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CHROMOGENIC

SUBSTRATES

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75

extracted twice with chloroform. Methanol is added to break the emulsion formed during the extraction. The crude product is purified by flash chromatography on a silica gel column using chloroform/methanol/water (65 : 25 : 1) as the eluting solvents. This thio-PE is obtained in 91% yield (127 mg, 0.23 mmol) as a white powder. Acknowledgments Supportfor this workwas providedby the NationalInstitutesof Health(GM-20,501)and National ScienceFoundation(DMB 89-17392).

[6] C h r o m o g e n i c S u b s t r a t e s a n d A s s a y o f P h o s p h o l i p a s e s A 2

By WONHWA CHO and FERENC J. KI~ZDY Introduction The high specificity of phospholipases A2 (PLA2) toward aggregated substrates renders the assay of these enzymes a particularly challenging analytical task. Sensitive assays require the use of aggregated substrates, such as micelles, mixed micelles, single bilayer vesicles, or monomolecular layers, where the activity of the enzyme depends critically on the physical state and the exact composition of the nonaqueous phase. For reproducible assays with such heterogeneous systems the experimental conditions must be strictly controlled since the presence of minor lipid impurities, the accumulation of small amounts of reaction products in the early phases of the reaction, or even slight changes in temperature or buffer composition may and often do elicit large changes in the rate of the enzymatic reaction. Homogeneous reaction kinetics, which are conducive to readily reproducible kinetics, could only be achieved with short-chain lecithins, such as dibutyryllecithin, toward which phospholipases A2 display a rather low specificity, and the assays based on these substrates are not sensitive enough for most purposes. The sensitivity of the spectrophotometric assay described herein is between the two extremes, and the method, by virtue of its simplicity, is readily adaptable to a variety of purposes ranging from routine analyses to investigations of mechanistic details of the enzymatic reaction. A variety of acyloxynitrobenzoic acids have been shown to be hydrolyzed by snake venom and pancreatic phospholipases A 2 . The reaction is Ca 2+-dependent, and the pH dependency of the catalysis is also consistent with a reaction mechanism making full use of the catalytic apparatus METHODS IN ENZYMOLOGY, VOL. 197

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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TABLE I SPECIFICITY OF PHOSPHOLIPASES A2 TOWARD COMPOUNDS 1 AND 2 a

k.t/ Km (X 10-3 M-I sec-1) Enzyme

Compound 1

Compound 2

Agkistrodon piscioorus piscivorus D-492 Crotalus atrox PLA23 Porcine pancreatic PLA2

2.91 - 0.05 7.01 -+ 0.28 0.34 -+ 0.03

18.9 - 0.3 46.4 - 1.3 2.3 - 0.2

o Assays were conducted at 37° in 10 mM Tris-HC1 buffer, pH 8.00, 0.1 M NaCI, 10 mM CaCI2, 1.6% acetonitrile.

observed in the hydrolysis of lecithins. 1 All phospholipases A 2 w e have tried reacted well with 4-nitro-3-(octanoyloxy)benzoic acid, but the specificity toward acyloxynitrobenzoic acids with the carboxylate in the meta or para position with respect to the scissile bond vary from enzyme to enzyme. Thus, for a novel enzyme some experimentation might be required in order to achieve optimal assay conditions. In Table I 2,3 the specificity constants, kcat/K m , are given for 4-nitro-3-(octanoyloxy)benzoic acid (1) and 3-nitro-4-(octanoyloxy)benzoic acid (2) with three representative phospholipases A 2. We have observed that at substrate concentrations in excess of 100 /.~M monomeric phospholipases A 2 a r e slowly and irreversibly activated during the hydrolysis of 4-nitro-3-(octanoyloxy)benzoate.4 Again, for a novel enzyme it should be ascertained experimentally that the reaction kinetics obey simple pseudo-first-order kinetics and that no irreversible activation complicates the assay. At So < 1 x 10 -4 M the condition So < Km is satisfied, and with S O>>E o the enzymatic reaction is first-order with respect to the substrate, with k~xp = kcat Eo/Km. The pH dependence of k~at/Km is bell-shaped, with a plateau at pH 7 to 8. Thus, the exact pH of the reaction mixture is not critical in this pH interval. For compound I the pKa of the phenol product is 7. l, and the apparent molar absorptivity of the product is slightly pH-dependent around pH 8; however, the calculation of the experimental first-order rate constant does not involve this quantity. 1 W. Cho, M. A. Markowitz, and F. J. K6zdy, J. Am. Chem. Soc. 110, 5166 (1988). 2 j. M. Maraganore, G. Merutka, W. Cho, W. Welch, F. J. K6zdy, and R. L. Heinrikson, J. Biol. Chem. 259, 13839 (1984). 3 W. Hachimori, A. M. Wells, and D. J. Hanahan, Biochemistry 10, 4084 (1971). 4 W. Cho, A. G. Tomasselli, R. L. Heinrikson, and F. J. K6zdy, J. Biol. Chem. 263, 11237 (1988).

