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Detection of intermediate oxidation states of adrenaline and noradrenaline by fluorescence spectrometric analysis
SRCHIVEY
OF
BIOCHEMISTRY
Detection
AND
BIOPHYSICS
of Intermediate
Noradrenaline
161, 116-130 (1963)
Oxidation
by Fluorescence
States
of Adrenaline
Spectrometric
and
Analysis’
W. H. HARRISON* From
the Departments
of Neurology
and Biochemistry, College of Physicians University, New York, IVew York
and Surgeons,
Columbia
Received November 16, 1962 Fluorescent intermediate oxidation states of adrenaline and noradrenaline, formed near physiological conditions of pH and concentration, were found detectable by direct fluorescence spectrometric analysis. Oxidizing agents or catalysts, such as Cu++, Fe+++, MnOz, tyrosinase, and ceruloplasmin were found to effect their formation. While the (non)identity of the products with any of the oxidation intermediates already known for adrenaline and noradrenaline has not yet been established, their properties indicate that they are reduced states of different reversible redox systems. They were found stable in the presence of the oxidation system in which their formation was effected, but unstable in the presence of oxidizing agents such as ferricyanide, in which case they are reversibly oxidized. The latter reversibility wits demonstrated by showing disappearance and reappearance of fluorescence upon alternate treatment with ferricyanide and ascorbic acid. When ferricyanide was used as the initial oxidizing agent, the products formed were nonfluorescent, but ascorbic acid effected their fluorescence; thus, a relationship between fluorescence and the reduced state was consistent in all experiments. The intermediates are believed closely related in that they were all found to be precursors of the same 3,5,6-t.rihydroxyindole derivatives. The results suggest new methods for the analysis of adrenaline and noradrenaline and their oxidation products which should aid in establishing whether minor oxidative pat,hways occur in civo. INTRODUCTION
It is now well established that the two major routes of adrenaline and noradrenaline metabolism are 0-methylation and oxidative deamination (1). There still remains the possibility of at1 occurrence of a minor pathway in which their dihydric phenolic moieties are oxidized to the corresponding o-quinones and the side chains cyclized to form trihydroxyindole derivatives. Catalysis of the latter type of reaction by various biological tissue and fluid preparations, certain oxidative enzymes, and metalloproteins has been demonstrated (2), but there is a lack of agreement among different investigators concerning whether the expected 1 This work was supported by the U. S. Public Health Service, Grant No. 2B-5216. 2 Supported by Training Grant No. BT-485.
products of oxidation are present or involved in physiological processes as well as whether the reaction is part of a normal or an abnormal metabolic pathway (3). This is in contrast to the convincing evidence supporting t.he involvement of the major pathways (1). Since most of the catecholamine metabolism can be accounted for by the two major pathways, an oxidative pathway, if it occurs, would involve a very small fraction of the already exceedingly small amounts of catecholamine found in biological systems. Because of this, detection of such products presents considerable difficulty for an analysis. From their chemical and physical properties (4), several oxidation products would be expected, each forming a part of a reduction-oxidation system, and the final equilibrium composition would 116
ADRENALINE-NORADRENALINE
be dictated by factors such as the pH of the system and the potential of the most concentrated or most active redox system. Consequently, any purification or concentration step in an analytical procedure which modifies the potential or pH would modify the relative amount of each intermediate in such an inherently unstable system. Phenomena of this type would most likely explain the wide divergence in available analytical data. This type of analytical problem requires either an extremely sensitive direct measuring technique or a method in which the redox potential is maintained during analysis at a physiological level in addition to control of pH and other variables. The investigation described in this paper represents the first phase of an approach to this problem. Fluorescence spectrometry was applied because of its extreme sensitivity. By this technique, evidence was obtained which indicates that relatively stable products, which fluoresce and are detectable by direct measurements, form on the treatment of adrenaline and noradrenaline with a variety of oxidizing agents at pH and concentration conditions as near physiological levels as the sensitivity of the fluorescence measurements will permit. This report includes a description of the oxidation reactions and some of the properties of the fluorescent products together with a discussion of the significance of the results. MATERIALS
AND METHODS
The fluorometric measurements were made with an Aminco-Bowman spectrophotofluorometer. Instrumental parameters such as cell and photomultiplier slit arrangements and photomultiplier tubes (IP21 or IP 28) were varied to give the sensitivity and resolution required by each experiment. Three-milliliter quartz cells supplied with the instrument were used in all experiments. In that most of the experiments were performed at room temperature, the activation cell shutter was kept closed except when making measurements in order to minimize temperature change and prevent resultant fluorescence intensity changes not related to the effect studied. The possibility that the fluorescence changes noted were the effect of irradiation or temperature change during the periods of measurement was checked in preliminary experiments by running duplicates at controlled
OXIDATION
117
temperatures; however, this possibility was ruled out in that final fluorescence patterns of the temperature-controlled runs were found to be similar to those subjected to the activation light at regular intervals. Use of a temperature control device in the cell compartment is required and planned for future studies in which quantitative titrations and kinetic measurements will be made. The wavelengths cited are uncorrected. The I-adrenaline and I-noradrenaline (bitartrates) and L-ascorbic acid were supplied by Nutritional Biochemicals Corp. Stock solutions (1 X 10u4%f) of Z-adrenaline and I-noradrenaline were prepared in 0.01 M HCl, and appropriate dilutions with distilled water were made in each experiment. Analytical reagent-grade chemicals were used in the preparation of the buffers, the metal salts, and the ethylenediamine tetraacetate (EDTA) solutions. Copper acetate (monohydrate), ferric chloride (hexahydrate), zinc acetate (dihydrate), manganese dioxide (85%), and potassium ferricyanide were used in the oxidation experiments. Several different lots of mushroom tyrosinase, supplied by Worthington Chemical Corp., were used; the reported activities, based on tyrosine oxidation, ranged from 500 to 600 units/mg. Stock solutions of tyrosinase were prepared in the buffers employed in each experiment at the concentrations noted. In separate experiments, dialysis of commercial tyrosinase or purificatZion with Sephadex did not give rise to an activity change in respect to formation of fluorescent oxidation products. The ceruloplasmin solution (Fraction IV-l) was supplied by Nutritional Biochemicals Corp. and was used without further treatment. RESULTS
At the outset of this investigation it appeared possible that the oxidation of adrenaline and noradrenaline might be followed by direct fluorescence-loss measurements at their characteristic excitation and emission peaks and that some of the oxidation products, trihydroxyindole derivatives, might be detectable by fluorescence measurements after treatment with reducing agents. Initially, it was assumed that nonfluorescent, o-quinoidal intermediates such as the aminochromes would be formed which when reduced to their “leuco” forms might fluoresce and be detectable. Although such compounds have not been reported to fluoresce, they would be expected to on the basis of Auorescence-structure relationships derived from
118
HARRISON
s
O
2
3
4
s
6
7
5 TIME
::
in MINUTES
E 2
theoretical considerations and empirical observations (5). This was tested by using ferricyanide as the oxidizing agent and ascorbic acid as the reducing agent; the results are given in Part I. In Parts II and III are described further tests with this approach in which Cu++, bound Cu++ (tyrosinase and ceruloplasmin), Fe*, and MnOz were used as oxidizing agents or catalysts. I. FERRICYANIDE
-50
FIG. 1. Fluorescence changes which result from various treatments of adrenaline and noradrenaline. The following are shown: time courses of fluorescence loss of adrenaline (A) and noradrenaline (x) during ferricyanide oxidation at pH 5.5 (A, and N,) and pH 3.0 (A6 and Nb) ; time courses of formation of fluorescent products which result when the products of ferricyanide oxidation at pH 6.0 are treated with ascorbic acid (A, and NJ and when the products of ferricyanide oxidation at pH 5.5 and pH 3.0 are treated simultaneously with ascorbic acid and alkali (A, and N, at pH 5.5 and Ad and Nd at pH 3.0). The subscripts refer to the following reaction mixtures which contained (in mpmoles) : (a) adrenaline or noradrenaline, 10; sodium acetate-acetic acid, 1 X 106; KsFe(CN)s, 380; 1.2 ml. final volume, pH 5.5. (b) adrenaline or noradrenaline, 10; sodium acetate-acetic acid, 1 X 106; ZnSOd, 434; K,Fe(CN)e, 380; 1.2 ml. final volume, pH 3.0. Fluorescence measurements for (a) and (b) were made at 280 rnp excitation and 330 rnp emission. (c) adrenaline and noradrenaline, 50; sodium acet,ate-acetic acid 5 X 106; KsFe(CN)G, 1900; 6.0 ml. final volume, pH 6.0. At the time intervals specified, l.O-ml. aliquots were transferred to quartz cuvettes containing 0.1 ml. of 0.57 M ascorbic acid. Fluorescence measurements were made at 400 rnM excitation and 515 rnk emission. (d) and (e) were run the same as (b) and (a), respectively, except that at the specified oxidation time a mixture containing 0.1 ml. of 0.57 M ascorbic acid and 0.9 ml. of 2.5 M NaOH was added. Each point for (d) and (e) was determined separately. Fluorescence measurements were made at 410 rnp excitation and 510 rnp emission.
OXIDATION
By fluorescence spectrometry, two steps in the ferricyanide oxidation of adrenaline and noradrenaline were detected. A fast step, occurring within 1% min., was observed by following decrease of the fluorescence of each amine. A slower step, occurring within 10 min., was found detectable and followed by measuring appearance of oxidation products which were found to exhibit fluorescence upon their reduction by ascorbic acid. Fast Step In preliminary experiments, the fluoressence of both amines was found to decrease rapidly and level off within a half minute during ferricyanide oxidations at pH’s 6 and 7. At pH 5.5, the reaction occurred within 1% min., and differences in the rates of the two amines were barely discernible (Fig. la). To show more clearly the differences in the reactivity of these two amines, it was necessary to follow the reaction at a pH at which the rates would be sufficiently slow to allow their measurement. This was demonstrated in an oxidation at pH 3, in the presence of Zn++, required as a catalyst (Fig. lb). It is believed pertinent that noradrenaline underwent the same changes as adrenaline at pH 3, although at a different rate, in view of the fact that it has been shown to be only slightly reactive at pH 3 in tests in which the degree of reactivity is estimated by measurement of fluorescent trihydroxyindole formed upon alkalinization (6). Slower Step Formation of oxidation products of adrenaline and noradrenaline during ferricyanide oxidation at pH 6 was demonstrated
ADRENALINE-NORADRENALINE
119
OXIDATION
by using the fact that they exhibit fluorescence upon reduction with ascorbic acid. The time courses for their formation are given in Fig. lc; for comparison with the fast step, the same time scale is used. The excitation spectra (Fig. 2) of each amine differed considerably but overlapped, and the fluorescence intensity of the noradrenaline derivative was considerably greater than that of the adrenaline derivative. Fluorescence was not exhibited when ascorbic acid was added at different intervals during the pH 3, Zn+f-catalyzed oxidation. A
1.0
0.5 ADRENALINE
300
400
350 WAVE LENGTH
Imp)
FIG. 2. Excitation spectra, before and after alkalinization, of the fluorescent products which result from ascorbic acid treatment of pH 6 oxidation products of adrenaline (A) and noradrenaline (N). Except for the pH, the oxidation reaction mixture was the same as described in Fig. 1 (a). Six minutes after addition of KaFe(CN)c, 0.1 ml. of 0.57 M ascorbic acid was added. The spectra of the adrenaline and noradrenaline oxidation products were obtained (solid line), and then 1.0 ml. of 2.5 M NaOH was added and the spectra were redetermined (dotted line). To obtain each spectrum, the emission intensity at 510 rnr was measured while scanning the excitation spectrum. The emission maximum for the noradrenaline product before alkalinization was found at 468 rnp (not shown) ; at its excitation and emission maxima the noradrenaline fluorescence intensity was 60% greater.
