PROSTAGLANDINS

AN IMPROVED SPECTROPHOTOMETRIC ASSAY FOR PROSTAGLARDIN SYNTBETASE BASED ON THE

OXIDATION OF EPINEPRRINE

Ming Sun

Departments of Ophthalmology and Biochemistry University of Rochester School of Medicine and Dentistry

Rochester, New York 14642

ABSTRACT

The spectrophotometric assay method for prostaglandin synthetase from Takeguchi and Sih (1) was improved by monitoring the absorption change at 320 nm instead of at 480 nm during the enzymatic synthesis. The measurement at 320 nm is more sensitive and more consistent than the A4so measurement. The improvement resulting from the measurement at 320 nm is attributed to a combination of factors, including a higher extinction coefficient, a more inclusive measurement of other epinephrine oxidative product(s) and lower interference due to the product of the further oxidation of adrenochrome. The validity of this spectrophotometric method was also verified in this report.

INTRODUCTION

Epinephrine is one of the most commonly used positive effecters in prostaglandin synthesis (1, 2, 3). In the enzyme catalyzed con- versions of arachidonic acid and certain other unsaturated fatty acid to their corresponding prostaglandins, L-epinephrine is oxidized to pink-colored adrenochrome which absorbs at 480 nm. Takeguchi and Sih have since developed a simple and rapid assay method for prosta- glandin synthetase based on adrenochrome formation (1).

Although the spectrophotometric measurement based on Adso adrenochrome absorption is convenient, the method is limited by its lack of sensitivity and stability, partially because A4so corresponds to a weak absorption band and adrenochrome is unstable, particularly at alkaline pH conditions. It is because of such drawbacks, that, even though the simplicity of the method suggested easy adaptation, the method has not yet been generally adopted.

Analysis of the spectral change during the course of oxidative conversion of epinephrine to adrenochrome and other product(s) reveals an alternate absorption region suitable for the spectropho- tometric assay of prostaglandin synthetase activity. By measuring the increase of the absorption at 320 nm instead of at 480 MI during the course of the enzymatic reaction, the sensitivity of the assay is enhanced at least 5 times over the A4go measurement. Also, the results obtained from the absorption measurement made at 320 nm were found very consistent with respect to the enzyme concentration, time course, substrate concentration and pH changes.

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MATERIALS AND METHODS

Arachidonic acid (all cis-5,8,11,14-eicosatetraenoic acid); 8,11,14-eicosatrienoic acid; docosahexaenoic acid; 11,14-eicosadi- enoic acid; linolenic acid, linoleic acid; oleic acid and l-epineph- rine were purchased from Sigma Chemical Co. Stock solutions of 1 mM concentration of these fatty acids were made in ethanol. Epinephrine solutions at concentrations of 20 or 50 mM were made in 0.1 N HCl and aliquot stored in the freezer. Bovine or sheep seminal vesicular microsomal fractions were used as the enzyme sources. The enzyme stock solution was prepared according to Anggard et al (4)) aliquot stored at -50°C until use. The assay me_dium consists of 0-20~1 of the diluted microsomal enzyme, 0.5-2 x 10 4 M epinephrine in a total volume of 2 ml in 50 mM tris-HCl or sodium carbonate buffer at desired pH. To initiate the reaction, the substrate-arachidonic acid or other fatty acids, at a concentration of 2-5 x lo-‘M was added to the assay mixture. The initial absorption change at 480. nm or at 320 nm were recorded on a Cary 14 Recording Spectrophotometer. Matched controls at same pH medium were run in the absence of the enzyme or in the presence of the same amount of the denature enzyme (by boiling for 3-5 minutes). Activity of PGS was expressed as AOD/min mg-pro- tein.

