A spectrophotometric assay for drosophila dopa decarboxylase

ANALYTICAL

BIOCHEMISTRY

56, 300-305

A Spectrophotometric Dopa

(1973)

Assay for Decarboxylase

Drosophila

Dopa decarboxylase (3,4-dihydroxy-L-phenylalanine-carboxy-lyase, EC 4.1.1.26) catalyzes the conversion of dopa (3,4-dihydroxy-n-phenylalanine) to dopamine (3,4-dihydroxyphenyl-ethylamine) . In Diptera, this enzyme is involved in synthesis of a compound, N-acetyl dopamine, responsible for hardening and darkening of the cuticle, and peaks of activity occur during molting, pupation, and eclosion (l-3). We are investigating genetic control of this enzyme in Drosophila melanogaster and required a rapid, simple, inexpensive, yet sensitive assay to survey different strains for variations in enzyme activity. Streffer has developed a spectrophotometric assay for mammalian aromatic L-amino acid decarboxylase in which dopamine is detected by its reaction with 2,4,6-trinitrobenzene I-sulfonic acid (TNB) to form 2,4,6-trinitrophenyl dopamine (TNP-dopamine) (4). Since the TNB reagent also reacts with other amino acids including the substrate, dopa, forming corresponding TNP derivatives, the TNB reaction is followed by a benzene extraction. TNP-dopamine is differentially soluble in benzene and its concentration is determined at its absorbance maximum in benzene of 340 nm. The specific procedure as outlined by Streffer has been used successfully in our laboratory. Recently, however, we have modified the technique by substitution of a KCN addition to stop the enzyme reaction rather than the acid precipitation used by Streffer. This obviates the need for the time-consuming and tedious neutralization necessary for the TNB reaction. The use of the entire enzyme reaction rather than an aliquot for estimation of the dopamine formed enables the benzene extraction to be run in the reaction tube. These modifications render the assay especially suitable for the processing of a large number of samples. L-Dopa, pyridoxal-5’-phosphate (PLP) , and TNB (picryl sulfonic acid) were obtained from Sigma Chemical Co., dopamine from Nutritional Biochemicals, and I-phenyl-2-thiourea (PTU) from Eastman Kodak. Newly emerged flies were collected and homogenized immediately or stored at -57°C. Homogenization was performed in ice-cold phosphate Copyright. All rights

300 @ 1973 by Academic Press. Inc. of reproduction in any form reserved.

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buffer, 0.1 M, pH 7.1 (50-100 flies/ml) containing sucrose, 0.3 M, and PTU, 0.5 mg/ml to inhibit phenol oxidase activity. The homogenate was centrifuged for 15 min at 17,500 rpm in a Sorvall RC-2 refrigerated centrifuge and the supernatant fraction centrifuged again at the same speed for 30 min. The resulting supernatant served as the enzyme preparation and was either assayed immediately or stored at -57°C. Each enzyme reaction mixture, final volume 0.25 ml, was prepared in a 14 by 100 mm ignition tube cooled in an ice bath and consisted of 0.1 ml dopa solution, .25 to 5 pmoles/ml; 0.1 ml PLP solution, O-5 pmoles/ ml, and 0.05 ml enzyme preparation. All solutions were made in 0.1 M phosphate buffer, pH 7.1, containing PTU, 0.5 mg/ml. After incubation for the enzyme reaction for 30 min at 42”C, tubes were returned to the ice bath and further enzyme activity prevented by the addition of 1 ml of 0.1 M phosphate buffer, pH 7.5, containing 2.5 m&r KCN and 4.26 mM TNB to each. A second incubation for 20 min at 4&42”C wa’s then performed to allow reaction of the TXB with the dopamine formed during the enzyme reaction. After incubation, 1.5 ml of spectrophotometric grade benzene was added to each sample and the tubes closed tight.ly with plastic caps. Extraction of the TNP-dopamine was performed by rapid mixing for about 15 SW on a test tube mixer (Vortex) and was followed by centrifugation for 15 min at maximum speed in a table top centrifuge to clear the benzene layer of precipitated protein. The benzene layer was aspirated and read against pure benzene at 340 nm in a Bec.kman DB spectrophotometer. Blanks were run for each enzyme reaction either by omitting the enzyme reaction incubation or by withholding the enzyme preparation until after incubation. Both methods yielded comparable results. OD values for the blanks (.O3-.05) were subtracted from readings (X1-.35) obtained for the enzyme reaction so that final value obtained was due only to dopamine formed during incubation of the enzyme preparation. Enzyme reactions were genera,lly run in triplicate and blanks in duplicate. Dopa decarboxylase activity was expressed as nmoles dopaminei min/fly and/or nmoles dopamine/min/mg protein. Protein was determined by the method of Lowry et nl. (5). To confirm identity of the product produced by the enzyme reaction with dopamine, spectrophotometric absorbance spectra were obtained for blanks to which dopamine was added and compared to those produced after incubation of the enzyme prepa,ration. Figure 1 illustrates that TNP-dopamine is characterized by three peaks of a.bsorhance in benzene; in addition to the major peak at 340 nm, there is a sharp peak at 280 nm and a small, broad peak with maximum absorbance at 420 nm. The absorbance spectrum produced by the TNP derivative of the enzyme

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1. Absorbance spectra of TNPdopamine and the TNP derivative of the product produced by the enzyme reaotion. A is a spectrum of a standard containing dopamine at 3.6 X lo4 M, and B is a spectrum produced after incubation of a Drosophila supernatant (70 ties/ml) for 30 min. FIG.