or NORADRENALINE
(mp molar,
FIG. 3. Relationship between adrenaline (A) or noradrenaline (N) concentration and the fluorescence exhibited by their oxidation products after treatment either with ascorbic acid (a) or with ascorbic acid followed by alkalinization (5 and c). The oxidations were carried out at pH 6.0 in 1.3 ml. final volume containing (in mpmoles) : adrenaline or noradrenaline, O-1.0; sodium acetate-acetic acid, 1 X 106; and K3Fe(CN)6 760. Six minutes after addition of KaFe(CN)e, 0.2 ml. of 0.57 M ascorbic acid was added in all cases. Fluorescence measurements for curve (a) for the noradrenaline product were made before alkalinization at 372 rnp excitation and 469 rnp emission. Curves (5) and (c) were determined after addition of 1.0 ml. of 2.5 M NaOH, and the fluorescence measurements were made at 410 rnp excitation and 510 rnp emission. Corrections were made for fluorescence intensities of controls not containing adrenaline or noradrenaline.
linear relationship between the fluorescence of the ascorbic acid-treated, pH 6 noradrenaline oxidation product and the concentration of noradrenaline was found (Fig. 3~2); however, the relationship was not linear in the case of adrenaline (not shown). The above oxidations were performed in 1 M acetate system; however, in separate experiments, fluorometrically identical products were formed at pH 7.0 in a 0.1 M acetate system, but their fluorescence in-
120
HARRISON
tensities were 50-75 % less. Ionic strength effects of this sort have been reported by others (5) and would be expected on theoretical grounds. of Intermediates to S ,5,6-Trihydroxyindoles
Conversion
Alkalinization of the ascorbic acid-treated, pH 6 oxidation products effected their conversion to products with the fluorescence characteristics of the 3,5,6-trihydroxyindoles of adrenaline and noradrenaline (Fig. 2); the conversion was completed immediately for both products. A linear relationship of the fluorescence of each product with concentration was shown (Figs. 36 and 3~). Simultaneous Addition of Alkali Ascorbic Acid
and
When alkali and ascorbic acid were added together to the pH 3, Zn++-catalyzed oxidation, a fluorescent 3,5,6-trihydroxyindole was formed from adrenaline but not from noradrenaline, and similar treatment of the pH 6 oxidation products resulted in 3,5,6trihydroxyindoles from both amines; these are the reactions involved in the trihydroxyindole procedure (6), and their time courses are included in Fig. 1 (d and e).
E$ect of Ascorbic Acid Concentration When the amount of ascorbic acid used was lowered to 0.02 mg., the fluorescence of the noradrenaline derivative became maximal and that of the adrenaline derivative negligible; the positions of the excitation and emission peaks of the noradrenaline derivative shifted (Table I). Further details on this effect and its possible application to a new analytical technique will be reported elsewhere. II. Cutl-, Fe+++ AND Mn09 OXIDATIOK Cu* and Fe+++ In a similar study, the mechanism of the Cu* and Fe+++ oxidation of adrenaline and noradrenaline was found to be considerably different from that of the ferricyanide oxidation. For example, on monitoring fluorescence changes by scanning both excitation and emission spectra during an adrenaline oxidation by Cuff at pH 7, t’svo steps were directly detectable (Fig. 4). As in the ferricyanide experiment, one step reflecting loss of the fluorescence of adrenaline was shown; however, in contrast, an additional concurrent direct formation of a fluorescent product was demonstrated. This fluorescent
TABLE FLUORESCENCE CHARACTERISTICS
OF
I
ADRENALINE AND NORADRENALIXE PRODCCTS
INTERMEDIATE
OXIDATION
Fluorescent products Oxidizing agent or catalyst
Adrenaline (Xmax) Excitation
Emission
Noradrenaline (X,,,) Excitation
iuncorrected)
cu++
Fe+++ Tyrosinase MnOz KsFe(CN)s KzFe(CN)6 Ceruloplasmin
fw
m/J
340 340 327, 390b 320, 3834 320, 400c
506 506 487O 5100 515c
323, 382
483
-
Emission
(uncorrected)
-
nw
36@’ 36Oa 358* 362” 320, 375c 300, 395c,J 360
,np
4900 490” 470h 468” 468” 47gcad 4iR
a Fluorescence increase with EDTA. b The values reported were from the experiment described in Fig. 6 in which an acetate-acetic acid system was used. Different maxima, particularly notable in the case with adrenaline, were obtained when phosphate buffer was used (Fig. 8). 5 Reducing agent required for fluorescence. J Formed at ascorbic acid concentrations at which adrenaline does not fluoresce.
ADRENALINE-NORADRENALINE
50 Hc----m-m-*
100 N
160
OXIDATION
165
170
175
121
I60
TIME in MINUTES
FIG. 4. Measurements of fluorescence changes during Cu++ oxidation of adrenaline (A) and noradrenaline (N). The fluorescence-loss measurements for both adrenaline and noradrenaline were made at 273 rnp excitation and 325 rnp emission. Measurements of the formation of fluorescent products from adrenaline were made at 340 rnp excitation and 506 rnp emission and from noradrenaline at 360 rnMexcitation and 490 rnp emission. The oxidations were carried out at pH 7.0 in 1.04 ml. final volume containing (in mpmoles) : adrenaline or noradrenaline 9.1; sodium acetate-acetic acid, 1 X 106; and Cu++, 40. The Cu* was added at zero time. At the times indicated (see arrows), 40 mpmoles ferricyanide in 0.01 ml. or 40 mfimoles ascorbic acid in 0.01 ml. or 40 mrmoles EDTA in 0.01 ml. were added, and fluorescence measurements were made at the times indicated.
product appeared to be in its reduced state in that addition of ascorbate did not affect the fluorescence. Its reducing properties were apparent in that it underwent oxidation by ferricyanide to a nonfluorescent derivative which was readily reconverted to the fluorescent state by a subsequent reduction with ascorbate (Fig. 4). Furthermore, successive oxidations and reductions by these agents could be repeated, and the changes followed fluorometrically (Fig. 4). The fluorescence characteristics of this product differed from the ferricyanide oxidation product having excitation and emission maxima of 340 and 506 rnp, respectively. When noradrenaline was substituted for adrenaline in the above oxidation, a fluorescence loss step was observed, but formation of a fluorescent product was just barely detectable (Fig. 4). However, on further examination, it was found that a potentially fluorescent product already in its reduced state, as in the adrenaline experiment, was formed but that its fluorescence
was quenched presumably by the oxidation agent, Cu*. This conclusion was reached from several lines of evidence. First, when the molar ratio of Cu* to noradrenaline in the oxidation was reduced from four to one, the intensity of the fluorescent product formed directly increased 12-fold to within 60% of the intensity of that obtained with adrenaline. Secondly, when EDTA in an amount equivalent to the Cu++ used in the oxidation was added at the end of the reaction, fluorescence comparable to that obtained from the adrenaline derivative was exhibited. Furthermore, alternate additions of equivalent amounts of Cu* and EDTA caused reversible exhibition of fluorescence. Also, when the Cuff was in sufficient quantity to quench the fluorescence, acidification to pH 3.0 caused appearance of fluorescence, and subsequent neutralization with alkali caused disappearance; this suggests that the quenching results from formation of a complex of the oxidation
122
HARRISON
product with Cu++ and that the complex can undergo a reversible dissociation by acidification. In systems in which the fluorescence quenching of oxidized noradrenaline by Cu++ was eliminated by EDTA or the oxidation was carried out with smaller quantities of Cut+-, the reducing properties of the fluorescent product and the ease of its interconversion to its oxidized and reduced states were identical to those for the adrenaline product. For example, successive oxidation and reduction by ferricyanide and ascorbate were demonstrated after EDTA complexing of Cu++ (Fig. 4). The fluorescence of the noradrenaline derivative differed from the adrenaline derivative, having excitation and emission maxima of 360 and 490 rnp, respectively. Importantly, in addition to its reducing properties, ascorbate, at pH 7.0 was shown to react in the noradrenaline oxidation mixture by complexing with Cu++ similar to EDTA. Thus ascorbate, when added to oxidized noradrenaline, effected formation of the fluorescent product presumably by a complexing reaction with Cu++; oxidation of the fluorescent product with ferricyanide caused disappearance of fluorescence which could be restored by addition of ascorbate; in the latter case, ascorbate functions as a reducing agent. Unless this dual function is considered, the effects of ascorbate on this system are easily misinterpreted. The above EDTA effect was not shown with the adrenaline derivative. To determine whether a very high level of Cu* would quench the fluorescent adrenaline product as it does the noradrenaline product at low levels, high ratios of Cuff to adrenaline were used in the oxidation, and the EDTA effect was checked. At a molar ratio of 20: 1 a slight effect was noted, the final fluorescence being increased by 20 % on addition of EDTA. When carried out at pH 5.0-7.5, formation of the fluorescent products during Cu++ oxidation of adrenaline and noradrenaline increased with increasing pH; no pH opt,imum was observed. At higher pH levels,
TIME in MINUTES
FIG. 5. Direct measurements of fluorescence changes during Fe+++ oxidation of adrenaline (A) and noradrenaline (N). The fluorescence loss measurements for both adrenaline and noradrenaline were made at 270 rnp excitation and 330 rnp emission. Measurements of the formation of fluorescent products from adrenaline were made at 340 rnp excitation and 506 rnp emission and from noradrenaline at 360 rnp excitation and 490 nm emission. The oxidations were carried out at pH 7.0 in 1.04 ml. final volume containing (in mpmoles) : adrenaline or noradrenaline, 9.1; sodium acetate-acetic acid, 1 X 105; and Fe++, 40. The Fe+++ was added at zero time. See text for properties of the fluorescent products formed.
unstable trihydroxyindole derivat’ives were formed. Oxidations by Fe+++ were similar to those by Cu* with respect to fluorescence loss of the amines (Fig. 5>, the direct formation of fluorescent products (Fig. 5), the quenching effect of the noradrenaline product by Fe+++, and the ease of interconversion of the oxidized and reduced forms of the products. The excitation and emission maxima of the adrenaline and noradrenaline products were 340 rnp, 506 ml*, and 360 rnp, 490 rnp, respectively; thus, they were identical with the Cu++ oxidation products. A distinct pH optimum, pH .5.9, was found for
ADRENALINE-NORADRENALINE
the Fe+++ oxidation of adrenaline but not for noradrenaline. Buffers used in the study included acetate, phosphate, and tris(hydroxymethyl)aminomethane (Tris). Quantitative but not qualitative differences in the reactions with Cu++ and Fe+++ were noted when different buffers were used. These included fluorescence intensity differences, different effects of EDTA on the fluorescence of the noradrenaline product, and different oxidation rates. For example, in oxidations in which the Cu++ to noradrenaline ratio was 4: 1, with 0.1 M acetate, pH 7.0, the quenching of the fluorescence of the noradrenaline derivative was complete; whereas with 0.1 M Tris, pH 7.0, it was 50%; and with 0.1 iVl phosphate buffer, pH 7.0, no quenching occurred; in all cases studied, quenching was eliminated by EDTA. MnOz Products formed by oxidation of adrenaline and noradrenaline with an excess of manganese dioxide in 0.1 M phosphate buffer, pH 7.0, were found to be fluorescent. The excitation and emission maxima of the adrenaline product were 320 rnp, 383 rnp (excitation), and 510 rnp (emission), respectively, while the noradrenaline product had a single excitation peak at 362 rnp and an emission peak at 468 rnp. Fluorescence of both products was increased by EDTA; thus apparently the Mn++ formed during the oxidation partially quenched the fluorescence by complexing with both products. The products were reversibly oxidized, as in previous cases, by alternate treatments with ferricyanide and ascorbate. Trihydroxyindole
Formation
On alkalinization to a 5% NaOH concentration, all of the above oxidation products converted to derivatives with the fluorescence characteristics of their respective 3,5,6-trihydroxyindoles. III.