Radioisotope assay by thin layer chromatography (TLC) was essen- tially following the established procedure (5) with slight modifica- tion. The reaction mixture contained 10 pmol of unlabeled and labeled arachidonic acid (5,6,8,9,11,12,14,15(n)3H labeled 135Ci/mmol or 1-14C labeled, 68 mCi/mmol; from Amersham Corp.); 1 mM of adrena- line and 5-10 mg of BSVM or SSVM enzyme in a total volume of 2 ml 0.1 M Tris-Hcl buffer pH 8.8. Aliquots 0.2 ml were taken out after the reaction mixture had stood at 20°C for 0, 1, 2, 3, 4, 6, and 8 min- utes. The reaction was stopped by acidification with .l ml of 2 N HCl or by pipetting the solution to a preheated test tube at 80°C and heated for an additional 3 minutes. The reaction mixtures were extracted twice with 3 ml portions of ethyl acetate. then dried under a flow of air in a ventilated residue was dissolved in a few drops of acetone. and different PGs were added as internal markers. spotted on a Silica gel G TLC plate. Organic acetate/acetic acid/2,2,4_trimethyl pentanelwater vol.) mixture was used as the developing solvent.

The extract was hood. Remaining Arachidonic acid

The mixture was layer of ethyl (11/2/5/10; by

Four 12 positive bands corresponding to arachidonic acid, PGE2, PGFz,, and PGD2 were marked on each developed TLC plate. These bands were then scraped off the plates and collected in glass scintillation vials; 0.5 ml of ethanol and 5 ml liquid scintillation cocktail (Research Products International Corp.) were added to each vial. The radioactivity was counted in a Beckman LS 233 Liquid Scintillation Systems.

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RESULTS

Epinephrine readily undergoes autoxidation at alkaline pH. Prostaglandin synthetase enhances the rate of this process in the presence of substrate, arachidonic acid or 8, 11, 14-eicosatrienoic acid. Fig. 1 shows the epinephrine spectral change due to the autox- idation. In sodium carbonate buffer at pH 9.7 the formation of pink-colored adrenochrome reached its maximum concentration at a time interval less than 20 minutes. The spectra at the visible region in Fig. 1 recorded the formation and further conversion of adrenochrome to a more stable product, as marked by the enhancement at first, and then gradual decrease of the absorptivity at 480 run. The absorption change at near-W region is in contrast with that at visible region, in that the former is increasing with time and the enhancement is much greater than the latter.

240 280 320 360 400 440 480 520 560

WAVELENGTH, NH.

Fig. 1 Epinephrine (2 X 10V4 M) absorption spectral change in 50 mM sodium carbonate buffer pH 9.7 during the course of autoxi- dation. Epinephrine was added to the buffer medium at the initial time 0: individual spectrum was scanned at indicated time.

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Figure 2 illustrates the time courses of prostaglandin synthetase activities at pH 8.7 and 9.7 in the presence of 10 PM arachidonic

.,d- A-A

v OO

I I I , I I

1 2 3 4 5 6

TIME, MIN.

Fig. 2 Time courses of prostaglandin synthetase activity, expressed as the change of the absorptivity of epinephrine: at 320 run, pH 9.7 -O-O-; at 320 nm, pH 8.7 -O-O-; at 480 nm, pH 9.7 -A-A- ; and at 480 MI, pH 8.7 -A-A-; Reaction medium contains 0.5 mg BSVM protein, 1 mM epinephrine and 10 PM arachidonic acid in 50 mM Tris-HCl pH 8.7 or in 50 mM sodium carbonate buffer pH 9.7.

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acid. The Y-axis, AA, represents the difference of the absorption between the enzymatic reaction and nonenzymatic autoxidation of epinephrine. It is noticeable that 1) the change of the absorption at 320 nm is much higher than that measured at 480 run; 2) the enzyme is more active at pH 9.7 than at pH 8.7; 3) although in all cases the time courses are not linear, the deviation from the 480 nm measure- ment is larger than that measured at 320 run.

