Comparative studies of enzymatic and non-enzymatic oxidation of 3,4-dihydroxyphenylalanine In vitro

Int.

138

3.

COMPARATIVE ENZYMATIC

Biochem.,

STUDIES

OXIDATION

138-144.

1972, 3,

[Scientechnica

OF ENZYMATIC

(Publishers)

Ltd.]

AND NON-

OF 3,4-DIHYDROXYPHENYLALANINE I?V VITRO

NGO Department

of Nuclear

Medicine

TRA77 and

AND

MARCEL

LAPLANTE

Radiobiology, Centre Quebec, Canada

(Rec&ed

6 December,

Hospitalier

Universitaire,

Sherbrooke,

I g7 I)

ABSTRACT I. No important

spontaneous decarboxylation of dopa is noted in 0. I M phosphate buffer, @H 7.0, and in a nitrogen atmosphere. 2. Dopa is decarboxylated by L-histidine decarboxylase and this amino-acid decarboxylase is a pyridoxal-phosphate-dependent enzyme. 3. Assay of dopa decarboxylase activity in isolated tissues with [carboxyl-l%]dopa using either a radiomanometric method or a continuous-flow ionization chamber method should be performed in a nitrogen atmosphere only.

THE decarboxylation of 3,4-dihydroxyphenylalanine (dopa) to dopamine by aromatic L-amino-acid decarboxylase, a pyridoxal-phosphate-(PLP-) dependent enzyme, has been described in animal tissues (Sandler 1969) and human postand Ruthven, mortem specimens (Lloyd and Hornykieand Prince, wicz, 1970; Vogel, McFarland, I 970). A so-called spontaneous decarboxylation of dopa was reported recently when [carboxyl-14C]dopa was incubated in phosphate buffer without any tissues (Vogel, rg6g; Vogel, Snyder, and Hare, 1970). It was suggested later that such a ‘ spontaneous decarboxylation ’ of dopa in vitro might contribute significantly to a rapid decarboxylation in uiuo (Hare, Vanna, Beasley, Chambers, and Vogel, rg71), resulting in low plasma dopa levels in patients with Parkinson’s disease after administration of large doses of this drug (Hare and others, 1971). However, we demonstrate here that this reaction is due to the well-known nonenzymatic oxidation of dopa to CO, and melanin rather than to the spontaneous decarboxylation of dopa to CO, and dopamine. A continuous-flow ionization chamber method was used for measurements of an

enzymatic

decarboxylation

and

a

non-enzymatic oxidation of dopa under various experimental conditions in vitro. METHODS

AND

MATERIALS

CHEMICALS

L-histidine decarboxylase (Cl. Wefchii) and PLP were purchased from Sigma Chemical Co. L-Dopa was obtained from Nutritional Biochemical Co. [Carboxyl-“C]dopa (specific activity: 3.4 mc. per mmole; radiochemical purity >gg per cent) was obtained fron New En,gland Nuclear Co. ‘Hydrogen peroxide (HgOZ) (50 per cent) was ourchased from Fisher Scientific Co. Phosohate buffers (0.1 M5 pH 7.0) were prepared ‘from distilled water and analytic-grade chemicals, and used in all exoeriments of this studv. Wistar male rats were purchased from Cana’dian Breeding Laboratories. 1

CONTINUOUS-FLOW

IONIZATION CHAMBER METHOD

Details of the ionization chamber method for an instantaneous and continuous measurement of 14C01 production from a [ %]biochemical have been published previouslv (Tran. Laplante, and LeBel, I 971). .&proximately 0.1’2 pc: [carboxvllLCldooa was added to 0.1 d\f ohosohate buff&. pH 3.0: at 37” C., and incubated’witd and withou; enzymes (Tran, 1971, 1972) or rat liver homogenates. The livers of male rats were homogenized with a ‘ Stir-R ’ (Tri-R Inst., Rockville eentre, New York) in 30 ml. of cold phosphate buffer, pH 7.0, and centrifuged at 30-v r.p.m. for IO minutes. Compressed gases with g5 per cent 0, and 5 per cent CO,, IOO per cent N2, or

OXIDATION

‘972,3

OF

3,+DIHYDROXYPHENYLALANINE

compressed air with approximately 20 per cent 0, and 80 per cent N, were passed continuously through the incubation chamber at a constant flow-rate ( IOOml. per minute). The gas flow then passed through an ionization chamber connected to a vibrating-reed electrometer which, in turn, was connected to a chart recorder.