CATALYSIS OF OXIDATION PROTEIN-C++ COMPLEX
BY A
Tyrosinase The direct fluorescence spectrometric method was used for studying the catalyzed
123
OXIDATION A 390/487
+50
N 358/470
0 : : E 0 3 Y
TIME in MINUTES
-50
-100
FIG. 6. Direct measurements of fluorescence changes during tyrosinase-catalyzed oxidation of adrenaline (A) and noradrenaline (N). The fluorescence loss measurements for both adrenaline and noradrenaline were made at 278 rnr excitation and 340 rn,uemission. Measurements of the formation of fluorescent products from adrenaline were made at 390 rnp excitation and 487 rnp emission and from noradrenaline at 353 rnp excitation and 470 rnp emission. The oxidations were carried out at pH 7.0 in 1.2 ml. final volume containing (in mpmoles) : adrenaline or noradrenaline, 10; sodium acetate-acetic acid, 1 X 105; and tyrosinase, 50 units (0.1 mg.). The tyrosinase was added at zero time. See text for the properties of the fluorescent products formed.
oxidation of adrenaline and noradrenaline by tyrosinase, a copper-containing protein; its activity is usually measured by manometric (7) or spectrophotometric (9) methods. An experiment which shows this is described in Fig. 6. The excitation and emission spectra of the fluorescent products formed from each amine are given in Fig. 7. The products appeared to be in the reduced state in that addition of ascorbate did not increase their fluorescence, whereas ferricyanide caused fluorescence disappearance. On long standing, e.g., for 24 hr., a fluorescence decrease of about 10 % resulted; however, the initial fluorescence could be restored by addition of ascorbate.
HARRISON 8
A
pHa5
r PH8.0 /.JH 8.5 pHT0 pHz5 pH6.5 pHa0 i
j1Hb.0
pHZ5 pHiTO @MS pH60
~,~ 10 303
350
400 WIVE
450 LENGTH
500
550
20
30 TIME
40
0
IO
20
30
40
in MINUTES
(mpl
FIG. 7. Excitation and emission spect,ra of oxidation products of adrenaline (A) and noradrenaline (N) formed in the tyrosinase reaction. The oxidation reaction mixtures are described in Fig. 6. Curve (a) is the excitation spectrum of oxidized adrenaline measured at 487 rnM emission; curves (b) and (c) are its emission spectra measured at 390 and 327 rnp excitation, respectively. Curve (d) is the excitation spectrum of oxidized noradrenaline measured at 470 rnr emission; curve (e) is its emission spectrum at 358 rnp excitation.
EDTA had no effect on either product in contrast to the previous experiments with Cd+, Fe+++, and MnOa oxidations. Differences between adrenaline and noradrenaline were apparent, particularly in the rate and extent of formation of their respective fluorescent products (Fig. 6). Reaction rates were found proportional to enzyme concentration under conditions at which substrate was not rate limiting. When catechol was used as a substrate in a separate experiment, only the fluorescence-loss step was observed; thus, the ethanolamine side chain in adrenaline or noradrenaline is required for formation of the fluorescent products. Because the acetic acid-acetate system is a poor buffer at physiological pH con-
FIG. 8. Direct measurements of fluorescence changes during tyrosinase-catalyzed oxidation of adrenaline (A) (Graph A) and noradrenaline (N) (Graph B) in phosphate buffer, pH 6.W3.5. The fluorescent products from adrenaline were measured at 378 rnp excitation and 503 rnp emission and from noradrenaline at 360 rnp excitation and 468 rnp emission. A second excitation peak for the adrenaline product at 330 rnp was also noted; the noradrenaline product gave a single peak. The oxidations were carried out at the designated pH in 1.2 ml. final volume containing (in mrmoles): adrenaline or noradrenaline, 10; phosphate buffer, 1 X 105; and tyrosinase, 50 units (0.1 mg.). The tyrosinase was added at zero time. After 90 min. the fluorescence of mixtures containing adrenaline were the following: pH 6.0, 0.6; pH 6.5, 0.1; pH 7.0, 0; pH 7.5, 0.6; pH 8.0, 1.3; pH 8.5, -. The values after 90 min. for the noradrenaline mixtures were the following: pH 6.0, 1.G; pH G.5, 1.6; pH 7.0, 2.8; pH 7.5, 5.8; pH 8.0, 7.7; pH 8.5, 22.5.
ditions, phosphate buffer was tested for use in obtaining pH optimum curves for the enzyme. It was not found entirely suitable, however, because whereas the fluorescent products were found to be stable after about 90 min. in the pH 7.0 acetate system, they were formed more rapidly and were very unstable in phosphate buffer ranging from pH 6.0 to 8.5 (Fig. 8). Chaix et ~7. (10) have reported a similar instability in
ADRENALINE-NORADRENALINE 60
-
A P h0sphots TRlS N N
\I 6
125
borate buffer on the fluorescence of adrenaline and noradrenaline is included in Fig. 9; this demonstrates that borate complex formation, known for the catechols (5), causes a fluorescence quenching. Kinetic data describing a study of tyrosinase-catalyzed oxidation of catecholamines by this method will be presented elsewhere.
A
5
OXIDATION
Ceruloplasmin
7
e
9
PH
FIG. 9. Fluorescence intensity of adrenaline and noradrenaline at a series of pHs in phosphate, Tris, and borate buffers. The buffer concentrations were 0.1 M, and the adrenaline or noradrenaline concentrations, 3.2 X 10e6 M.
phosphate buffer. Because the advantages of the acetic acid-acetate system necessitated its use in many cases, it was necessary to adjust all reactants to neutral pH because of its poor buffering capacity. Results similar to those found with the acetate system were obtained when the reaction was run in 0.1 M Tris buffer, pH 7.0. In this case, the products were stable in contrast to the instability noted in phosphate buffer. The mechanism by which the different buffers influence this reaction is obscure. A check of the effect of Tris and phosphate buffers at different pH’s on the fluorescence of untreated adrenaline and noradrenaline (Fig. 9) showed that these buffer systems are not inert, but instead exert considerable effect. Whether the regions of fluorescence change shown in Fig. 9 indicate differences in the ionization state of these amines or whether other effects are being detected is not known; however, it appears that fluorescence is a sensitive indicator of some interaction of the buffer system and the reactant. Similar effects on the oxidation products formed might be expected as well. The effect of
In systems containing 10 mpmoles adrenaline or noradrenaline, 100 pmoles sodium phosphate buffer, pH 7.0, and 0.01 ml. ceruloplasmin solution in 1.11 ml. total volume, fluorescent oxidation products with the excitation and emission maxima given in Table I were formed, reaching a maximum level after 25 min. Addition of 41.1 mpmoles EDTA enhanced the fluorescence of both products, and no shift in maxima occurred. Further studies with ceruloplasmin will be presented elsewhere. Preparation of Intermediates The chromes of adrenaline and noradrenaline were prepared and compared with the oxidized forms of the above fluorescent intermediate oxidation products in respect to the fluorescence characteristics they exhibit upon alternate treatments with ascorbic acid and ferricyanide. A modification of the method developed by Schayer (11) for the preparation of adrenochrome, which involves Ag,O oxidation in methanol, was used; determination of fluorescence loss across the oxidation step was used as a criterion for establishing conditions for complete oxidation of each amine. Both chromes were reduced reversibly, presumably to their leucoaminochromes, as shown by alternate treatments with ascorbic acid and ferricyanide; the reversibility was judged by the disappearance and appearance of the red color of their solutions. However, neither reduced form exhibited the fluorescence peaks shown for the above intermediates. Since it is essential to establish, definitively, the relationships of the aminochromes to the fluorescent intermediates, a more detailed study with this approach will be made. When the concentrations of the amines in the reaction mixtures of the various oxi-
126
HARRISON
dations were increased above the micromolar level in order to obtain more concentrated solutions of their fluorescent products for isolation purposes, it was found that the products were no longer detectable; the possibility that they were not observed because of “concentration quenching” effects was always eliminated by diluting the samples appropriately. As an example, when the amine concentration in the Cu++ oxidation was increased tenfold, only redcolored ‘Laminochromes” and some fluorescent trihydroxyindole were detected; when the solution was treated with ascorbic acid, the color disappeared but the fluorescent intermediates were still not detectable. The same effect was noted when the amine concentration in the ferricyanide oxidation was increased; formation of chromes, some trihydroxyindole, and, in some cases, black precipitates resulted. In that it is important to isolate and characterize these intermediates, other approaches will be tried. DISCUSSION
By a direct fluorescence spectrometric method, stable, fluorescent products have been shown to result from oxidations, performed near physiological pH and concentrations, of adrenaline and noradrenaline with CL++, Fe++, MnOz, tyrosinase, and ceruloplasmin. While the evidence is insufficient for identification of these products, several of their properties have been ascertained. For example, the products are unstable in the presence of an oxidizing agent such as ferricyanide in which case their fluorescence disappears. However, the reversibility of this oxidation was established by demonstrating the reappearance of their fluorescence upon reduction with ascorbate. When ferricyanide was employed for the formation of the oxidation products, the products formed were nonfluorescent; but when an amount of ascorbate, sufficient to reduce both the ferricyanide and the oxidation product was added, fluorescent products resulted. Thus the relationship between fluorescence and the reduced state was consistent in all experiments. From a study of their reactivity in alkali, the products of every type of oxidation studied
were found on the basis of their fluorescence to be precursors of their respective 3,5,6trihydroxyindole derivatives. The products exhibited different fluorescence characteristics (Table I) depending on the type of oxidation agent or catalyst used. Whether these differences reflect major differences in structure or whether the metal ions in the media or other factors influence the positions of the excitation and emission peaks remains in doubt; their excitation and emission spectra did, however, overlap in every case. The EDTA effects found with the noradrenaline but not the adrenaline product must reflect a fundamental difference in structure or complexing ability of the two molecules, but its nature is not clear. Because of pertinence to the interpretations of this study, a summary of the various interconversions and disproportionation reactions known for the oxidation products of adrenaline (4) (12), together with possible alternative pathways, are described in Scheme I; noradrenaline is believed to undergo the same reactions although this has not been demonstrated in all cases (6). Compounds V, IIIa, and VII, shown in Scheme I, have been isolated and identified (7, 4, 12). Compounds II, III, VI and VIII have not been isolated. HarleyMason (12) has shown that reduction of adrenochrome (V) gives rise to equal amounts of 111~ and VII by isolating and identifying them. Structure VI was postulated by Harley-Mason for a precursor of compound VII which he detected and found to have a characteristic ultraviolet absorption spectrum and instability in alkali; he described the reaction leading to compounds IIIa and VII as a disproportionation reaction in which intermediates, III and VI are formed from a common intermediate semiquinone with structure IV. He also showed that an alternative, alkali-catalyzed direct conversion of adrenochrome (V) to its trihydroxyindole derivative (VII) could occur in the absence of a reducing agent. Adrenoquinone (II) has been assumed to convert upon oxidation to adrenochrome (V) via an oxidative cyclization (arrow 2) ; however, the alternative pathway, i.e. an intramolecular oxidative cyclization to leucoadrenochrome
ADRENALINE-NORADRENALINE
OXIDATION
127
O= O-
’ ox- ’
-
IO” N &i 3
Possible interconversions of the oxidation products of adrenaline (I). The products shown are adrenoquinone (II), leucoadrenochrome (III), 5,6-dihydroxy-1-methylindole (IIIa), adrenochrome (V), 3,5,6-trihydroxy-1-methylindole (VII) and its o-quinone state, (VIII), and structures (IV) and (VI), postulated by Harley-Mason (12).