Adrenochrome is not the stable final oxidative product in the autoxidation of epinephrine. It undergoes further oxidation. The conversion is marked by the disappearance of Adsa peak and the in- crease of Aaoo peak. An isosbestic point is located at 418 nm (6, 7). Fig. 3 shows the time course of adrenochrome conversion to a more stable product. Two solutions both contained equal amount of adrenochrome and enzyme, with the lower one in the presence of 10 FM arachidonic acid. It is clear that the rate of the decrease of adrenochrome absorption at 480 nm is faster in the presence of the enzyme substrate than that in its absence. Thus, adrenochrome may be used also, as a cofactor during the enzymatic synthesis of prosta- glandins. Fig. 4 shows the profiles of the enzyme concentration- dependent activity measured at 320 run and 480 nm in 50 mM tris buffer pH 8.7. Both AAszo and hAdso were found to increase with the in- crease of the enzyme concentrations. However , at an interval of 20 minutes, while hA4ao has registered no further enzyme activity as compared with 10 minutes incubation, AAs has almost doubled. The correlation is better at the lower enzyme concentration range as indicated in this figure.

Table I compares the rates of epinephrine conversion in the presence of different unsaturated fatty acids as the enzyme sub- strate. BSVM prostaglandin synthetase shows to have certain sub- strate preference, although there are considerable reactivity with several fatty acids other than arachiodonic acid. Among these fatty acids, 8, 11, 14-eicosatrienoic acid (20:3) is the next most active fatty acid (72% as active as arachidonic acid). Linolenic acid (18:3), the precursor or arachidonic acid, only attained about 14% of the activity. In general, the measurements made at 320 nm were parallel to those measured at 480 nm, with the former being more sensitive.

Substrate concentration dependent studies indicate decline of the enzyme activity at the arachidonic concentration greater than 10m4M (Fig. 5). Measurements made at 320 nm and at 480 run were found to be consistent with each other.

To verify the epinephrine assay, prostaglandin synthetase was also assayed using the TLC and radioisotope method as stated in the method section. Good separation between arachidonic acid and dif- ferent PGs was achieved in the solvent system employed. The authen- tic internal markers were located at Rfs 0.94, 0.40, 0.29, and 0.18 for arachidonic acid, PGDp, PGEz, and PGFz,, respectively. It was found that the rates of arachidonic acid consumption and the total PG

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production (PGE2+PGF2 +PGD2 + unmarked bands) over a period from 0 to 8 min. were consist nt with the AAaso time course (Fig. 2). 8 The profiles were similar to that reported by Nugteren and Hazelhof (5) therefore not presented here.

0,3

s G?

0.2

0.1 !

0- 0 1 2 3 4 5 6 7

__-" TIME (MINUTES)

Fig. 3 Conversion of adrenochrome to its oxidative product, measured by the decrease of the absorptivity at 480 nm in 50 mM tris- HCl pH 8.7 buffer, in the presence of 0.5 mg BSVM and 10 pM of arachidonic acid ( -A-A- ); and in the absence of ara- chidonic acid ( -O-O- ).

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Fig. 4

0.8

0.6

4 0.4 a

0.2

0

0 1,o 2.0 3.0

CONCENTRATION, FIG/ML

Dependence of enzyme concentration of epinephrine conversion to its oxidative products: measured at 480 nm, 10 minutes after the initial addition of arachidonic acid -A-A-A-; at 5 minutes, -o-o-; and at 320 run, 5 minutes after the addition of the substrate -- o--o---; 10 minutes after the addition of the substrate -- A--A--. Arachidonic acid concentration was 4xr@ M, epinephrine concentration was at 0.5 mM, in 50 mM Tris-HCl pH 8.1 buffer media.

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TABLE I. BSVM Catalyzed Fatty Acids Conversion To Their Corresponding Products Using Epinephrine as Cofactor.

