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Investigation on the electrochemical properties of catecholamines and melanins
Bioelectrochemistry and Bioenergetics 7 (198o) 741-748 J. Electroanal. Chem. 116 (198o) 741-748
Elsevier Sequoia S.A., Lausanne - Printed in Italy Short Communications
3 2 9 - I n v e s t i g a t i o n on the Electrochemical Properties of Catecholamines a n d Melanins * b y J. MATYSIK and M. PRZEGALI~SKI Institute of Chemistry, Maria Sklodowska University P1. M. Sk|odowskiej 3, Lublin, Poland Revised manuscript received September i lth 198o
Summary The melanization process was investigated using electrochemical methods. On the a.c. and multisweep curves the particular stages occur as separated, consecutively rising and decreasing peaks, corresponding to gradual association of precursor molecules. An interesting effect is the rise of d.c. waves at potentials characteristic for H202 (appearing usually if 02 is present in the solution) in deoxygenated solutions of melanins. The differences in the curves of catechol-melanin and indole-melanin permit to distinguish these compounds using polarographic methods. The result of practical importance is the possibility of a fast deteimination of small amounts of adrenaline and noradrenaline b y use of multisweep methods.
Introduction An essential structural element of catecholamines and melanins is the system of hydroxyl (or quinone) groups in the ortho position at the aromatic ring [I]. Such a structure ensures their strong chelating and reversible red-ox properties [2, 3]- It is just these properties that are mainly responsible for tile functions of catecholamines and melanins in living organisms and they also make these compounds convenient objects for electrochemical studies [4, 5]The methods of the investigations included d.c., a.c., s.w. (square wave) polarography, multisweep and electrocapillary measurements since * Presented at the 5th International Symposium on Bioelectrochemistry, 3-8 September 1979, Weimar (D.D.R.).
o3o2-4598/8o/o74I-O748
© 198o Elsevier Sequoia S.A.
742
Matysik and Przegaliflski
these techniques permit a very quick or even continuous recording of the r e d - o x processes and adsorption effects. The present investigations dealt with catechol-melanin and indolemelanin as well as with adrenaline and noradrenaline as soluble compounds occurring in living organisms, containing the essential structural element similar to that of melanins.
Esperimental
Melanins were obtained without use of enzymes, b y the air oxidation of catechol and DOPA in solution o,I M KOH. The product was precipitated with hydrochloric acid at pH----2 and purified b y dialysis. The d.c. studies were carried out using the polarograph LP-6o, (LABoRATORNI P~ISTROJE), a.c.-using polarograph P L P - A C , IChF, P.A.N., s.w.-using polarograph O H - I o 4 , RADELKIS, the multisweep measurements were made on a Digiscope polarograph OP-3, TELPOD at sweep rates from 2 to 20 V s -1. The oscillopolarographic observations were done on the Polaroscope K~I~IK P-576, to obtain the relationship d U / d t = f ( U ) . The electrocapillary curves of melanins were obtained from averaged drop-time of 50 drops of Hg at corresponding potentials. The electrolyte used was a I M water solution of K O H or I M NH4NO3N H 4 0 H buffer, p H ---- 8,5. The characteristics of the D.M.E. were 2.1 mg s -1, and 2. 4 s drop time. As the reference electrode served a S.C.E. The measurements were made in three (digiscope, s.w., a.c. polarography) or two electrode system, at 293 K. If necessary, the oxygen was removed from the solutions using nitrogen.
Results and discussion
It turned out that melanization of catecholamines is a convenient object for the study. Simultaneously with the solution turning brown, the multisweep polarography curves registered the stages of the process as consecutive rising and falling of reversible signals (peaks), corresponding most probably to the degrees of polymerization (Fig. I). Considering that on the a.c. polarograms at the potential of the mentioned signals the well pronounced peak occur b u t no polarographic waves were observed on the d.c. curves we conclude that the a.c. and multisweep peaks are capacitive and not faradaic. The a.c. and electrocapillary curves show that the signals are connected to electrode processes taking place in the interval of the adsorption of the compound (Fig. 2, 3). The first changes in the curves become visible already after seconds and the period of quick changes lasts for about I h. It turned out that the rate of the melanization process is quickened b y the presence of heavy metal ions.