(III) followed by oxidation to adrenochrome (V) (arrow 1) cannot be excluded. The latter pathway would be similar to the mechanism shown for the formation of 2,3-dihydro4,6,7-trihydroxyindole from 2,4,5-t& hydroxyphenethylamine which occurs via intramolecular oxidative cyclization of the intermediate p-quinone (18). Koelle and Friedenwald (13) showed that at millimolar concentration levels, adrenochrome (V) is reduced directly to leucoadrenochrome (III) and that the disproportion&ion reaction which was demonstrated by Harley-Mason (12) with 0.275 M adrenochrome does not occur. Identification of the fluorescent prod ucts reported in this investigation as any of the intermediates described in Scheme I
or as different structures must await further study of their properties. On considering the intermediates of Scheme I, the o-quinoidal structures can be excluded since such structures do not fluoresce (5) ; and structure 111~ can also be excluded because it does not give rise to a trihydroxyindole derivative upon alkalinization. The experiments in this although preliminary in investigation, nature, indicated that ascorbic acid-treated presumably leucoaminoaminochromes, chromes, do not exhibit the fluorescence shown by the fluorescent intermediates. On the other hand, the properties reported for intermediate VI by Harley-Mason (12) have similarities to the intermediates detected in this study; for example, intermediate VI is
128
HARRISON
converted in alkali to compound VII and has an ultraviolet absorption maximum at 347 mp which is close to the excitation maxima (340 mp) of the products from Cu++ and Fe+++ oxidations of adrenaline (Table I). A more detailed comparison is planned. Since the concentrations in this study were closer to physiological levels and the methods of analysis direct rather than involving additional treatments such as alkalinization which might modify the products --a possibility in other methods -it is believed that by this technique products more closely representing the true products of these oxidation reactions are detected. And, since in this study, although not identified, the products have been shown to be measurable, it’ would appear that methods similar t,o the type employed in this study would offer a number of advantages for deterrnnation of products formed in z&o and of possible oxidation products in biological tissues and fluids. In this respect, it is believed significant that these findings indicate that, oxidation at physiological conditions may give rise to reduced as well as oxidized states of reversible redox systems. In that most tests (14) for oxidation products have been designed for the detect#ion of o-quinoidal forms, e.g., adrenochrome, the question arises whether some of the products are in t,he reduced form and consequently not accounted for; this is particularly possible when an alumina purification step is used since the reduced form is selectively adsorbed on the alumina and separated from the oxidized form. Among the properties found in this study which might be exploited for the differentiation and measurement of the oxidation products of adrenaline and noradrenaline are their fluorescence spectra differences and the metal cation quenching effect, specific to the noradrenaline oxidation product. The same properties would serve equally well for the development of new, more specific methods for analysis of the unoxidized amines themselves, in procedures which would include oxidation steps of the type studied. Further investigation is required to determine which of the various systems studied are best for the above-suggested applications. Certain aspects of the mechanism by
which the catecholamines are oxidized and the fluorescent products or their precursors are formed were revealed in this study. For example, the time relationships between the different fluorometrically detectable steps were shown to vary wit.h the type of oxidation. In the ferricyanide oxidation, in about 2 min. the fluorescence loss was completed and maximal formation of the intermediate which could be converted to 3,5,6trihydroxyindole by alkali was attained, and 8 min. later the intermediate which fluoresced only on addition of ascorbate was formed maximally; this suggests that several steps are involved in this type oxidation. In the oxidations with CL? and Fe+++, and (tyrosinase) in which case bound Cutf fluorescent products were formed directly, the time periods for the fluorescence lossand-gain steps overlapped. These differences obviously indicate different mechanisms. For example, the ferricyanide oxidation appears distinctly different from the free or bound metal-ion oxidations. This might be expected in that ferricyanide ion is a fully coordinated very stable Fe+++ complex (16), thus eliminating the possibility of its involvement in a chelating reaction with the catecholamine. Thus this fluorescence loss must reflect only an oxidation reaction. On the other hand, coordination is possible in the free or bound metal-ion oxidations. It is likely t,hat complexing or oxidation reactions or both, occurring simultaneously, give rise to the fluorescence loss when free or bound metal ions are used; it appears to be a specific fluorescence-quenching effect, in that the rates for adrenaline and noradrenaline differed. Chelation and oxidation were indicated from studies of Chaix et al. (17). Also, studies in progress of oxidations by Cu++ and Fe+++ indicate that both complexing and oxidation do occur during the fluorescence loss step; one indication of this comes from the fact that a fluorescence decrease occurs in the absence of oxygen and is a function of the concentration of the metal cation. However, in such cases the fluorescence is not restored with EDTA thus indicating the occurrence of an underlying reduction-oxidation reaction in the complex. This is in contrast to the fluorescence quenching by chelation without oxidation
ADRENALINE-NORADRENALINE
indicated in the experiment described above, e.g., Fig. 4, in which case the fluorescence of the noradrenaline oxidation product was shown to be quenched by Cu++; in that case EDTA did restore the fluorescence and oxidation had not occurred; thus both cases can occur. Both chelation and oxidation may occur in a similar fashion during fluorescence loss in the tyrosinase oxidation, but in this case the step in which the fluorescent product is formed must be rate controlled by certain specific features of this enzyme. One explanation is that the fluorescence change during the first period represents chelation and oxidation of an amount of catecholamine required to activate the enzyme in such a way that it can catalyze the second step. It is interesting to compare this fluorescence loss step with the induction period shown by others (8) by oxygen uptake studies made during oxidation of catecholamines by tyrosinase. While direct comparisons between oxygen uptake and fluorescence loss were not made in this study, the similarities in time relationships are highly suggestive that the changes shown by fluorimetry in this study occur during that phase. An extensive quantitative examination by fluorescence and fluorescence polarization techniques of different protein-metal-ion complex-induced oxidations of the catecholamines is required for further clarification. In that certain enzymes and metal-protein complexes have been shown to catalyze such oxidations (2), it is interesting to speculate on the possible formation and participation of such redox systems in the functional processes of these important and apparently multifunctional catecholamines. A recent report of the activity of adrenochrome on oxidative phosphorylation (19) showed that, at micromolar levels, it decreased both oxygen uptake and phosphorylation, thus demonstrating a general toxic effect without an uncoupling action. Other toxic effects relating to glycolysis have also been noted for adrenochrome and noradrenochrome (20). However, since it is highly likely that the fluorescent substances found in this study may more closely relate to biologically formed products than the aminochromes,
OXIDATION
129
the specific involvement of oxidation products should not be excluded until these products are tested. That reactive intermediate oxidation products, not necessarily identical with the aminochromes, are formed was indicated from a recent study by Walaas and Walaas (21). Using ceruloplasmin as the catalytic agent, they demonstrated by a spectrophotometric method a coupled reduction-oxidation reaction between oxidation products of noradrenaline and adrenaline with reduced pyridine nucleotides (DPKH and TPNH) ; the noradrenaline product was found markedly more reactive than that of the adrenaline. Adrenochrome itself did not exhibit a similar react’ivity. The nature of the intermediates which participate in the reaction with the nucleotides was not ascertained, but Walaas and Walaas suggested that they were either free radicals or unstable uncyclized quinones. Although there is no obvious relationship between the active intermediates studied by Walaas and Walaas and those reported in this paper, both studies indicate formation of active products which are not identical to the products already characterized and reported in the literature. Further work should establish whether isolable intermediates or free radicals, e.g., semiquinones, are the products detected in these and other studies. ACKNOWLEDGMENT The author is most pleased to acknow-ledge his gratefulness to Professor David Nachmansohn for the opportunity of working in his laboratory and for his advice and encouragement during this investigation. REFERENCES 1. AXELROD, J., in “Adrenergic Mechanisms” (J. R. Vane, G. E. W. Wolstenholme, and M. O’Connor, eds.), p. 24. Little, Brown and Co., Boston, 1960. 2. ZELLER, E. A., Pharmacol. Rev. 11, 387 (1959). 3. LA BROSSE, E. H., MANN, J. D., AND KETTY, S. S., J. Psychiat. Res. 1, 68 (1961). 4. HEACOCK, R. A., Chem. Rev. 69, 181 (1959). “Fluorescence Assay in 5. UDENFRIEND, S., Biology and Medicine.” Academic Press, New York, 1962. 6. EULER, U. S. v., “Noradrenaline.” C. C Thomas, Springfield, Ill., 1956. 7. GREEN, D. E., AND RICHTER, D., Biochem. J. 31, 596 (1937).
130
HARRISON
8. DAWSON, C. R., AND TARPLEY, W. B., in “The Enzymes”, (J. B. Sumner and K. Myrblck, eds.), Vol. II, part I, p. 454. Academic Press, New York, 1951. 9. SUSSMAN, A. S., Arch. Biochem. Biophys. 96, 407 (1961). 10. CHAIX, P., CHAUVET, J., AND JEZEQUEL, J., Biochim. Biophys. Acta 4, 471 (1950). 11. SCHAYER, R. W., J. Am. Chem. Sot. 74, 2441 (1952). 12. HARLEY-MASON, J., J. Chem. Sot. 1960, 1276. 13. KOELLE, G. B., AND FRIEDENWALD, J. S., Arch. Biochem. Biophys. 32, 370 (1951). 14. SZARA, S., AXELROD, J., AND PERLIN, S., Am. J. Psychiat. 115, 162 (1958).
15. GUDBJARNASON, S., AND BING, R. J., Biochim. Biophys. Acta 60, 158 (1962). of the Coordi16. BAILAR, J. C., JR., “Chemistry nation Compounds.” Reinhold Publ. Corp., New York, 1956. 17. CHAIX, P., MORIN, G., AND JEZEQUEL, J., Biochim. Biophys. Acta 6, 472 (1950). 18. SENOH, S., AND WITKOP, B., J. Am. Chem. Sot. 81, 6231 (1959). 19. KRALL, A. R., SIEGEL, G. J., AND GOZANSKY, D. M., Federation Proc. 21, 55F (1962). 20. HOCHSTEIN, P., AND COHEN, G., J. Neurothem. 6, 370 (1960). 21. WALAAS, E., AND WALAAS, O., Arch. Biochem. Biophys. 96, 151 (1961).