reaction was identical to that of TNP-dopamine and absorbance of all three peaks increased both with duration of the enzyme incubation anti concentration of the enzyme preparation. A standard curve for dopamine (Fig. 2) produced by standards containing increasing concentrations of dopamine with concomitantly decreasing concentrations of dopa was used to quantify the results of the enzyme assays. The molar extinction coefficient of TNP-dopamine was found, in agreement with Streffer, to be 1.24 X 10’ mol-1 cm-l. When performed according to the conditions described above, the TNB reaction is sensitive to as little as 6 nmoles dopamine and linear through a concentration of 90 nmoles. We found, however, that unless dopa is included in the reaction, readings for dopamine are reduced by as much as 50% by the presence of PLP. This phenomenon has been noted previously by McCaman, McCaman, and Lees in an assay in which dopa decarboxylase activity was measured by the amount of labeled dopamine extracted by liquid cation exchange, and attributed to nonenzymic Schiff base formation between PLP and dopamine (3). Our data indicates that a sufficiently high concentration of dopa relative to PLP prevents this interact,ion and allows detection of dopamine by the TNB reaction, but at excess cofactor concentration, or at low concentrations of dopa, com-

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FIG. 2. Standard curve for dopamine. Readings from standards containing increasing concentrations of dopamine with concomitantly decreasing concentrations of dopa, minus the control blank (0 dopamine and 1.2 x lo-’ M dopa).

plex formation results in erroneous readings for dopamine. For this reason substrate concentration curves must be run at low PLP concentration. Incubation of dopamine with the enzyme preparation resulted in a loss of approximately 10% of the product detectable by the TNB reagent. However, this loss also occurred in t,he presence of a boiled enzyme preparation and therefore may be the result of Schiff base formation wit.h endogenous cofactor. This interaction would be avoided in the usual enzyme reaction by the presence of substrate. An enzyme concentration curve obtained under standard conditions (dopa 1.2 X 10m3M, PLP 6 X lo-” M, 30 min incubation at 42°C) is shown in Fig. 3. Activity is linear over an approximate tenfold range of protein concentration and the sensitivity such that the average activity of a single fly can easily be determined. Generally 25-50 flies are used

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FIG. 3. Enzyme concentration curve. Dopa concentration was 1.2 X IOmJM; pyridoxal phosphate, 6 X 1OA M. The homogenate was made with 100 flies/ml buffer. Protein concentration was determined on the 0.5 dilution.

in preparing homogenates in order to obtain a valid estimate of average activity. For the usual assay, a concentration of 50 flies/ml homogenizing ,buffer is used which corresponds to the 0.5 dilution on the enzyme concentration curve. The insect enzyme is much more limited in substrate specificity than the mammalian (6)) and only dopa was tested as a substrate in t.he course of this work. Inhibition of the enzyme reaction was noted at. substrate concentrations higher than 1.2 X 1O-3M and it was determined that this phenomenon was real and not due simply to exhaustion of the TNB reagent by excess dopa. Inhibition of mammalian n-amino acid decarboxylase ha,s also been noted at, high substrate concentrations (7). The enzyme preparation was found to possessan appreciable amount

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of activity without. exogenous cofactor, and addition of PLP stimulated the reaction by about fivefold. Though ferric ions are often included as an additional cofactor in assays of the insect enzyme (2,6), they had no stimulatory effect, on the crude enzyme preparations used in this work. Though the specific assay conditions described here were defined for t,he Drosophila epidermal enzyme, the general assay procedure should bc applicable to dopa decarboxylase from other sources providing the enzyme reaction can bc run in the pH range 6-7.ri. The pH optimum of the Drosophila enzyme is pH 7.1(2). For mammalian L-amino acid decarboxylase, the pH optimum is over pH 8, but both dopa and dopamine are highly unstable at basic pH, so when dopa is used as a substrate, the reaction is generally run in the pH range appropriate for this assay procedure (81. For homogenates prepared from mammalian sources, PTU should be replaced by isonicotinic acid 2-isopropylhydrazide iiproniazid) , I .O mM, or some other monoamine oxidase inhibitor (4). ACKNOWLEDGMENTS This work was supported by a XIH Research Grant 1 ROl GM1924241 to TRFW. AFS was supported by the PHS Training Grant 5-TOl-GM01450 and .JCS by the NSF Grant GU 1531. The authors wish to thank Dr. Reginald Garrett for many helpful discussions during the course of this work. REFERENCES 1. SEKERIS, 2. LUNAN,

C. E. AND KARLSON, K. D. AND MITCHELL,

3. MCCAMAN,

W.,

M.

242. 4. STREFFER.

C.

LOWRY.

8. CHRISTENSON, Biophys.

Btichim.

265. (1963) AND J.

141,

R. Biophys.

ROSEBROWH.

Hoppe

CLARK,

G., 356.

J..

IV.

W.

DAIRMAN.

Pharmacol. Rev. 18, 89. K. (1969) AI& Biochem. Biophys. 132, E., AND LEES, G. J. (1972) Anal. Biochem.

(1966)

H.

MCCAMAN.

(1967)

0. H.. Che,m. 193, 6. SEKERIS. C. E. 7. SCHDTT. H. F.

5

P.

Actn 139, 193. FARR, A. L, AND RANDALL.

Seyler 2. Phy&l. Chews. G. (1952) 1. Biol. Chem. W., AND UDENFRIEND,

R.

J. (1951)

J. Rio{.

332, 70. 198, 449. S.

(1970)

Arch.

&o&em.

ALLEN F. SHERALD JOHN C. SPARROW THEODORE R. F. WRIGHT University

of

Virginia

L%301 May

Received

Virginia,

Chnrlottesville,

93, 1973;

accepted

July

$3, 1.w~

450. 45,