Electrochemical
Properties
of Catecholamines
and
Melar~ins
743
'30
'20
•
q
'10
1
,,I -1.5
U(V) -1.0
-0.5
-10
2
"-30
"-40 Fig. I. lgiultisweep c u r v e s of a d r e n a l i n e 1 . 5 × i o -¢ M in N I - I , N O 3 + N I - I , O t { , p H ~ 8, 5, in t h e p r e s e n c e of air. Sweep r a t e ~ 2o V s - k i. t h e s u p p o r t i n g e l e c t r o l y t e ; 2. t h e s u p p o r t i n g e l e c t r o l y t e + a d r e n a l i n e , a f t e r o m i n ; 3- t h e s u p p o r t i n g e l e c t r o l y t e + a d r e n a l i n e , a f t e r i o m i n ; 4. t h e s u p p o r t i n g e l e c t r o l y t e + a d r e n a l i n e , a f t e r i h.
A strange effect was the occurrence of the d.c. polarographic waves of indole-melanine in deoxidized I M KOH solution at U,A = - o , 9 5 V and U5 = - 1 , 3 8 V which are characteristic for the presence [6] of HO2and H202 (Fig. 4). The formation of H202 in the reaction between quinones and O H - ions was described by LAZAROV and coworkers [7]
Q + OH- ~ Q-~+ OH"
744
Matysik
and
Przegalifiski
l:
F i g . 2. a.c. p o l a r o g r a m s .
1
I. I M KOI-I, d e o x i d i z e d s o l u t i o n ; 2. a s I + c a t e c h o l - m e l a n i n 2 0 0 tzg c m 3, a m p l i t u d e = IO m V , f r e q u e n c y = 76 I-Iz.
U(V)
-1;6.
-1.'2
'
-018
-0'.4
010
40 •
1
3.5
3.0
F i g . 3. E l e c t r o c a p i l l a r y c u r v e s ( d r o p t i m e vs. U ) . I. I 2V/KOFI, d e o x i d i z e d s o l u t i o n ; 2. a s I + catechol-melanin; 3. a s I + i n d o l e melanin.
2.5 U t\/)
-@
-~is
-0%
-0%
Electrochemical
Properties
of C a t e c h o l a m i n e s
alld Melallins
745
(where Q = quinone form) and the course of this reaction should be forced b y following irreversible dimerization : 2
OH" -+ H202
however, this explanation makes objections due to energetic reasons. -1.9
-15
-1.1
-0.7
u(v)
.G
-o'3
-2 %
--3
-4 Fig. 4. d.c. p o l a r o g r a m s . I. I M K O H , deoxidized s o l u t i o n ; 2. as I + indole m e l a n i n IOO Ixg c m -a.
The similarity between the polarographic behaviour of the deoxidized melanine solutions and H202 solutions in I M K O H occurs also in the other methods. S.w. peaks corresponding to H202 and to melanine solutions are located at the same potential values, they have similar asymetric shape and identical half-wawe width (24 ° mV). In the a.c. polarograms of both compounds no peaks (corresponding to the Hi02 electroreduction) o c c u r e d . On the oscillopolarograms, at potentials corresponding to s.w. polarographic waves, in both cases there did not appear any oscillopolarographic indentation. B y electroreduction of the TI+I and pb+2 in deoxidized melanine solutions effects of hidden limiting currents occurred characteristic for the reduction of these ions in the presence of 03 or H~O2. Since in the d.c. curves of catechole-melanin only a wave corresponding to H202
Matysik
746
and
Przegalifiski
reduction occurs, it offers a possibility for differentiation between the two melanins (Fig. 5). Another practical result of the oscillopolarographic and multisweep investigations m a y be seen in the possibility of a quick determination of low concentration of hydrolyzing salts of heavy metals in the presence of catecholamines as well as a possibility of quantitative determination of catecholamines (Fig. 6) (adrenaline, noradrenaline DOPA) after introduction of some inorganic cations to the solution E8].
~.9.. '
1.5 J
.
1.1 .
.
.
o . ~ U(V)'
-013
--2
--3
'-4
-5
Fig. 5d.c. p o l a r o g r a m s . I. M IKOI-I, deoxidized s o l u t i o n ; 2. as i + catechol m e l a n i n 2oo a g c m -3.
In order to verify the results as an analytical method, we examined pharmaceutical solutions of adrenaline for injections. 0,27 cm 3 of the content of the injection was diluted b y 9,73 cm3 ammonium buffer and after addition of Cu ~+ to concentration 1.5 × lO -5 M, the solution was examined using the multisweep polarograph. The results of these examinations were very promising, however, the method brought no positive results in underproteinized organic fluids. The mechanism of forming of oscillographic signals in the Men+-catecholamine system already at low substrate concentration is based on the course of reversible r e d - o x processes of catecholamine complexed cations under continuous complex adsorption, enhancing the concentration at the electrode surface.
