experimental errors these parameters were determined using the least squares
method. A typical plot is shown in Fig. 1. In that the
donors studied (D = norharmane, harmane, harmol
and harmine) have delocalized electronic ,structures,
they can be involved in ~t-electron interactions with
typical acceptors as well as with FAD [30, 311.
Figure 2 shows the absorption spectra of mixed
solutions of FAD and varying concentrations of
norharmane in water at 5C. The negative bands of the
spectra are due to the fact that the FAD concentration

10

I
0.06

I
0.08

I
0.10

I
0.12

Fig. 1. Plots of A/[D]0 vs A for the FAD-norharmane


complexat various temperatures:5C (o), 15C (A),25C (O)
and 45C ( ) .

in the sample cell is lower than that in the reference cell


because FAD is involved in complex formation in the
sample cell. As expected, the presence of two characteristic isosbestic points in a family of absorption curves
suggests the formation ofa 1 : 1 complex between FAD
and norharmane (Fig. 2).
The corresponding FOSTER-HAMMICK-WARDLEY
plots and absorption spectra of the other complexes
between FAD and fl-carboline derivatives studied are
similar to Figs 1 and 2 and are therefore not included.
The computed values of KDA at four different
temperatures (5, 15, 25 and 45C) were used to
calculate AG for the complex formation. Using the
'Can't Hoff equation, a plot of In KDA against 1/Twas
made to determine the change in enthalpy. AS values
were calculated from AG and AH .
The equilibrium constants of these complexes determined by Eqn. (2) and thermodynamic parameters
and as well as the corresponding standard error for
95 % confidence interval values are shown irL Table 2.
Fluorescence spectroscopy
The fact that a linear STERN-VOLMERplot was
obtained for all the fl-carboline derivatives in aqueous
solution enables calculation of the quenching constant
K from the slope of the linear plot. As a typical
example, the STERN-VOLMER plot for the
FAD-norharmane system is shown in Fig. 3. The
progressive changes of the fluorescence spectra of
FAD caused by adding norharmane are indicated in
Fig. 4 (which refers to 5C). Similar behaviour to that

Complexes of FAD and fl-carboline derivatives

767

0.4

0.3

0.2

0.1

-0.1

Temperature 5 C

-0.2
1
J
450

I
500

I
550

I
600

~. (nm)

Fig. 2. Absorption spectra of FAD-norharmane solutions. Concentration of FAD: 1.00 x 10 -4 M;


concentration of norharmane (M): (1) 12.15 x 10 -s, (2) 9.72 x 10 -3, (3) 7.29 x 10 -3, (4) 5.83 x 10 -3, (5) 4.86
x 10 -s, (6)3.88 x 10 -3.

2-

,/,Ji/"
1

I
2

I
I
3
4
(D)0 x 103

Temperot

I
5

Fig. 3. Plots of Io/I vs [D]o for the FAD-norharmane


complex at various temperatures: 5C (e), 15C (&), 25C ( 0 )
and 45C ( ).

s h o w n in Figs 3 a n d 4 was found for each o f the


complexes between F A D a n d fl-carboline derivatives
at other temperatures.
The t h e r m o d y n a m i c quantities o f these complexes
were obtained from the quenching constants at four
temperatures (5, 15, 25 a n d 45C) by the usual method.

450

500

550

600

650

ure

5"C

700

X (nm)

Fig. 4. Quenching of FAD fluorescence caused by added


norharmane. Concentration of FAD: 1.00 x 10 -4 M; concentration of norharmane (M): (2) 0.6 x 10 -3, (3) 1.0 x 10 -3,
(4) 1.6x 10 -3 , (5)2.0x 10 -3 , (6)2.4x 10 -3 , (7)3.0x 10 -3 ,
(8) 4.0 x 10 -3, (9) 5.0 x 10 -3.

The values of the quenching constants o f these complexes determined by Eqn. (3) a n d the t h e r m o d y n a m i c
parameters are indicated in Table 3.

ARMANDO CODOI~ER el OL

768

+1 +1

+1+1+1+1

~+1+1

+1+1 4-1-1-1

<

+1 4"1 +1 +1

+1 +1 +1 +1

+1+1+1+1

+1 +1 +1 +1

<

::1

+1 +1 +1 +1

+1 +1 +1 +1

+1+1+1+1

i
k2

+14-1 +1 +1

+1 +1 +1 +1

m~

+1 +1 4-1 +1

+1 +1 +1 +1

~'~

Complexes of FAD and fl-carboline derivatives


DISCUSSION

Whereas most of the experimental work [32] establishes that there is a straight line correspondence
between the value of KDA and 2m~x for charge-transfer
complexes, in the sense that an increase in the stability
of the complex involves an increase in ~-max,it can be
seen from the data reported in Table 2 that this is not
true for the complexes between F A D and the flcarboline derivatives studied in this work. This feature
can be attributed to the unusual behaviour of ftavins,
as argued by ORTIZ [33].
The shape of the charge-transfer bands is usually
broad but it can be seen from Fig. 2 that the band for
the complex between F A D and norharmane is sharp.
This phenomenon can be attributed to the presence of
more acceptor (FAD) in the reference than in the
sample cell, leading to a negative overlap between the
charge-transfer band and the band characteristic of
FAD.
An important feature which arises from Table 2 is
the considerable difference in equilibrium constants
between the adducts of the various fl-carboline derivatives with norharmane. This can be attributed to the
different chemical structures of the molecules; a slight
difference occurring when the R 2 substituent (see
Table 1) changes from H to CH3 (slightly different
polarity between substituents) and a large difference
when R 1 changes in the follow order: H to O H to
O C H 3, since for these substituent groups the ordering
of polarity is O C H 3 > O H > H.
On the other hand, the values of A H and AS
obtained for the complexes between fl-carboline derivatives and F A D confirm the previous arguments,
because there is an important influence on the thermodynamic parameters by the R1 substituent group as
can be seen from columns 9 and 10 in Table 2.
As expected, the quenching constants decrease with
increase in temperature, as shown in Fig. 3.
With respect to the emission spectra given in Fig. 4,
we observe typical fluorescence bands at 5C for the
complex between F A D and norharmane. The fluorescence band of F A D is quenched when donor (norharmane) is added (see curves 1-9).
The variation of the quenching constant for complexes of F A D with fl-carboline derivatives with
respect to the different substituents R 1 and R 2 given in
Table 3 confirms the above arguments based on absorption methods, although the values obtained from
the STERN-VOLMER equation are higher than those
obtained from the FOSTER-HAMMICK-WARDLEY
equation. This is as expected, since the equilibrium
constant derived from the STERN--VOLMER equation
involves not only the thermodynamic equilibrium
constant formation but all the mechanisms involved
on the quenching fluorescence. It is usual that the
values of K obtained from Eqn. (3) are somewhat
higher than those of KDA obtained from Eqn. (2).
The variation of the values of AG , A H and AS
listed in Table 3 are in good agreement with the

769

variation expected on the basis of the arguments given


above for the absorption results.

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