Substrates Aszo/min. mg. Adso/min. mg. Arachidonic Acid 0.107 0.072 8,11,14-eicosatrienoic acid 0.150 0.057 11,14-eicosadienoic acid 0.087 0.036 linoleic acid 0.054 0.027 linolenic acid 0.030 0.006 oleic acid 0.00 0.00 docosahexenoic acid 0.024 0.005

The assay medium contains 5 X 10m4 M epinephrine and 10m4 M fatty acid as substrate in a total volume of 1 ml in 50 mM Tris-HCl buffer pH 8.7.

0,5 -

, 1 I I

0 4 8 12 16 20 24

CARAI M x10-5

Fig. 5 Dependence on arachidonic acid concentration of prostaglandin synthetase activities, expressed by the enhancement of the absorptivLty at 320 nm (-o-) and at 480 MI (-A-) 5 minutes after the addition of the substrate arachidonic acid. The reaction medium contains 1 mM epinephrine; 0.5 mg of BSVM protein in a total of 1 ml 50 mM Tris-HCl buffer pH 8.7.

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DISCUSSION

Various aromatic compounds, such as hydroquinone, catechol- amines, tryptophan and serotonin, were known to be able to serve as cofactors in prostaglandin biosynthesis (8, 9). Recently, it has been demonstrated that these compounds may stimulate the conversion of arachidonic acid to PGG and to PGH (3,lO). In the process of the transformation, the cofactor, such as epinephrine could be conceiv- ably oxidized to adrenochrome, accompanied by the change of the absorption spectrum. The cofactors are therefore mainly to serve to provide a reducing equivalent during the enzymatic synthesis of prostaglandins. To fulfill such a function, there is probably a requirement of high oxidation-reduction protential as well as the specific structural requirement for these compounds. We may expect, therefore, that the higher the oxidation-reduction potential, the better the compound will be as the enzyme cofactor (e.g. epinephrine, E”=.808; p-aminophenol, EOz0.779). The exceptions of this generaliz- ation should reflect the structural requirement and the steric specificity of the enzyme complex.

Adrenochrome is not the only and the final product from the oxidative conversion of epinephrine (7) - Comparison between the spectra of adrenochrome and epinephrine product(s) indicates that the ratio between AszofA4ao of epinephrine product is higher than the ratio obtained from the adrenochrome absorption. The increase of the absorptivity in the near-W region could be attributed to three different absorbing species besides adrenochrome. These are the intermediates formed during the oxidative conversion of epinephrine to adrenochrome, oxidative product(s) other than adrenochrome and the product from the further oxidation of adrenochrome. The formation of these unidentified compounds as well as adrenochrome are all subject to superoxide dismutase inhibition (7). They result from the autox- idation of epinephrine, and presumably are produced during the en- zymatic synthesis of prostaglandins when epinephrine is present as the cofactor. Thus, the increase of sensitivity and consistency of A320 measurement could be attributed to a more inclusive measurement of the oxidative conversion of epinephrine to its oxidative products.

Both the enzyme coupled epinephrine oxidation and its autoxi- dation are facilitated at alkaline pH (6,7). Prostaglandin synthe- tase shows a higher activity at pH 9.7 in 50 mM sodium carbonate buffer than at pH 8.7 in tris-buffer (Fig. 2). The time course studies, however, indicate a rapid decline of the absorptivity at 480 MI at pH 9.7, after a short initial period (1 min.) of increase. Our studies also show that the more active the enzyme fraction is, the shorter the time will be to reach to the A4ao maximum. It is diffi- cult, therefore, to assess the enzyme activity, because of such a short initial period, and it is unreliable to monitor the absorption change at time intervals after the initial period. We have shown that the disappearance of the adrenochrome absorption at 480 nm is

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largely due to the same oxidative process; and that adrenochrome is just as unstable as epinephrine; furthermore, both compounds can serve as cofactors in prostaglandin synthesis (Fig. 3).