Electrochemical
P r o p e r t i e s of C a t e c h o l a m i n e s alld 1Viclanins
747
"40 .,SO .20 ~z~.:.10 K, -1.6
-1.2 , ~ - 0;8
I
-0.4 I U(V)
--0.0
/
I
.-10
•-20
'-30 .-40 Fig. 6. Multisweep curves. I. 1. 5 × IO -s M Cu +~ in N t - I 4 N O 3 + N H 4 O t { , ptL = 8. 5 in the presence of air ; 2. as I in the presence of adrenaline 5 × lO-5 M sweep rate ~ 20 V s -x.
This is implied by the increase of signals during the drop time of the D.M.E. and by the fact that the potentials of the indentations or peaks are characteristic not for catecholamines but for the metal used. It seems that in the adsorbed layer melanine retains both the chelating and red-ox systems, which, together with the flat orientation of the aromatic ring at the D.M.E. (and the ~ electrons interactions), makes possible ion adsorption and electron transmission between the electrode surface and metal ions.
References [11 G . A . SWAN,
Progress in the Chemistry of Organic Natural Products, S p r i n g e r -
Verlag, W i e n , N e w Y o r k
(1974) T. SARNA a n d S. ~UKIEWlCZ, Folia Histochem. Cytochem. 10 (1972) 265 [3] H . G . CASSIDY a n d A . A . KUN, Oxydation-Reduction Polymers, I n t e r s c i e n c e
[2]
Publ.,
New York
(1965)
748 [4] [5] [6] [7] [8]
Matysik and Przegaliflski V. HORAK and J.R. GILLETTE, Mol. Pharmacol. 7 (1971) 429 M. PRZEGALIi~SKI, P h . D . Thesis, UMCS, Lublin (1976) J. CHODKOWSKI, Rozn. Chem. 27 (1953) 309 S. LAZAROV. A. TROFINOV and T. PoPov, Z. Phys. Chem. 11 (1967) 145 IV[. PRZEGALII~SKI and J. ~¢[ATYSIK, Talanta 27 (198o) 920
Elsevier Sequoia S.A., Lausanne - Printed in Italy Short Communications
3 2 9 - I n v e s t i g a t i o n on the Electrochemical Properties of Catecholamines a n d Melanins * b y J. MATYSIK and M. PRZEGALI~SKI Institute of Chemistry, Maria Sklodowska University P1. M. Sk|odowskiej 3, Lublin, Poland Revised manuscript received September i lth 198o
Summary The melanization process was investigated using electrochemical methods. On the a.c. and multisweep curves the particular stages occur as separated, consecutively rising and decreasing peaks, corresponding to gradual association of precursor molecules. An interesting effect is the rise of d.c. waves at potentials characteristic for H202 (appearing usually if 02 is present in the solution) in deoxygenated solutions of melanins. The differences in the curves of catechol-melanin and indole-melanin permit to distinguish these compounds using polarographic methods. The result of practical importance is the possibility of a fast deteimination of small amounts of adrenaline and noradrenaline b y use of multisweep methods.
Introduction An essential structural element of catecholamines and melanins is the system of hydroxyl (or quinone) groups in the ortho position at the aromatic ring [I]. Such a structure ensures their strong chelating and reversible red-ox properties [2, 3]- It is just these properties that are mainly responsible for tile functions of catecholamines and melanins in living organisms and they also make these compounds convenient objects for electrochemical studies [4, 5]The methods of the investigations included d.c., a.c., s.w. (square wave) polarography, multisweep and electrocapillary measurements since * Presented at the 5th International Symposium on Bioelectrochemistry, 3-8 September 1979, Weimar (D.D.R.).
o3o2-4598/8o/o74I-O748
© 198o Elsevier Sequoia S.A.
742
Matysik and Przegaliflski
these techniques permit a very quick or even continuous recording of the r e d - o x processes and adsorption effects. The present investigations dealt with catechol-melanin and indolemelanin as well as with adrenaline and noradrenaline as soluble compounds occurring in living organisms, containing the essential structural element similar to that of melanins.