OF
BIOCHEMISTRY
Detection
AND
BIOPHYSICS
of Intermediate
Noradrenaline
161, 116-130 (1963)
Oxidation
by Fluorescence
States
of Adrenaline
Spectrometric
and
Analysis’
W. H. HARRISON* From
the Departments
of Neurology
and Biochemistry, College of Physicians University, New York, IVew York
and Surgeons,
Columbia
Received November 16, 1962 Fluorescent intermediate oxidation states of adrenaline and noradrenaline, formed near physiological conditions of pH and concentration, were found detectable by direct fluorescence spectrometric analysis. Oxidizing agents or catalysts, such as Cu++, Fe+++, MnOz, tyrosinase, and ceruloplasmin were found to effect their formation. While the (non)identity of the products with any of the oxidation intermediates already known for adrenaline and noradrenaline has not yet been established, their properties indicate that they are reduced states of different reversible redox systems. They were found stable in the presence of the oxidation system in which their formation was effected, but unstable in the presence of oxidizing agents such as ferricyanide, in which case they are reversibly oxidized. The latter reversibility wits demonstrated by showing disappearance and reappearance of fluorescence upon alternate treatment with ferricyanide and ascorbic acid. When ferricyanide was used as the initial oxidizing agent, the products formed were nonfluorescent, but ascorbic acid effected their fluorescence; thus, a relationship between fluorescence and the reduced state was consistent in all experiments. The intermediates are believed closely related in that they were all found to be precursors of the same 3,5,6-t.rihydroxyindole derivatives. The results suggest new methods for the analysis of adrenaline and noradrenaline and their oxidation products which should aid in establishing whether minor oxidative pat,hways occur in civo. INTRODUCTION
It is now well established that the two major routes of adrenaline and noradrenaline metabolism are 0-methylation and oxidative deamination (1). There still remains the possibility of at1 occurrence of a minor pathway in which their dihydric phenolic moieties are oxidized to the corresponding o-quinones and the side chains cyclized to form trihydroxyindole derivatives. Catalysis of the latter type of reaction by various biological tissue and fluid preparations, certain oxidative enzymes, and metalloproteins has been demonstrated (2), but there is a lack of agreement among different investigators concerning whether the expected 1 This work was supported by the U. S. Public Health Service, Grant No. 2B-5216. 2 Supported by Training Grant No. BT-485.
products of oxidation are present or involved in physiological processes as well as whether the reaction is part of a normal or an abnormal metabolic pathway (3). This is in contrast to the convincing evidence supporting t.he involvement of the major pathways (1). Since most of the catecholamine metabolism can be accounted for by the two major pathways, an oxidative pathway, if it occurs, would involve a very small fraction of the already exceedingly small amounts of catecholamine found in biological systems. Because of this, detection of such products presents considerable difficulty for an analysis. From their chemical and physical properties (4), several oxidation products would be expected, each forming a part of a reduction-oxidation system, and the final equilibrium composition would 116
ADRENALINE-NORADRENALINE
be dictated by factors such as the pH of the system and the potential of the most concentrated or most active redox system. Consequently, any purification or concentration step in an analytical procedure which modifies the potential or pH would modify the relative amount of each intermediate in such an inherently unstable system. Phenomena of this type would most likely explain the wide divergence in available analytical data. This type of analytical problem requires either an extremely sensitive direct measuring technique or a method in which the redox potential is maintained during analysis at a physiological level in addition to control of pH and other variables. The investigation described in this paper represents the first phase of an approach to this problem. Fluorescence spectrometry was applied because of its extreme sensitivity. By this technique, evidence was obtained which indicates that relatively stable products, which fluoresce and are detectable by direct measurements, form on the treatment of adrenaline and noradrenaline with a variety of oxidizing agents at pH and concentration conditions as near physiological levels as the sensitivity of the fluorescence measurements will permit. This report includes a description of the oxidation reactions and some of the properties of the fluorescent products together with a discussion of the significance of the results. MATERIALS
AND METHODS
The fluorometric measurements were made with an Aminco-Bowman spectrophotofluorometer. Instrumental parameters such as cell and photomultiplier slit arrangements and photomultiplier tubes (IP21 or IP 28) were varied to give the sensitivity and resolution required by each experiment. Three-milliliter quartz cells supplied with the instrument were used in all experiments. In that most of the experiments were performed at room temperature, the activation cell shutter was kept closed except when making measurements in order to minimize temperature change and prevent resultant fluorescence intensity changes not related to the effect studied. The possibility that the fluorescence changes noted were the effect of irradiation or temperature change during the periods of measurement was checked in preliminary experiments by running duplicates at controlled
OXIDATION
117
temperatures; however, this possibility was ruled out in that final fluorescence patterns of the temperature-controlled runs were found to be similar to those subjected to the activation light at regular intervals. Use of a temperature control device in the cell compartment is required and planned for future studies in which quantitative titrations and kinetic measurements will be made. The wavelengths cited are uncorrected. The I-adrenaline and I-noradrenaline (bitartrates) and L-ascorbic acid were supplied by Nutritional Biochemicals Corp. Stock solutions (1 X 10u4%f) of Z-adrenaline and I-noradrenaline were prepared in 0.01 M HCl, and appropriate dilutions with distilled water were made in each experiment. Analytical reagent-grade chemicals were used in the preparation of the buffers, the metal salts, and the ethylenediamine tetraacetate (EDTA) solutions. Copper acetate (monohydrate), ferric chloride (hexahydrate), zinc acetate (dihydrate), manganese dioxide (85%), and potassium ferricyanide were used in the oxidation experiments. Several different lots of mushroom tyrosinase, supplied by Worthington Chemical Corp., were used; the reported activities, based on tyrosine oxidation, ranged from 500 to 600 units/mg. Stock solutions of tyrosinase were prepared in the buffers employed in each experiment at the concentrations noted. In separate experiments, dialysis of commercial tyrosinase or purificatZion with Sephadex did not give rise to an activity change in respect to formation of fluorescent oxidation products. The ceruloplasmin solution (Fraction IV-l) was supplied by Nutritional Biochemicals Corp. and was used without further treatment. RESULTS
At the outset of this investigation it appeared possible that the oxidation of adrenaline and noradrenaline might be followed by direct fluorescence-loss measurements at their characteristic excitation and emission peaks and that some of the oxidation products, trihydroxyindole derivatives, might be detectable by fluorescence measurements after treatment with reducing agents. Initially, it was assumed that nonfluorescent, o-quinoidal intermediates such as the aminochromes would be formed which when reduced to their “leuco” forms might fluoresce and be detectable. Although such compounds have not been reported to fluoresce, they would be expected to on the basis of Auorescence-structure relationships derived from
118
HARRISON
s
O
2
3
4
s
6
7
5 TIME
::
in MINUTES
E 2
theoretical considerations and empirical observations (5). This was tested by using ferricyanide as the oxidizing agent and ascorbic acid as the reducing agent; the results are given in Part I. In Parts II and III are described further tests with this approach in which Cu++, bound Cu++ (tyrosinase and ceruloplasmin), Fe*, and MnOz were used as oxidizing agents or catalysts. I. FERRICYANIDE
-50
FIG. 1. Fluorescence changes which result from various treatments of adrenaline and noradrenaline. The following are shown: time courses of fluorescence loss of adrenaline (A) and noradrenaline (x) during ferricyanide oxidation at pH 5.5 (A, and N,) and pH 3.0 (A6 and Nb) ; time courses of formation of fluorescent products which result when the products of ferricyanide oxidation at pH 6.0 are treated with ascorbic acid (A, and NJ and when the products of ferricyanide oxidation at pH 5.5 and pH 3.0 are treated simultaneously with ascorbic acid and alkali (A, and N, at pH 5.5 and Ad and Nd at pH 3.0). The subscripts refer to the following reaction mixtures which contained (in mpmoles) : (a) adrenaline or noradrenaline, 10; sodium acetate-acetic acid, 1 X 106; KsFe(CN)s, 380; 1.2 ml. final volume, pH 5.5. (b) adrenaline or noradrenaline, 10; sodium acetate-acetic acid, 1 X 106; ZnSOd, 434; K,Fe(CN)e, 380; 1.2 ml. final volume, pH 3.0. Fluorescence measurements for (a) and (b) were made at 280 rnp excitation and 330 rnp emission. (c) adrenaline and noradrenaline, 50; sodium acet,ate-acetic acid 5 X 106; KsFe(CN)G, 1900; 6.0 ml. final volume, pH 6.0. At the time intervals specified, l.O-ml. aliquots were transferred to quartz cuvettes containing 0.1 ml. of 0.57 M ascorbic acid. Fluorescence measurements were made at 400 rnM excitation and 515 rnk emission. (d) and (e) were run the same as (b) and (a), respectively, except that at the specified oxidation time a mixture containing 0.1 ml. of 0.57 M ascorbic acid and 0.9 ml. of 2.5 M NaOH was added. Each point for (d) and (e) was determined separately. Fluorescence measurements were made at 410 rnp excitation and 510 rnp emission.
OXIDATION
By fluorescence spectrometry, two steps in the ferricyanide oxidation of adrenaline and noradrenaline were detected. A fast step, occurring within 1% min., was observed by following decrease of the fluorescence of each amine. A slower step, occurring within 10 min., was found detectable and followed by measuring appearance of oxidation products which were found to exhibit fluorescence upon their reduction by ascorbic acid. Fast Step In preliminary experiments, the fluoressence of both amines was found to decrease rapidly and level off within a half minute during ferricyanide oxidations at pH’s 6 and 7. At pH 5.5, the reaction occurred within 1% min., and differences in the rates of the two amines were barely discernible (Fig. la). To show more clearly the differences in the reactivity of these two amines, it was necessary to follow the reaction at a pH at which the rates would be sufficiently slow to allow their measurement. This was demonstrated in an oxidation at pH 3, in the presence of Zn++, required as a catalyst (Fig. lb). It is believed pertinent that noradrenaline underwent the same changes as adrenaline at pH 3, although at a different rate, in view of the fact that it has been shown to be only slightly reactive at pH 3 in tests in which the degree of reactivity is estimated by measurement of fluorescent trihydroxyindole formed upon alkalinization (6). Slower Step Formation of oxidation products of adrenaline and noradrenaline during ferricyanide oxidation at pH 6 was demonstrated
ADRENALINE-NORADRENALINE
119
OXIDATION
by using the fact that they exhibit fluorescence upon reduction with ascorbic acid. The time courses for their formation are given in Fig. lc; for comparison with the fast step, the same time scale is used. The excitation spectra (Fig. 2) of each amine differed considerably but overlapped, and the fluorescence intensity of the noradrenaline derivative was considerably greater than that of the adrenaline derivative. Fluorescence was not exhibited when ascorbic acid was added at different intervals during the pH 3, Zn+f-catalyzed oxidation. A
1.0
0.5 ADRENALINE
300
400
350 WAVE LENGTH
Imp)
FIG. 2. Excitation spectra, before and after alkalinization, of the fluorescent products which result from ascorbic acid treatment of pH 6 oxidation products of adrenaline (A) and noradrenaline (N). Except for the pH, the oxidation reaction mixture was the same as described in Fig. 1 (a). Six minutes after addition of KaFe(CN)c, 0.1 ml. of 0.57 M ascorbic acid was added. The spectra of the adrenaline and noradrenaline oxidation products were obtained (solid line), and then 1.0 ml. of 2.5 M NaOH was added and the spectra were redetermined (dotted line). To obtain each spectrum, the emission intensity at 510 rnr was measured while scanning the excitation spectrum. The emission maximum for the noradrenaline product before alkalinization was found at 468 rnp (not shown) ; at its excitation and emission maxima the noradrenaline fluorescence intensity was 60% greater.