Precaution must be exercised in assaying prostaglandin syn- thetase activity using this epinephrine oxidation procedure, since epinephrine is readily autoxidixable at alkaline pH, or when it is exposed to the near-W light. We also noted that in many enzyme q icrosomal preparations there were small amount of fatty acid con- taminations, included arachidonic acid and/or its precursors. Also frequently, there were many other types of oxygenases present in the crude tissue homogenate and microsomal preparation. These endogenous factors may also cause the rapid oxidation of epinephrine. Prosta- glandin synthetase activities could be differentiated from these non-specific oxidative enzymes actions by the addition of the sub- strate arachidonic acid and measuring the enhancement of epinephrine oxidation rate.

The validity of this enzyme assay method was therefore estab- lished from the facts that the activity, represented as AA320 is 1) enzyme concentration dependent (Fig. 4); 2) substrate concentration dependent (Fig. 5); 3) incubation time dependent (Fig. 2); 4) sub- strate specific (Table I), and finally, by comparison with the veri- f ied procedure. Although it is more sensitive, A330 measurements were closely matched with the measurements made at Adso.

In conclusion, we have demonstrated here that the spectropho- tometric measurement at A330 is more sensitive and consistent than that at A430 for assaying the activity of prostaglandin synthetase. This method is convenient and sensitive for general screening of prostaglandin synthetase activity and for rapid location of the active enzyme fractions in the preparative chromatography or in other purification procedures.

ACKNOWLEDGEMENTS

I am very grateful to Dr. S. Zigman and to Dr. H. l-l. Tai for their generous supply of some of the material and for their valuable comments and advice on this work. I also want to thank Dr. J. E. Pike of the Upjohn Company for the supply of prostaglandins.

This work was supported by The Public Health Service Grant # EY-00459 .and by a medical research grant from Rochester Eye and Human Parts Bank.

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1)

708

Takeguchi, C. and C. J. Sih. A Rapid Spectrophotometric Assay for Prostaglandin Synthetase: Application to The Study of Non- Steroidal Anti-inflammatory Agents. Prostaglandins 2:169. 1972.

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2) Wlodawer, P. and B. Samuelsson. On The Organization and Mechanism of Prostaglandin Synthetase. J. Biol. Chem. 248:4673. 1973.

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3) Tai, H. H. Mechanism of Prostaglandin Biosynthesis in Rabbit Kidney Medulla, A Rate Limiting Step and The Differential Stimulatory Actions of L-Adrenaline and Glutathione. J. Biochem. 160:577. 1976. -

4) Anggard, E., F. M. Matschinsky, and B. Samuelsson. Prosta- glandins: Enzymatic Analysis. Science 163: 479-480. 1969. -

5) Nugteren, D. H. and E. Hazelhof. Isolation and Properties of Intermediates in Prostaglandin Biosynthesis. Biochim. Biophy. Acta 326: 448. 1973. -

6) Misra, H. P. and I. Fridovich. The Role of Superoxide Anion in the Autoxidation of Epinephrine and a Simple Assay for Super- oxide Dismutase. J. Biol. Chem. 247: 3170. 1972. -

7) Sun, M. and S. Zigman. Improved Spectrophotometric Assay for Superoxide Dismutase Based on Eninenhrine Autoxidation. Anal. B&hem. 89:000, 1978. -

8) Sih, C. J., C. Takeguchi, and P. J. Foss. Mechanism of glandin Biosynthesis III. Catecholamine and Serotonin enzymes. J. Amer. Chem. Sot. 92: 6670. 1970. -

9) Takeguchi, C., E. Kohno, and C. J. Sih. Mechanism of

Prosta- as Co-

Prosta- glandin Biosynthesis I. Characterization and Assay of Bovine Prostaglandin Synthetase. Biochem. 10:2372. 1971. -

10) Miyamoto, T., N. Ogino, S. Yamamoto, and 0. J. Nayaishi. Puri- fication of Prostaglandin Endoperoxide Synthetase from Bovine Vesicular Gland Microsomes. J. Biol. Chem. 251: 2629. 1976. -

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