Esperimental
Melanins were obtained without use of enzymes, b y the air oxidation of catechol and DOPA in solution o,I M KOH. The product was precipitated with hydrochloric acid at pH----2 and purified b y dialysis. The d.c. studies were carried out using the polarograph LP-6o, (LABoRATORNI P~ISTROJE), a.c.-using polarograph P L P - A C , IChF, P.A.N., s.w.-using polarograph O H - I o 4 , RADELKIS, the multisweep measurements were made on a Digiscope polarograph OP-3, TELPOD at sweep rates from 2 to 20 V s -1. The oscillopolarographic observations were done on the Polaroscope K~I~IK P-576, to obtain the relationship d U / d t = f ( U ) . The electrocapillary curves of melanins were obtained from averaged drop-time of 50 drops of Hg at corresponding potentials. The electrolyte used was a I M water solution of K O H or I M NH4NO3N H 4 0 H buffer, p H ---- 8,5. The characteristics of the D.M.E. were 2.1 mg s -1, and 2. 4 s drop time. As the reference electrode served a S.C.E. The measurements were made in three (digiscope, s.w., a.c. polarography) or two electrode system, at 293 K. If necessary, the oxygen was removed from the solutions using nitrogen.
Results and discussion
It turned out that melanization of catecholamines is a convenient object for the study. Simultaneously with the solution turning brown, the multisweep polarography curves registered the stages of the process as consecutive rising and falling of reversible signals (peaks), corresponding most probably to the degrees of polymerization (Fig. I). Considering that on the a.c. polarograms at the potential of the mentioned signals the well pronounced peak occur b u t no polarographic waves were observed on the d.c. curves we conclude that the a.c. and multisweep peaks are capacitive and not faradaic. The a.c. and electrocapillary curves show that the signals are connected to electrode processes taking place in the interval of the adsorption of the compound (Fig. 2, 3). The first changes in the curves become visible already after seconds and the period of quick changes lasts for about I h. It turned out that the rate of the melanization process is quickened b y the presence of heavy metal ions.
Electrochemical
Properties
of Catecholamines
and
Melar~ins
743
'30
'20
•
q
'10
1
,,I -1.5
U(V) -1.0
-0.5
-10
2
"-30
"-40 Fig. I. lgiultisweep c u r v e s of a d r e n a l i n e 1 . 5 × i o -¢ M in N I - I , N O 3 + N I - I , O t { , p H ~ 8, 5, in t h e p r e s e n c e of air. Sweep r a t e ~ 2o V s - k i. t h e s u p p o r t i n g e l e c t r o l y t e ; 2. t h e s u p p o r t i n g e l e c t r o l y t e + a d r e n a l i n e , a f t e r o m i n ; 3- t h e s u p p o r t i n g e l e c t r o l y t e + a d r e n a l i n e , a f t e r i o m i n ; 4. t h e s u p p o r t i n g e l e c t r o l y t e + a d r e n a l i n e , a f t e r i h.
A strange effect was the occurrence of the d.c. polarographic waves of indole-melanine in deoxidized I M KOH solution at U,A = - o , 9 5 V and U5 = - 1 , 3 8 V which are characteristic for the presence [6] of HO2and H202 (Fig. 4). The formation of H202 in the reaction between quinones and O H - ions was described by LAZAROV and coworkers [7]
Q + OH- ~ Q-~+ OH"
744
Matysik
and
Przegalifiski
l:
F i g . 2. a.c. p o l a r o g r a m s .
1
I. I M KOI-I, d e o x i d i z e d s o l u t i o n ; 2. a s I + c a t e c h o l - m e l a n i n 2 0 0 tzg c m 3, a m p l i t u d e = IO m V , f r e q u e n c y = 76 I-Iz.
U(V)
-1;6.
-1.'2
'
-018
-0'.4
010
40 •
1
3.5
3.0
F i g . 3. E l e c t r o c a p i l l a r y c u r v e s ( d r o p t i m e vs. U ) . I. I 2V/KOFI, d e o x i d i z e d s o l u t i o n ; 2. a s I + catechol-melanin; 3. a s I + i n d o l e melanin.
2.5 U t\/)
-@
-~is
-0%
-0%
Electrochemical
Properties
of C a t e c h o l a m i n e s
alld Melallins
745
(where Q = quinone form) and the course of this reaction should be forced b y following irreversible dimerization : 2
OH" -+ H202
however, this explanation makes objections due to energetic reasons. -1.9
-15
-1.1
-0.7
u(v)
.G
-o'3
-2 %
--3
-4 Fig. 4. d.c. p o l a r o g r a m s . I. I M K O H , deoxidized s o l u t i o n ; 2. as I + indole m e l a n i n IOO Ixg c m -a.