or NORADRENALINE
(mp molar,
FIG. 3. Relationship between adrenaline (A) or noradrenaline (N) concentration and the fluorescence exhibited by their oxidation products after treatment either with ascorbic acid (a) or with ascorbic acid followed by alkalinization (5 and c). The oxidations were carried out at pH 6.0 in 1.3 ml. final volume containing (in mpmoles) : adrenaline or noradrenaline, O-1.0; sodium acetate-acetic acid, 1 X 106; and K3Fe(CN)6 760. Six minutes after addition of KaFe(CN)e, 0.2 ml. of 0.57 M ascorbic acid was added in all cases. Fluorescence measurements for curve (a) for the noradrenaline product were made before alkalinization at 372 rnp excitation and 469 rnp emission. Curves (5) and (c) were determined after addition of 1.0 ml. of 2.5 M NaOH, and the fluorescence measurements were made at 410 rnp excitation and 510 rnp emission. Corrections were made for fluorescence intensities of controls not containing adrenaline or noradrenaline.
linear relationship between the fluorescence of the ascorbic acid-treated, pH 6 noradrenaline oxidation product and the concentration of noradrenaline was found (Fig. 3~2); however, the relationship was not linear in the case of adrenaline (not shown). The above oxidations were performed in 1 M acetate system; however, in separate experiments, fluorometrically identical products were formed at pH 7.0 in a 0.1 M acetate system, but their fluorescence in-
120
HARRISON
tensities were 50-75 % less. Ionic strength effects of this sort have been reported by others (5) and would be expected on theoretical grounds. of Intermediates to S ,5,6-Trihydroxyindoles
Conversion
Alkalinization of the ascorbic acid-treated, pH 6 oxidation products effected their conversion to products with the fluorescence characteristics of the 3,5,6-trihydroxyindoles of adrenaline and noradrenaline (Fig. 2); the conversion was completed immediately for both products. A linear relationship of the fluorescence of each product with concentration was shown (Figs. 36 and 3~). Simultaneous Addition of Alkali Ascorbic Acid
and
When alkali and ascorbic acid were added together to the pH 3, Zn++-catalyzed oxidation, a fluorescent 3,5,6-trihydroxyindole was formed from adrenaline but not from noradrenaline, and similar treatment of the pH 6 oxidation products resulted in 3,5,6trihydroxyindoles from both amines; these are the reactions involved in the trihydroxyindole procedure (6), and their time courses are included in Fig. 1 (d and e).
E$ect of Ascorbic Acid Concentration When the amount of ascorbic acid used was lowered to 0.02 mg., the fluorescence of the noradrenaline derivative became maximal and that of the adrenaline derivative negligible; the positions of the excitation and emission peaks of the noradrenaline derivative shifted (Table I). Further details on this effect and its possible application to a new analytical technique will be reported elsewhere. II. Cutl-, Fe+++ AND Mn09 OXIDATIOK Cu* and Fe+++ In a similar study, the mechanism of the Cu* and Fe+++ oxidation of adrenaline and noradrenaline was found to be considerably different from that of the ferricyanide oxidation. For example, on monitoring fluorescence changes by scanning both excitation and emission spectra during an adrenaline oxidation by Cuff at pH 7, t’svo steps were directly detectable (Fig. 4). As in the ferricyanide experiment, one step reflecting loss of the fluorescence of adrenaline was shown; however, in contrast, an additional concurrent direct formation of a fluorescent product was demonstrated. This fluorescent
TABLE FLUORESCENCE CHARACTERISTICS
OF
I
ADRENALINE AND NORADRENALIXE PRODCCTS
INTERMEDIATE
OXIDATION
Fluorescent products Oxidizing agent or catalyst
Adrenaline (Xmax) Excitation
Emission
Noradrenaline (X,,,) Excitation
iuncorrected)
cu++
Fe+++ Tyrosinase MnOz KsFe(CN)s KzFe(CN)6 Ceruloplasmin
fw
m/J
340 340 327, 390b 320, 3834 320, 400c
506 506 487O 5100 515c
323, 382
483
-
Emission
(uncorrected)
-
nw
36@’ 36Oa 358* 362” 320, 375c 300, 395c,J 360
,np
4900 490” 470h 468” 468” 47gcad 4iR
a Fluorescence increase with EDTA. b The values reported were from the experiment described in Fig. 6 in which an acetate-acetic acid system was used. Different maxima, particularly notable in the case with adrenaline, were obtained when phosphate buffer was used (Fig. 8). 5 Reducing agent required for fluorescence. J Formed at ascorbic acid concentrations at which adrenaline does not fluoresce.
ADRENALINE-NORADRENALINE
50 Hc----m-m-*
100 N
160
OXIDATION
165
170
175
121
I60
TIME in MINUTES
FIG. 4. Measurements of fluorescence changes during Cu++ oxidation of adrenaline (A) and noradrenaline (N). The fluorescence-loss measurements for both adrenaline and noradrenaline were made at 273 rnp excitation and 325 rnp emission. Measurements of the formation of fluorescent products from adrenaline were made at 340 rnp excitation and 506 rnp emission and from noradrenaline at 360 rnMexcitation and 490 rnp emission. The oxidations were carried out at pH 7.0 in 1.04 ml. final volume containing (in mpmoles) : adrenaline or noradrenaline 9.1; sodium acetate-acetic acid, 1 X 106; and Cu++, 40. The Cu* was added at zero time. At the times indicated (see arrows), 40 mpmoles ferricyanide in 0.01 ml. or 40 mfimoles ascorbic acid in 0.01 ml. or 40 mrmoles EDTA in 0.01 ml. were added, and fluorescence measurements were made at the times indicated.
product appeared to be in its reduced state in that addition of ascorbate did not affect the fluorescence. Its reducing properties were apparent in that it underwent oxidation by ferricyanide to a nonfluorescent derivative which was readily reconverted to the fluorescent state by a subsequent reduction with ascorbate (Fig. 4). Furthermore, successive oxidations and reductions by these agents could be repeated, and the changes followed fluorometrically (Fig. 4). The fluorescence characteristics of this product differed from the ferricyanide oxidation product having excitation and emission maxima of 340 and 506 rnp, respectively. When noradrenaline was substituted for adrenaline in the above oxidation, a fluorescence loss step was observed, but formation of a fluorescent product was just barely detectable (Fig. 4). However, on further examination, it was found that a potentially fluorescent product already in its reduced state, as in the adrenaline experiment, was formed but that its fluorescence
was quenched presumably by the oxidation agent, Cu*. This conclusion was reached from several lines of evidence. First, when the molar ratio of Cu* to noradrenaline in the oxidation was reduced from four to one, the intensity of the fluorescent product formed directly increased 12-fold to within 60% of the intensity of that obtained with adrenaline. Secondly, when EDTA in an amount equivalent to the Cu++ used in the oxidation was added at the end of the reaction, fluorescence comparable to that obtained from the adrenaline derivative was exhibited. Furthermore, alternate additions of equivalent amounts of Cu* and EDTA caused reversible exhibition of fluorescence. Also, when the Cuff was in sufficient quantity to quench the fluorescence, acidification to pH 3.0 caused appearance of fluorescence, and subsequent neutralization with alkali caused disappearance; this suggests that the quenching results from formation of a complex of the oxidation
122
HARRISON
product with Cu++ and that the complex can undergo a reversible dissociation by acidification. In systems in which the fluorescence quenching of oxidized noradrenaline by Cu++ was eliminated by EDTA or the oxidation was carried out with smaller quantities of Cut+-, the reducing properties of the fluorescent product and the ease of its interconversion to its oxidized and reduced states were identical to those for the adrenaline product. For example, successive oxidation and reduction by ferricyanide and ascorbate were demonstrated after EDTA complexing of Cu++ (Fig. 4). The fluorescence of the noradrenaline derivative differed from the adrenaline derivative, having excitation and emission maxima of 360 and 490 rnp, respectively. Importantly, in addition to its reducing properties, ascorbate, at pH 7.0 was shown to react in the noradrenaline oxidation mixture by complexing with Cu++ similar to EDTA. Thus ascorbate, when added to oxidized noradrenaline, effected formation of the fluorescent product presumably by a complexing reaction with Cu++; oxidation of the fluorescent product with ferricyanide caused disappearance of fluorescence which could be restored by addition of ascorbate; in the latter case, ascorbate functions as a reducing agent. Unless this dual function is considered, the effects of ascorbate on this system are easily misinterpreted. The above EDTA effect was not shown with the adrenaline derivative. To determine whether a very high level of Cu* would quench the fluorescent adrenaline product as it does the noradrenaline product at low levels, high ratios of Cuff to adrenaline were used in the oxidation, and the EDTA effect was checked. At a molar ratio of 20: 1 a slight effect was noted, the final fluorescence being increased by 20 % on addition of EDTA. When carried out at pH 5.0-7.5, formation of the fluorescent products during Cu++ oxidation of adrenaline and noradrenaline increased with increasing pH; no pH opt,imum was observed. At higher pH levels,
TIME in MINUTES
FIG. 5. Direct measurements of fluorescence changes during Fe+++ oxidation of adrenaline (A) and noradrenaline (N). The fluorescence loss measurements for both adrenaline and noradrenaline were made at 270 rnp excitation and 330 rnp emission. Measurements of the formation of fluorescent products from adrenaline were made at 340 rnp excitation and 506 rnp emission and from noradrenaline at 360 rnp excitation and 490 nm emission. The oxidations were carried out at pH 7.0 in 1.04 ml. final volume containing (in mpmoles) : adrenaline or noradrenaline, 9.1; sodium acetate-acetic acid, 1 X 105; and Fe++, 40. The Fe+++ was added at zero time. See text for properties of the fluorescent products formed.
unstable trihydroxyindole derivat’ives were formed. Oxidations by Fe+++ were similar to those by Cu* with respect to fluorescence loss of the amines (Fig. 5>, the direct formation of fluorescent products (Fig. 5), the quenching effect of the noradrenaline product by Fe+++, and the ease of interconversion of the oxidized and reduced forms of the products. The excitation and emission maxima of the adrenaline and noradrenaline products were 340 rnp, 506 ml*, and 360 rnp, 490 rnp, respectively; thus, they were identical with the Cu++ oxidation products. A distinct pH optimum, pH .5.9, was found for
ADRENALINE-NORADRENALINE
the Fe+++ oxidation of adrenaline but not for noradrenaline. Buffers used in the study included acetate, phosphate, and tris(hydroxymethyl)aminomethane (Tris). Quantitative but not qualitative differences in the reactions with Cu++ and Fe+++ were noted when different buffers were used. These included fluorescence intensity differences, different effects of EDTA on the fluorescence of the noradrenaline product, and different oxidation rates. For example, in oxidations in which the Cu++ to noradrenaline ratio was 4: 1, with 0.1 M acetate, pH 7.0, the quenching of the fluorescence of the noradrenaline derivative was complete; whereas with 0.1 M Tris, pH 7.0, it was 50%; and with 0.1 iVl phosphate buffer, pH 7.0, no quenching occurred; in all cases studied, quenching was eliminated by EDTA. MnOz Products formed by oxidation of adrenaline and noradrenaline with an excess of manganese dioxide in 0.1 M phosphate buffer, pH 7.0, were found to be fluorescent. The excitation and emission maxima of the adrenaline product were 320 rnp, 383 rnp (excitation), and 510 rnp (emission), respectively, while the noradrenaline product had a single excitation peak at 362 rnp and an emission peak at 468 rnp. Fluorescence of both products was increased by EDTA; thus apparently the Mn++ formed during the oxidation partially quenched the fluorescence by complexing with both products. The products were reversibly oxidized, as in previous cases, by alternate treatments with ferricyanide and ascorbate. Trihydroxyindole
Formation
On alkalinization to a 5% NaOH concentration, all of the above oxidation products converted to derivatives with the fluorescence characteristics of their respective 3,5,6-trihydroxyindoles. III.