0

10

50

60

70

80

90

103

110

120

FIG. I.-Composite data on the rates of WO, production from [carboxyl-W]dopa incubated with or without PLP and H,O, in a 0.1 M phosphate buffer in N,: 02, and air atmospheres. The ordinate represents the percentage of incubated 14C produced as WO, per minute, and the abscissa represents the time (in minutes) after the administration of [carboxyl-14C]dopa. Each point represents the mean of the “CO, production for each group of 4 experiments at the given time, and the length of the vertical bar through each point represents &I standard error of the mean. For the purpose of comparing various curves obtained from either the non-enzvmatic oxidation or the enzymatic decarboxylatibn of dopa, we have utilized T,,, and the total fraction of the incubated IpC appearing as “CO, within the first I 20 minutes following the incubation of [carboxylThese characteristics have been W]dopa. defined previously (Tran and others, ig7r ; Thenen, Gawthorne, and Stokstad, I 970). SPECTRAL

MEASUREVIENT

Visible and ultra-violet absorption measurements were made with an Acta III Beckman recording spectrophotometer equipped with a

‘39

TRAN

140

AND

o-3.o-absorbance expanded scale. Non-radioactive dopa was added to 0’ I ,Vf phosphate buffer, PH 7.0, incubated at 37” C., and gassed with ~5 per cent 0, and 3 per cent CO,. The spectra were scanned repeatedly (Tran, Similarly, 1971). a cuvette of non-radioactive dopa solution was scanned repeatedly when exposed to the air only. 301

ht. 3. Biochrm.

LAPLANTE

RESULTS data showing the rate of t4C0. production from [carboxyl-14C]dopa incubated with or \vithout PLP and H,O,, in the presence of pure Nz, g5 per cent O?, and j per cent CO,, and air atmospheres. There

Fig. I represents

f 1

PLfSO.01unit L-histidine dearbox @se 95%QJ, 5%~

10

-0

20

30

40

FIG. n.-Composite

data on the rates of WO, concentrations of PLP and L-histidine

various

G” 3

50

60 70 Time, mm.

20

30

4i

SC

6C

70

eo

90

‘00

1,G

90

100

110 120

production from [carboxyl-“C]dopa incubated with decarboxylase in N, and 0,. See Fig. I for details.

.L-__~L__-I-

10

80

120

‘lme,mln

FIG. 3.-Composite data on the rates of WO, production from [carboxyl-‘T]dopa incubated with rat liver homogenates in 0, and N,. See Fig. I for details.

was a very smaIl amount of WO, produced in nitrogen. However, it is clearly seen that r4C0, _ production occurred largely when the incubation chamber was gassed with air or with a mixture of 95 per cent 0, and 5 per cent CO,. -An inhibition of t4COz production was noted in the presence of 06 m;tf PLP. WO * production was distinctly increased when 0.25 mAW H?O, as added to the incubation chamber. Fig. 2 represents data showing changes in rates of t%O, production from [carboxylW]dopa whin incubated with 0.01 unit L-histidine decarboxylase and 0.1, 06, and I *o m&I PLP, in the presence of N, and 0, atmospheres. Similarly, Fig. 3 shows the changes in rates of14COz production from [carboxyl-14C]dopa by rat liver homogenates

OXIDATION

1972%3

OF 3,4-DIHYDROXYPHENYLALANINE

in 0,

and iYZ atmospheres. As shown in 1-3, a twofold decreased 14C02 production from [carboxyl-W]dopa was obtained in NZ as compared with those obtained in 0,. Fig. 4 shows that the absorption spectrum was slightly changed, with the maximum