CHROMOGENIC SUBSTRATES AND ASSAY OF PLA2

[6]

77

Assay Method Substrates. Acyloxynitrobenzoic acids are readily synthesized from commercially available acyl chlorides and hydroxynitrobenzoic acids. ~ The method is illustrated with the synthesis of 4-nitro-3-(octanoyloxy)benzoic acid. 3-Hydroxy-4-nitrobenzoic acid (183 mg, 1.0 mmol) and N , N diisopropylethylamine (259 mg, 2.0 mmol) are dissolved in 2 ml of dry tetrahydrofuran and added dropwise to a stirred solution of octanoyl chloride (163 rag, 1.0 mmol) in 10 ml of dry tetrahydrofuran at 0°. The mixture is slowly warmed to room temperature and stirred overnight. The precipitate is filtered and washed with three 10-ml aliquots of ether. The combined filtrates are concentrated in vacuo and dissolved in 50 ml of ether/hexane, 2 : 1 (v/v). The organic layer is washed with two 50-ml aliquots of 10 mM HCI and with two 50-ml aliquots of water, dried with anhydrous NaeSO4, and evaporated in vacuo. The residue is fractiovated by flash chromatography using Merck kieselgel 60 and hexane-ether-acetic acid, 20:10:1 (v/v/v), and the eluate is monitored by TLC [Rf 0.44 on a silica gel plate with hexane-ether-acetic acid, 20: 10:1 (v/v/v)]. The appropriate fractions are pooled and evaporated in vacuo. The product is recrystallized from hexane-ethyl acetate, 10:1 (v/v), mp 142°-143 °. The purity of the product is readily ascertained spectrophotometrically after alkaline hydrolysis of a weighed sample of the ester; for the phenolate ion of 3-hydroxy4-nitrobenzoic acid the e425 value is 4990 M-~ sec-1 and for that of 4hydroxy-3-nitrobenzoic acid the e,110 v a l u e is 4550 M - l s e c - 1 . Reagents

Assay buffer: Tris-HCl, I0 mM, pH 8.00, containing I0 mM CaCI2 and 0.1 M NaCI Substrate stock solution: Compound 1, 3.1 mM (0.96 mg/ml), in dry acetonitrile Enzyme stock solution: Ideally in the range of 1 x 10 -5 to 1 x 10 -4 M, (0.1-2 mg/ml), dissolved in the assay buffer; however, any buffer which is mixed in a 1 : 60 ratio with the assay buffer does not appreciably change the pH of the latter. The enzyme is dissolved directly in the assay buffer and used without dilution as the assay medium. Enzymes of concentrations as low as 1 /~g/ml can be assayed. A s s a y Procedure. Three milliliters of the assay buffer is equilibrated at 37° in a thermostatted 1 cm path length cuvette in the sample compartment of the spectrophotometer. A 50-/zl aliquot of the substrate stock solution is added from the flattened tip of a glass rod, and the solution is vigorously stirred. In monitoring the reaction at 410 nm a very slight

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[6]

buffer-catalyzed hydrolysis might be observed at this point, depending on the exact value of the pH. The enzymatic reaction is initiated by the addition of 50 ~1 of enzyme stock solution, and the increase in absorbance at 410 nm is recorded. Complete hydrolysis should produce a total change of about 0.2 absorbance units (AU). Ideally, the reaction should be allowed to continue for at least four half-lives. Analysis of Results. The experimental first-order rate constant can be calculated from the absorbance versus time data using any of the commercially available nonlinear least-squares fit programs. The program M U L T I : written in BASIC, has been found convenient to use with data collected in a digital form by an on-line microcomputer. If Ai and Af are the absorbance readings at the beginning and the end of the enzymatic reaction, respectively, then the absorbance A at time t is given by A = A i + ( A f - Ai)(1 - e-kexp t) The first few, usually noisy, points are discarded, and a minimum of 20