CATALYSIS OF OXIDATION PROTEIN-C++ COMPLEX
BY A
Tyrosinase The direct fluorescence spectrometric method was used for studying the catalyzed
123
OXIDATION A 390/487
+50
N 358/470
0 : : E 0 3 Y
TIME in MINUTES
-50
-100
FIG. 6. Direct measurements of fluorescence changes during tyrosinase-catalyzed oxidation of adrenaline (A) and noradrenaline (N). The fluorescence loss measurements for both adrenaline and noradrenaline were made at 278 rnr excitation and 340 rn,uemission. Measurements of the formation of fluorescent products from adrenaline were made at 390 rnp excitation and 487 rnp emission and from noradrenaline at 353 rnp excitation and 470 rnp emission. The oxidations were carried out at pH 7.0 in 1.2 ml. final volume containing (in mpmoles) : adrenaline or noradrenaline, 10; sodium acetate-acetic acid, 1 X 105; and tyrosinase, 50 units (0.1 mg.). The tyrosinase was added at zero time. See text for the properties of the fluorescent products formed.
oxidation of adrenaline and noradrenaline by tyrosinase, a copper-containing protein; its activity is usually measured by manometric (7) or spectrophotometric (9) methods. An experiment which shows this is described in Fig. 6. The excitation and emission spectra of the fluorescent products formed from each amine are given in Fig. 7. The products appeared to be in the reduced state in that addition of ascorbate did not increase their fluorescence, whereas ferricyanide caused fluorescence disappearance. On long standing, e.g., for 24 hr., a fluorescence decrease of about 10 % resulted; however, the initial fluorescence could be restored by addition of ascorbate.
HARRISON 8
A
pHa5
r PH8.0 /.JH 8.5 pHT0 pHz5 pH6.5 pHa0 i
j1Hb.0
pHZ5 pHiTO @MS pH60
~,~ 10 303
350
400 WIVE
450 LENGTH
500
550
20
30 TIME
40
0
IO
20
30
40
in MINUTES
(mpl
FIG. 7. Excitation and emission spect,ra of oxidation products of adrenaline (A) and noradrenaline (N) formed in the tyrosinase reaction. The oxidation reaction mixtures are described in Fig. 6. Curve (a) is the excitation spectrum of oxidized adrenaline measured at 487 rnM emission; curves (b) and (c) are its emission spectra measured at 390 and 327 rnp excitation, respectively. Curve (d) is the excitation spectrum of oxidized noradrenaline measured at 470 rnr emission; curve (e) is its emission spectrum at 358 rnp excitation.
EDTA had no effect on either product in contrast to the previous experiments with Cd+, Fe+++, and MnOa oxidations. Differences between adrenaline and noradrenaline were apparent, particularly in the rate and extent of formation of their respective fluorescent products (Fig. 6). Reaction rates were found proportional to enzyme concentration under conditions at which substrate was not rate limiting. When catechol was used as a substrate in a separate experiment, only the fluorescence-loss step was observed; thus, the ethanolamine side chain in adrenaline or noradrenaline is required for formation of the fluorescent products. Because the acetic acid-acetate system is a poor buffer at physiological pH con-
FIG. 8. Direct measurements of fluorescence changes during tyrosinase-catalyzed oxidation of adrenaline (A) (Graph A) and noradrenaline (N) (Graph B) in phosphate buffer, pH 6.W3.5. The fluorescent products from adrenaline were measured at 378 rnp excitation and 503 rnp emission and from noradrenaline at 360 rnp excitation and 468 rnp emission. A second excitation peak for the adrenaline product at 330 rnp was also noted; the noradrenaline product gave a single peak. The oxidations were carried out at the designated pH in 1.2 ml. final volume containing (in mrmoles): adrenaline or noradrenaline, 10; phosphate buffer, 1 X 105; and tyrosinase, 50 units (0.1 mg.). The tyrosinase was added at zero time. After 90 min. the fluorescence of mixtures containing adrenaline were the following: pH 6.0, 0.6; pH 6.5, 0.1; pH 7.0, 0; pH 7.5, 0.6; pH 8.0, 1.3; pH 8.5, -. The values after 90 min. for the noradrenaline mixtures were the following: pH 6.0, 1.G; pH G.5, 1.6; pH 7.0, 2.8; pH 7.5, 5.8; pH 8.0, 7.7; pH 8.5, 22.5.
ditions, phosphate buffer was tested for use in obtaining pH optimum curves for the enzyme. It was not found entirely suitable, however, because whereas the fluorescent products were found to be stable after about 90 min. in the pH 7.0 acetate system, they were formed more rapidly and were very unstable in phosphate buffer ranging from pH 6.0 to 8.5 (Fig. 8). Chaix et ~7. (10) have reported a similar instability in
ADRENALINE-NORADRENALINE 60
-
A P h0sphots TRlS N N
\I 6
125
borate buffer on the fluorescence of adrenaline and noradrenaline is included in Fig. 9; this demonstrates that borate complex formation, known for the catechols (5), causes a fluorescence quenching. Kinetic data describing a study of tyrosinase-catalyzed oxidation of catecholamines by this method will be presented elsewhere.
A
5
OXIDATION
Ceruloplasmin
7
e
9
PH
FIG. 9. Fluorescence intensity of adrenaline and noradrenaline at a series of pHs in phosphate, Tris, and borate buffers. The buffer concentrations were 0.1 M, and the adrenaline or noradrenaline concentrations, 3.2 X 10e6 M.
phosphate buffer. Because the advantages of the acetic acid-acetate system necessitated its use in many cases, it was necessary to adjust all reactants to neutral pH because of its poor buffering capacity. Results similar to those found with the acetate system were obtained when the reaction was run in 0.1 M Tris buffer, pH 7.0. In this case, the products were stable in contrast to the instability noted in phosphate buffer. The mechanism by which the different buffers influence this reaction is obscure. A check of the effect of Tris and phosphate buffers at different pH’s on the fluorescence of untreated adrenaline and noradrenaline (Fig. 9) showed that these buffer systems are not inert, but instead exert considerable effect. Whether the regions of fluorescence change shown in Fig. 9 indicate differences in the ionization state of these amines or whether other effects are being detected is not known; however, it appears that fluorescence is a sensitive indicator of some interaction of the buffer system and the reactant. Similar effects on the oxidation products formed might be expected as well. The effect of
In systems containing 10 mpmoles adrenaline or noradrenaline, 100 pmoles sodium phosphate buffer, pH 7.0, and 0.01 ml. ceruloplasmin solution in 1.11 ml. total volume, fluorescent oxidation products with the excitation and emission maxima given in Table I were formed, reaching a maximum level after 25 min. Addition of 41.1 mpmoles EDTA enhanced the fluorescence of both products, and no shift in maxima occurred. Further studies with ceruloplasmin will be presented elsewhere. Preparation of Intermediates The chromes of adrenaline and noradrenaline were prepared and compared with the oxidized forms of the above fluorescent intermediate oxidation products in respect to the fluorescence characteristics they exhibit upon alternate treatments with ascorbic acid and ferricyanide. A modification of the method developed by Schayer (11) for the preparation of adrenochrome, which involves Ag,O oxidation in methanol, was used; determination of fluorescence loss across the oxidation step was used as a criterion for establishing conditions for complete oxidation of each amine. Both chromes were reduced reversibly, presumably to their leucoaminochromes, as shown by alternate treatments with ascorbic acid and ferricyanide; the reversibility was judged by the disappearance and appearance of the red color of their solutions. However, neither reduced form exhibited the fluorescence peaks shown for the above intermediates. Since it is essential to establish, definitively, the relationships of the aminochromes to the fluorescent intermediates, a more detailed study with this approach will be made. When the concentrations of the amines in the reaction mixtures of the various oxi-
126
HARRISON
dations were increased above the micromolar level in order to obtain more concentrated solutions of their fluorescent products for isolation purposes, it was found that the products were no longer detectable; the possibility that they were not observed because of “concentration quenching” effects was always eliminated by diluting the samples appropriately. As an example, when the amine concentration in the Cu++ oxidation was increased tenfold, only redcolored ‘Laminochromes” and some fluorescent trihydroxyindole were detected; when the solution was treated with ascorbic acid, the color disappeared but the fluorescent intermediates were still not detectable. The same effect was noted when the amine concentration in the ferricyanide oxidation was increased; formation of chromes, some trihydroxyindole, and, in some cases, black precipitates resulted. In that it is important to isolate and characterize these intermediates, other approaches will be tried. DISCUSSION
By a direct fluorescence spectrometric method, stable, fluorescent products have been shown to result from oxidations, performed near physiological pH and concentrations, of adrenaline and noradrenaline with CL++, Fe++, MnOz, tyrosinase, and ceruloplasmin. While the evidence is insufficient for identification of these products, several of their properties have been ascertained. For example, the products are unstable in the presence of an oxidizing agent such as ferricyanide in which case their fluorescence disappears. However, the reversibility of this oxidation was established by demonstrating the reappearance of their fluorescence upon reduction with ascorbate. When ferricyanide was employed for the formation of the oxidation products, the products formed were nonfluorescent; but when an amount of ascorbate, sufficient to reduce both the ferricyanide and the oxidation product was added, fluorescent products resulted. Thus the relationship between fluorescence and the reduced state was consistent in all experiments. From a study of their reactivity in alkali, the products of every type of oxidation studied
were found on the basis of their fluorescence to be precursors of their respective 3,5,6trihydroxyindole derivatives. The products exhibited different fluorescence characteristics (Table I) depending on the type of oxidation agent or catalyst used. Whether these differences reflect major differences in structure or whether the metal ions in the media or other factors influence the positions of the excitation and emission peaks remains in doubt; their excitation and emission spectra did, however, overlap in every case. The EDTA effects found with the noradrenaline but not the adrenaline product must reflect a fundamental difference in structure or complexing ability of the two molecules, but its nature is not clear. Because of pertinence to the interpretations of this study, a summary of the various interconversions and disproportionation reactions known for the oxidation products of adrenaline (4) (12), together with possible alternative pathways, are described in Scheme I; noradrenaline is believed to undergo the same reactions although this has not been demonstrated in all cases (6). Compounds V, IIIa, and VII, shown in Scheme I, have been isolated and identified (7, 4, 12). Compounds II, III, VI and VIII have not been isolated. HarleyMason (12) has shown that reduction of adrenochrome (V) gives rise to equal amounts of 111~ and VII by isolating and identifying them. Structure VI was postulated by Harley-Mason for a precursor of compound VII which he detected and found to have a characteristic ultraviolet absorption spectrum and instability in alkali; he described the reaction leading to compounds IIIa and VII as a disproportionation reaction in which intermediates, III and VI are formed from a common intermediate semiquinone with structure IV. He also showed that an alternative, alkali-catalyzed direct conversion of adrenochrome (V) to its trihydroxyindole derivative (VII) could occur in the absence of a reducing agent. Adrenoquinone (II) has been assumed to convert upon oxidation to adrenochrome (V) via an oxidative cyclization (arrow 2) ; however, the alternative pathway, i.e. an intramolecular oxidative cyclization to leucoadrenochrome
ADRENALINE-NORADRENALINE
OXIDATION
127
O= O-
’ ox- ’
-
IO” N &i 3
Possible interconversions of the oxidation products of adrenaline (I). The products shown are adrenoquinone (II), leucoadrenochrome (III), 5,6-dihydroxy-1-methylindole (IIIa), adrenochrome (V), 3,5,6-trihydroxy-1-methylindole (VII) and its o-quinone state, (VIII), and structures (IV) and (VI), postulated by Harley-Mason (12).