I4I

and air atmospheres, or H,O,, but not in the presence of N, atmospheres. The result indicates, therefore, that there is no important spontaneous decarboxylation of dopa in 0.1 M phosphate buffer, pH 7.0, and in N,, whereas there is a considerable non-enzymatic oxidation of dopa to melanin occurring

Tables

Table II.-

T, ~x AND CUMULATIVE "CO, PRODUCTIONDURINGTHE INITIAL120 MINUTESFROM [CARBOXYL-%]DOPA INCUBATED WITH PLP .+ND L-HISTIDINEDECARBOXYLASEIX N, AND 0, ATMOSPHERES

I EXPERIMENTAL CONDITIONS (0. I 2 MC [carboxyl-‘*Cl dopa+o.oI unit L-hi&dine decarboxylase)

I

NO. OF EXPERIMENTS

Nitrogen atmosphere

0’1

I ‘0

57’75kI.42 42.2514.32 36.62 &o-23

0.6

35.00*1.24

0.6

Oxygen atmosphere

4.973 zto.458 5.188*0.102 6.616io.973 16.512io.485

Table III.-Tm,,, AND CUMULATIVE WO, PRODUCTIONDURINGTHE INITIAL I 20 MINUTESFROM [CARBOXYL-~*C]DOPA INCUBATEDWITH RAT LIVER HOMOGENATES IN N, AND 0, ATMOSPHERES

I ATMOSPHERE

I No. OF EXPERIMENTS

Nitrogen Oxygen

4 4

I (minu~~S.E.) 14.83io.66 27.66i3.19

absorbances at 210 nm. and 280 nm., when the cuvette containing IO-~ mole dopa solution was exposed to the air. The absorption spectrum of dopa was indeed observed to change noticeably after the introduction of 0 2 into the dopa solution, as shown in Fig. 5, Within IO-I 20 minutes there were gradual increases at the 2lo-nm. and 28o-nm. peaks. DISCUSSION In previous studies measurements of liberated 14C0, from [carboxyl-14C]dopa suggested that dopa was easily ’ decarboxylated ’ in phosphate buffer without the addition of tissue homogenates (Vogel, 1969; Vogel, Snyder, and Hare, 1970). We observe here that such a very large amount of 14C0, can be detected in the presence of OZ

W PRODUCTIONIN 120 MINUTES (percentage&S.E. per g. tissue) 2-856fo.252 5.363 ko.788

in 0, (Swan, 1963 ; Rizzoli and Galzigna, 1971) or in H,O,. This non-enzymatic oxidation of dopa by H,O, is probably due to its ions, HO-s or 0,w2, formed in the phosphate buffer. The formation of polyphenols from dopa in air or OZ can be seen in our study by changes in the absorption spectrum of this substrate at typical peaks at ZIO nm. and 280 nm. (Hegedus and Altschule, 1970 ; Sassetti and Fudenberg, I g7 I ) . This is supported further by the observed formation of black dopa melanin from I-IO x IO-~ mM non-radioactive dopa incubated with 0, for IO hours, or H,O, in the phosphate buffer (Tran and Laplante, 1972). In addition, such a nonenzymatic oxidation of dopa was inhibited significantly by a relatively large dose of PLP,

142

TRAN

AND

possibly due to the formation of a DopaPLP complex (Fellman and Roth, I g7 I). Interestingly, we observed that both the non-enzymatic oxidation of dopa in 0, and the spontaneous decarboxyiation of dopa in