The similarity between the polarographic behaviour of the deoxidized melanine solutions and H202 solutions in I M K O H occurs also in the other methods. S.w. peaks corresponding to H202 and to melanine solutions are located at the same potential values, they have similar asymetric shape and identical half-wawe width (24 ° mV). In the a.c. polarograms of both compounds no peaks (corresponding to the Hi02 electroreduction) o c c u r e d . On the oscillopolarograms, at potentials corresponding to s.w. polarographic waves, in both cases there did not appear any oscillopolarographic indentation. B y electroreduction of the TI+I and pb+2 in deoxidized melanine solutions effects of hidden limiting currents occurred characteristic for the reduction of these ions in the presence of 03 or H~O2. Since in the d.c. curves of catechole-melanin only a wave corresponding to H202
Matysik
746
and
Przegalifiski
reduction occurs, it offers a possibility for differentiation between the two melanins (Fig. 5). Another practical result of the oscillopolarographic and multisweep investigations m a y be seen in the possibility of a quick determination of low concentration of hydrolyzing salts of heavy metals in the presence of catecholamines as well as a possibility of quantitative determination of catecholamines (Fig. 6) (adrenaline, noradrenaline DOPA) after introduction of some inorganic cations to the solution E8].
~.9.. '
1.5 J
.
1.1 .
.
.
o . ~ U(V)'
-013
--2
--3
'-4
-5
Fig. 5d.c. p o l a r o g r a m s . I. M IKOI-I, deoxidized s o l u t i o n ; 2. as i + catechol m e l a n i n 2oo a g c m -3.
In order to verify the results as an analytical method, we examined pharmaceutical solutions of adrenaline for injections. 0,27 cm 3 of the content of the injection was diluted b y 9,73 cm3 ammonium buffer and after addition of Cu ~+ to concentration 1.5 × lO -5 M, the solution was examined using the multisweep polarograph. The results of these examinations were very promising, however, the method brought no positive results in underproteinized organic fluids. The mechanism of forming of oscillographic signals in the Men+-catecholamine system already at low substrate concentration is based on the course of reversible r e d - o x processes of catecholamine complexed cations under continuous complex adsorption, enhancing the concentration at the electrode surface.
Electrochemical
P r o p e r t i e s of C a t e c h o l a m i n e s alld 1Viclanins
747
"40 .,SO .20 ~z~.:.10 K, -1.6
-1.2 , ~ - 0;8
I
-0.4 I U(V)
--0.0
/
I
.-10
•-20
'-30 .-40 Fig. 6. Multisweep curves. I. 1. 5 × IO -s M Cu +~ in N t - I 4 N O 3 + N H 4 O t { , ptL = 8. 5 in the presence of air ; 2. as I in the presence of adrenaline 5 × lO-5 M sweep rate ~ 20 V s -x.
This is implied by the increase of signals during the drop time of the D.M.E. and by the fact that the potentials of the indentations or peaks are characteristic not for catecholamines but for the metal used. It seems that in the adsorbed layer melanine retains both the chelating and red-ox systems, which, together with the flat orientation of the aromatic ring at the D.M.E. (and the ~ electrons interactions), makes possible ion adsorption and electron transmission between the electrode surface and metal ions.
References [11 G . A . SWAN,
Progress in the Chemistry of Organic Natural Products, S p r i n g e r -
Verlag, W i e n , N e w Y o r k
(1974) T. SARNA a n d S. ~UKIEWlCZ, Folia Histochem. Cytochem. 10 (1972) 265 [3] H . G . CASSIDY a n d A . A . KUN, Oxydation-Reduction Polymers, I n t e r s c i e n c e
[2]
Publ.,
New York
(1965)
748 [4] [5] [6] [7] [8]
Matysik and Przegaliflski V. HORAK and J.R. GILLETTE, Mol. Pharmacol. 7 (1971) 429 M. PRZEGALIi~SKI, P h . D . Thesis, UMCS, Lublin (1976) J. CHODKOWSKI, Rozn. Chem. 27 (1953) 309 S. LAZAROV. A. TROFINOV and T. PoPov, Z. Phys. Chem. 11 (1967) 145 IV[. PRZEGALII~SKI and J. ~¢[ATYSIK, Talanta 27 (198o) 920