(III) followed by oxidation to adrenochrome (V) (arrow 1) cannot be excluded. The latter pathway would be similar to the mechanism shown for the formation of 2,3-dihydro4,6,7-trihydroxyindole from 2,4,5-t& hydroxyphenethylamine which occurs via intramolecular oxidative cyclization of the intermediate p-quinone (18). Koelle and Friedenwald (13) showed that at millimolar concentration levels, adrenochrome (V) is reduced directly to leucoadrenochrome (III) and that the disproportion&ion reaction which was demonstrated by Harley-Mason (12) with 0.275 M adrenochrome does not occur. Identification of the fluorescent prod ucts reported in this investigation as any of the intermediates described in Scheme I
or as different structures must await further study of their properties. On considering the intermediates of Scheme I, the o-quinoidal structures can be excluded since such structures do not fluoresce (5) ; and structure 111~ can also be excluded because it does not give rise to a trihydroxyindole derivative upon alkalinization. The experiments in this although preliminary in investigation, nature, indicated that ascorbic acid-treated presumably leucoaminoaminochromes, chromes, do not exhibit the fluorescence shown by the fluorescent intermediates. On the other hand, the properties reported for intermediate VI by Harley-Mason (12) have similarities to the intermediates detected in this study; for example, intermediate VI is
128
HARRISON
converted in alkali to compound VII and has an ultraviolet absorption maximum at 347 mp which is close to the excitation maxima (340 mp) of the products from Cu++ and Fe+++ oxidations of adrenaline (Table I). A more detailed comparison is planned. Since the concentrations in this study were closer to physiological levels and the methods of analysis direct rather than involving additional treatments such as alkalinization which might modify the products --a possibility in other methods -it is believed that by this technique products more closely representing the true products of these oxidation reactions are detected. And, since in this study, although not identified, the products have been shown to be measurable, it’ would appear that methods similar t,o the type employed in this study would offer a number of advantages for deterrnnation of products formed in z&o and of possible oxidation products in biological tissues and fluids. In this respect, it is believed significant that these findings indicate that, oxidation at physiological conditions may give rise to reduced as well as oxidized states of reversible redox systems. In that most tests (14) for oxidation products have been designed for the detect#ion of o-quinoidal forms, e.g., adrenochrome, the question arises whether some of the products are in t,he reduced form and consequently not accounted for; this is particularly possible when an alumina purification step is used since the reduced form is selectively adsorbed on the alumina and separated from the oxidized form. Among the properties found in this study which might be exploited for the differentiation and measurement of the oxidation products of adrenaline and noradrenaline are their fluorescence spectra differences and the metal cation quenching effect, specific to the noradrenaline oxidation product. The same properties would serve equally well for the development of new, more specific methods for analysis of the unoxidized amines themselves, in procedures which would include oxidation steps of the type studied. Further investigation is required to determine which of the various systems studied are best for the above-suggested applications. Certain aspects of the mechanism by
which the catecholamines are oxidized and the fluorescent products or their precursors are formed were revealed in this study. For example, the time relationships between the different fluorometrically detectable steps were shown to vary wit.h the type of oxidation. In the ferricyanide oxidation, in about 2 min. the fluorescence loss was completed and maximal formation of the intermediate which could be converted to 3,5,6trihydroxyindole by alkali was attained, and 8 min. later the intermediate which fluoresced only on addition of ascorbate was formed maximally; this suggests that several steps are involved in this type oxidation. In the oxidations with CL? and Fe+++, and (tyrosinase) in which case bound Cutf fluorescent products were formed directly, the time periods for the fluorescence lossand-gain steps overlapped. These differences obviously indicate different mechanisms. For example, the ferricyanide oxidation appears distinctly different from the free or bound metal-ion oxidations. This might be expected in that ferricyanide ion is a fully coordinated very stable Fe+++ complex (16), thus eliminating the possibility of its involvement in a chelating reaction with the catecholamine. Thus this fluorescence loss must reflect only an oxidation reaction. On the other hand, coordination is possible in the free or bound metal-ion oxidations. It is likely t,hat complexing or oxidation reactions or both, occurring simultaneously, give rise to the fluorescence loss when free or bound metal ions are used; it appears to be a specific fluorescence-quenching effect, in that the rates for adrenaline and noradrenaline differed. Chelation and oxidation were indicated from studies of Chaix et al. (17). Also, studies in progress of oxidations by Cu++ and Fe+++ indicate that both complexing and oxidation do occur during the fluorescence loss step; one indication of this comes from the fact that a fluorescence decrease occurs in the absence of oxygen and is a function of the concentration of the metal cation. However, in such cases the fluorescence is not restored with EDTA thus indicating the occurrence of an underlying reduction-oxidation reaction in the complex. This is in contrast to the fluorescence quenching by chelation without oxidation
ADRENALINE-NORADRENALINE
indicated in the experiment described above, e.g., Fig. 4, in which case the fluorescence of the noradrenaline oxidation product was shown to be quenched by Cu++; in that case EDTA did restore the fluorescence and oxidation had not occurred; thus both cases can occur. Both chelation and oxidation may occur in a similar fashion during fluorescence loss in the tyrosinase oxidation, but in this case the step in which the fluorescent product is formed must be rate controlled by certain specific features of this enzyme. One explanation is that the fluorescence change during the first period represents chelation and oxidation of an amount of catecholamine required to activate the enzyme in such a way that it can catalyze the second step. It is interesting to compare this fluorescence loss step with the induction period shown by others (8) by oxygen uptake studies made during oxidation of catecholamines by tyrosinase. While direct comparisons between oxygen uptake and fluorescence loss were not made in this study, the similarities in time relationships are highly suggestive that the changes shown by fluorimetry in this study occur during that phase. An extensive quantitative examination by fluorescence and fluorescence polarization techniques of different protein-metal-ion complex-induced oxidations of the catecholamines is required for further clarification. In that certain enzymes and metal-protein complexes have been shown to catalyze such oxidations (2), it is interesting to speculate on the possible formation and participation of such redox systems in the functional processes of these important and apparently multifunctional catecholamines. A recent report of the activity of adrenochrome on oxidative phosphorylation (19) showed that, at micromolar levels, it decreased both oxygen uptake and phosphorylation, thus demonstrating a general toxic effect without an uncoupling action. Other toxic effects relating to glycolysis have also been noted for adrenochrome and noradrenochrome (20). However, since it is highly likely that the fluorescent substances found in this study may more closely relate to biologically formed products than the aminochromes,
OXIDATION
129
the specific involvement of oxidation products should not be excluded until these products are tested. That reactive intermediate oxidation products, not necessarily identical with the aminochromes, are formed was indicated from a recent study by Walaas and Walaas (21). Using ceruloplasmin as the catalytic agent, they demonstrated by a spectrophotometric method a coupled reduction-oxidation reaction between oxidation products of noradrenaline and adrenaline with reduced pyridine nucleotides (DPKH and TPNH) ; the noradrenaline product was found markedly more reactive than that of the adrenaline. Adrenochrome itself did not exhibit a similar react’ivity. The nature of the intermediates which participate in the reaction with the nucleotides was not ascertained, but Walaas and Walaas suggested that they were either free radicals or unstable uncyclized quinones. Although there is no obvious relationship between the active intermediates studied by Walaas and Walaas and those reported in this paper, both studies indicate formation of active products which are not identical to the products already characterized and reported in the literature. Further work should establish whether isolable intermediates or free radicals, e.g., semiquinones, are the products detected in these and other studies. ACKNOWLEDGMENT The author is most pleased to acknow-ledge his gratefulness to Professor David Nachmansohn for the opportunity of working in his laboratory and for his advice and encouragement during this investigation. REFERENCES 1. AXELROD, J., in “Adrenergic Mechanisms” (J. R. Vane, G. E. W. Wolstenholme, and M. O’Connor, eds.), p. 24. Little, Brown and Co., Boston, 1960. 2. ZELLER, E. A., Pharmacol. Rev. 11, 387 (1959). 3. LA BROSSE, E. H., MANN, J. D., AND KETTY, S. S., J. Psychiat. Res. 1, 68 (1961). 4. HEACOCK, R. A., Chem. Rev. 69, 181 (1959). “Fluorescence Assay in 5. UDENFRIEND, S., Biology and Medicine.” Academic Press, New York, 1962. 6. EULER, U. S. v., “Noradrenaline.” C. C Thomas, Springfield, Ill., 1956. 7. GREEN, D. E., AND RICHTER, D., Biochem. J. 31, 596 (1937).
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8. DAWSON, C. R., AND TARPLEY, W. B., in “The Enzymes”, (J. B. Sumner and K. Myrblck, eds.), Vol. II, part I, p. 454. Academic Press, New York, 1951. 9. SUSSMAN, A. S., Arch. Biochem. Biophys. 96, 407 (1961). 10. CHAIX, P., CHAUVET, J., AND JEZEQUEL, J., Biochim. Biophys. Acta 4, 471 (1950). 11. SCHAYER, R. W., J. Am. Chem. Sot. 74, 2441 (1952). 12. HARLEY-MASON, J., J. Chem. Sot. 1960, 1276. 13. KOELLE, G. B., AND FRIEDENWALD, J. S., Arch. Biochem. Biophys. 32, 370 (1951). 14. SZARA, S., AXELROD, J., AND PERLIN, S., Am. J. Psychiat. 115, 162 (1958).
15. GUDBJARNASON, S., AND BING, R. J., Biochim. Biophys. Acta 60, 158 (1962). of the Coordi16. BAILAR, J. C., JR., “Chemistry nation Compounds.” Reinhold Publ. Corp., New York, 1956. 17. CHAIX, P., MORIN, G., AND JEZEQUEL, J., Biochim. Biophys. Acta 6, 472 (1950). 18. SENOH, S., AND WITKOP, B., J. Am. Chem. Sot. 81, 6231 (1959). 19. KRALL, A. R., SIEGEL, G. J., AND GOZANSKY, D. M., Federation Proc. 21, 55F (1962). 20. HOCHSTEIN, P., AND COHEN, G., J. Neurothem. 6, 370 (1960). 21. WALAAS, E., AND WALAAS, O., Arch. Biochem. Biophys. 96, 151 (1961).