3.0

LAPLANTE

350

450

Fro. 4.---Ultra-violet and visible spectra of dopa during the course of oxidation in 0,. The complete cuvette mixture (3.0 ml.) contained potassium phosphate buffer, 0.1 M, PH 7.0, and IO-* mole dopa. Absorption spectra were taken immediately (A), and after IO (B), 20 (C), 30 (D), 60 (E), and 120 (F) minutes. N, were completely inhibited by boiled human plasma and erythrocytes (Tran and Lapiante, 1g72), and by boiled rat liver homogenates {Vogel, McFarland, and Prince, 1970; Lapiante, Tran, and LeBei, 197x). Such a non-enzymatic oxidation of dopa was easily reversed by Cu2+ in O,, but not in N, (Laplanteandothers, 197 I) .Werecentlyfound a two-fold increase in ‘*CO, production from [carboxyl-YI]dopa incubated with rat liver homogenates, in 0.1 M phosphate buffer, PH 7.0, and in an 0, atmosphere, as compared

Biochtm.

to values obtained in N,. Similar results were noted with this l%-labelled dopa and L-histidine decarboxylase in the presence of PLP in 0, and N, respectively. These observations suggest the occurrence of an

CC’--250

1nt. J.

_.._

XC FIG. 5.-Ultra-violet and visible spectra of L-phenylalanine decarboxylase-PLP complex. The complete cuvette mixture (3.0 ml.) contained potassium phosphate buffer, WI M, fiH 7.0; ~3 mg. L-phenylalan~e decarboxylase; and IO--~ mole PLP. The cuvette mixtures were equilibrated for 7 minutes at 37” C. and absorption spectra were taken at o, (A): 8 (a), 20 (C), 45 (D), 75 (E), and 120 (F) minutes. enzymatic decarboxylation of dopa in N, and, on the other hand, the occurrence of both an enzymatic decarboxylation and a non-enzymatic oxidation of dopa in 0,. This suggests also that measurement of dopa decarboxylase activity in isolated tissues with [carboxyl-14C]dopa, using either a modified standard Warburg technique or a continuous-flow ionization chamber, shoufd be performed in Nz only.

OXIDATION OF 3,4_DIHYDROXYPHENYLALANINE

‘972, 3

lt is of interest to note, finally, that aromatic L-amino-acid decarboxylase of the hog kidney slightly decarboxylated L-histidine (Christenson, Dairman, and Udenfriend, whereas we report here that L‘970), histidine decarboxylase can easily decarboxylate dopa. As shown in Fig. 2, a gradually increased 1%0, production and a shortened .r,,, were obtained when larger quantities of PLP were added to the phosphate buffer containing the same amount of enzyme in a N, atmosphere. This indicates that PLP is reallv a co-factor for the activation of Lhistidine decarboxylase for dopa decarboxylation. Our overall results demonstrate that under the conditions employed dopa can undergo an enzymatic and a non-enzymatic reaction. SUMMARY The present paper describes experiments that examine various patterns of enzymatic and non-enzymatic oxidation of dopa in vitro. The results obtained from both a spectrophotometric method and a continuous-flow ionization chamber method with [carboxyl14C]dopa confirm that dopa oxidized nonenzymatically to melanin in 0.1 A4 phosphate No buffer, PH 7.0, and in the presence of 0,. important spontaneous decarboxylation of dopa is, however, found in this phosphate buffer and in the presence of N,. Other results show that L-histidine decarboxylase decarboxylates dopa easily in the presence of pyridoxal phosphate. Also, they suggest that measurement of dopa decarboxylase activity in isolated tissues with [carboxyl-%]dopa, using either a modified standard Warburg technique or a continuous-flow ionization chamber method, should be performed in N, only. ACKNOWLEDGEMENTS

This work was supported by the University of Marcel Sherbrooke Medical School Fund. Laplante is the recipient of a studentship from the Medical Research Council of Canada.

CHRISTENSON,

J.,

REFERENCES C.. DAIRMAN, W.,

FRIEND, S. (1970).