A,t data pairs are analyzed for one experiment. The parameters to be optimized for A i , A f , and kexp. If pure enzyme is available, the method is first calibrated with a known enzyme concentration, and the value of kcat/Km is calculated from kexp. Then, with this value, any further assay yields the enzyme concentration in the reaction mixture by E o -- k¢xp/(kcat/ K m) and the concentration of the enzyme stock solution by E s = E o × 0.05/3.10. Alternatively, kex p c a n be determined graphically from the slope of a of log(Af - A) versus time. With very low enzyme concentrations, the initial rate is calculated from the initial rate of absorbance change, Va = AA/At, corrected for the nonenzymatic, buffer-catalyzed rate. Under these conditions, the exact pH and the exact substrate concentration, So, must be known in order to convert the data to a rate constant according to the equation kcatE° - W a ( 1 "[-_10(7"I0-pH)~

4990 S O

/

Scope and Limitations. The assay described in this chapter provides a rapid, accurate, and convenient method for measuring phospholipase A 2 concentrations as low as 1 × 10 -8 M. Since nitrophenyl esters are susceptible to hydrolysis by a variety of esterases and peptidases, it is recommended that, when working with crude extracts and partially purified enzymes, appropriate control experiments be carried out, where the 10 mM C 2+ of the assay buffer is replaced by 0.1 mM EDTA, and that the 5 K. Yamaoka, Y. Tanigawara, T. Nakagawa, and T. Uno, J. Pharmacobio-Dyn. 4, 879 (1981).

[7]

ASSAYS FOR PLASMALOGEN-HYDROLYZING

ENZYMES

79

kexp of this control be subtracted from the value of kexp of the enzymatic reaction. Finally, the adherence of the progress curve of the reaction to first-order kinetics should be verified for each novel e n z y m e in order to ascertain that S O~ Km and that no irreversible activation occurs under the chosen experimental conditions.

[7] C o u p l e d

Enzyme Assays for Phospholipase with Plasmalogen Substrates

Activities

B y M. S. JURKOWITZ-ALEXANDER, Y. HIRASHIMA, and

L. A. HORROCKS Introduction Ether-linked glycerophospholipids of the plasmalogen type (l-alk-l'enyl-sn-2-acylglycerophosphocholine or -ethanolamine) are present in virtually all mammalian cell membranes. ~ Among the enzymes known to function in plasmalogen metabolism in mammalian tissues are the hydrolytic e n z y m e s lysoplasmalogenase 2-5 and plasmalogen-selective phospholipase A 2 .6,7 Phospholipase A 2 (PLA2) catalyzes the hydrolysis of plasmalogen at the sn-2 position, producing free fatty acid and lysoplasmalogen [Eq. (1)]. Plasmalogen + H20 PLA2~lysoplasmalogen + fatty acid

(1)

Lysoplasmalogenase catalyzes the hydrolysis of the alkenyl ether bond of lysoplasmalogen at the sn-1 position, forming free aldehyde and glycerophosphocholine [Eq. (2)]. Both of these enzymes have been purified recently. Plasmalogen-selective phospholipase A2 was purified from the cytosol of sheep platelets s and canine myocardium, 6 and lysoplasmalogenase was purified from liver microsomes. 5,9 i L. A. Horrocks, in "Ether Lipids: Chemistry and Biology" (F. Snyder, ed.), p. 177. Academic Press, New York, 1972. z H. R. Warner and W. E. M. Lands, J. Biol. Chem. 236, 2404 (1961). 3j. Gunawan and H. Debuch, J. Neurochem. 39, 693 (1982). 4 j. Gunawan and H. Debuch, Hoppe-Seyler's Z. Physiol. Chem. 362, 445 (1981). 5 M. Jurkowitz-Alexander, H. Ebata, J. S. Mills, E. J. Murphy, and L. A. Horrocks, Biochim. Biophys. Acta 1002, 203 (1989). 6 S. L. Hazen, R. J. Stuppy, and R. W. Gross, J. Biol. Chem. 265, 10622(1989). 7 R. A. Wolf and R. W. Gross, J. Biol. Chem. 260, 7295 (1985). s L. A. Loeb and R. W. Gross, J. Biol. Chem. 260, 10467(1986). 9 M. S. Jurkowitz-Alexander and L. A. Horrocks, this volume [46].

METHODS IN ENZYMOLOGY, VOL. 197

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Chromogenic substrates and assay of phospholipases A2.

[6] CHROMOGENIC SUBSTRATES AND ASSAY OF PLA 2 75 extracted twice with chloroform. Methanol is added to break the emulsion formed during the extra...
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