‘ Preparation

and UDENand properties

‘43

of a homogeneous aromatic L-amino acid decarboxylase from hog kidney ‘, Archs Biochem. Biophys., 141, 356-367. FELLMAX, J. H., and ROTII, E. S. (rg7r), ‘ Inhibition of tyrosine aminotransferase activity by L-3,+-dihydroxyphenylalanine ‘, Biochemistry, xo, 408-414. HARE, T. A., VANNA, S.. BEASLEY, B., CHAMBERS, R., and VOGEL, W. H. (IgTr), ‘Amino acid and dopa levels in plasma and urine from Ldopa-treated patients with Parkinson’s disease ‘, 3. Lab. clin. Med.? 77, 319-325. HEGEDUS, R. L.? and ALTSCHULE. M. D. (IgTo), ‘ The formation of rheomelanins in human blood plasma from catecholamines, from ~ L-dopa and from some of their derivatives ‘, Archs int. Physiol. Biochim., 78, 443-459. LAPLANTE, M., TRAN, N., and LEBEL, E. (197x), ’ Influence du Cut+ sur I’oxydation nonenzymatic de DOPA dam le foie de rat ‘, Proc. Can: Fedn Biol. Socs, 14, 22. LLOYD. K.. and HORNYKIEWICZ. 0. iroto). ’ Parkinson’s disease: activity of L-dopa- ‘de: carboxylase in discrete brain regions ‘, Science, N.T., x70, 1212-1213. RIZZOLI, A. A., and GALZIGNA, L. (1g71), ‘ Effects of L-dopa suspension and solution on spontaneous activity in mice ‘, II Farmaco, 26, 37-39. SANDLER, M., and RUTHVEN, C. R. J. (rg6g), ‘ The biosynthesis and metabolism of the catecholamines ‘, Progress in Medicinal Chemistry ied. Ellis. E. G. P.. and West. G. B.). DD. 200” IA 265. London: Plenum Press. ’ SA&TI, R. J., and FUDENBERG, H. H. (IgTr), ‘ Aloha-methvldona melanin. Svnthesis and stabilization ‘in htro ‘. Biochem. Pharmac., 20, 57-66. ‘ Chemical structure of SWAN, G. A. (rg6S), melanins ‘, Ann. N. 2: Acad. Sci., IOO, IOO.+IOI~. THENEN, S. W., GAWTHORNE, J: M., and STOICSTAD. E. L. R. (~oto). ‘ The oxidation of ~ j-methyl -idC-tetrahydrofolate and histidine-2in vitamin B,,-deficient rats ‘, “C to =co, Proc. Sot. exp. Biol. Med., 134, 199-203. TRAN, N. (rg~r), ‘ Interaction of aromatic L-amino-acid decarboxylases with pyridoxal phosphate ‘, Int. 3. Biochem., 2, 700-704. between TM% N. (19721, ‘ Complex-formation hydrogen peroxide and L-phenylalanine decarboxylase ‘, Znt. 3. Biochem., 3, 61-65. TRAN, N., and LAPLANTE, M. (rg72), unpublished data. TRAN, N., LAPLANTE, M., and LEBEL, E. (rg71), ‘ Altered catabolism of I%-formate by erythrocytes of folic acid-deficient rats: a possible in vitro means for differential diagnosis of megaloblastic anemias in man? ’ , 3. nucl. Med., 12, 222-226. VOGEL, W. H. (I g6g), ‘ Non-enzymatic decarboxylation of dihvdroxvphenvlalanine ‘, Xaturwirsenschaften, 56,462. ’I,

,,

‘44

TFUN AND LAPLANTE

VOGEL, W. H., ~~FARLMD, H., and PRINCE, L. N. (rgjo), ‘ Decarboxylation of 3,4dihvdroxvohenvlalanine in various human adit angfetai tissues ‘, Biochmn. Phannac., xg, 618-620. VOGEL, W. H., SNYDER, R., and HARE, T. A. enzymatic decarboxylation of (197oh ‘ The

DOPA in human liver homogenates exp. Biol. Med., 1% 477-481:

Kg Word Index: dopa decarboxylase,

‘, Proc Sac.

3,4-Dihydroxyphenylalaninet L-histidine decarboxylase.