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ANALYTICAL BIOCHEMISTRY ARTICLE NO. 241, 167–172 (1996) 0394 Continuous Assay for Acid Phosphatase Using Phenyl Phosphate Ewa Luchter-Wasylewska Institute of Medical Biochemistry, Jagiellonian University, Collegium Medicum, Kopernika 7, 31-034 Krakow, Poland Received April 1, 1996 A continuous spectrophotometric assay for the determination of the initial rate of an acid phosphatasecatalyzed reaction in an acidic environment, using phenyl phosphate as a substrate, is presented. The method is
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  ANALYTICAL BIOCHEMISTRY 241, 167–172 (1996) ARTICLE NO . 0394 Continuous Assay for Acid Phosphatase UsingPhenyl Phosphate Ewa Luchter-Wasylewska  Institute of Medical Biochemistry, Jagiellonian University, Collegium Medicum,Kopernika 7, 31-034 Krakow, Poland  Received April 1, 1996 calteau reagent (4) or alkaline aminoantipyrine and A continuous spectrophotometric assay for the de- ferricyanide in an Emerson reaction (5, 6). Phenol can termination of the initial rate of an acid phosphatase- be spectrophotometrically measured at 287 nm as the catalyzed reaction in an acidic environment, using phenolate ion after alkalization of the sample (7), or it phenyl phosphate as a substrate, is presented. The can be detected electrochemically (8). The determina- method is based on the continuous determination of  tion of phosphate released from phenyl phosphate in phenol, a product of the enzymatic hydrolysis, by the the phosphatase-catalyzed hydrolysis using many col- kinetic measurement of its absorbance. The method orimetric methods is performed (9–12). allows for the direct estimation of acid phosphatase For the correct estimation of kinetic constants of  activity in an acidic solution. This is possible without the enzyme-catalyzed reaction, only continuous one- interrupting the reaction by alkalization or precipita- step methods should be employed. In reverse mi- tion required for commonly used end-point colorimet- celles, the procedures based on alkalization or precip- ric detection procedures for phosphate or phenol, and itation should not be used because of the turbidity of  without the use of any coupled assays. The method has the sample. been developed for acid phosphatase activity determi- Reverse micelles (water-in-oil microemulsions) are nation in an aqueous solution and in sodium bis(2- formed by water microdroplets surrounded by a ethylhexyl)sulfosuccinate (AOT)–isooctane –water re- monolayer of surfactant molecules arranged with verse micelles in a broad pH range (pH 3.8 to 8.8). The their polar heads toward the water pool and their proposed procedure has been used for the determina- hydrophobic tails in contact with the apolar solvent. tion of kinetic constants (  K   m and k cat ) for human pros- The dimension of the water cavity depends on the tatic acid phosphatase in aqueous solutions, and in molar ratio of water and surfactant, abbreviated as AOT–isooctane –water reverse micelles, at pH 3.8, 4.5,and 5.7.  1996 Academic Press, Inc. w 0 , and is comparable to the diameter of a proteinmolecule. Proteins hosted in reverse micelles possesstheir biological activity. Reverse micelles resemblethe microenvironment that enzymes find in the celland are simple but efficient models for studying bio-Acid phosphatases (EC, the enzymes thatlogical molecules in the membrane-like environment.nonspecifically hydrolyze low and high molecular phos-Macroscopic homogeneity and transparency allow forphomonoesters in an acidic environment, are widelythe use of spectroscopic techniques for catalytic anddistributed in mammalian body fluids and tissues andstructural studies of macromolecules entrapped inin plants and microorganisms (1). Most methods forreverse micelles. Sodium bis(2-ethylhexyl)sulfosucci-acid phosphatase activity measurements are discontin-nate (AOT) 1 –isooctane–water reverse micelles areuous. They are based on the colorimetric determinationthe best-characterized reverse micelles (13–15).of phosphate or phenol/alcohol, products released in theHuman prostatic acid phosphatase (PAP) catalyzingenzyme-catalyzed reactions (1–3).the hydrolysis of low and high molecular phosphoestersPhenyl phosphate has been used as the acid phospha-tase substrate for about 50 years (1–3). Measurementsof phenol produced through enzymatic hydrolysis are 1 Abbreviations used: AOT, sodium bis(2-ethylhexyl)sulfosuccinate performed colorimetrically using many end-point (aerosol OT); w 0 , molar ratio of water to detergent ( w 0 Å [H 2 O]/ [AOT]); PAP, human prostatic acid phosphatase. methods; for example, these which utilize Folin –Cio- 167 0003-2697/96 $18.00Copyright  1996 by Academic Press, Inc.All rights of reproduction in any form reserved.  EWA LUCHTER-WASYLEWSKA 168is a glycoprotein composed of two subunits (16–21). ous-buffered stock solution of either enzyme or sub-strate to 1 ml of 0.1 M AOT solution in isooctane andSince Gutman’s report it has been a well-known diag-nostic marker of prostatic cancer (1–3, 16, 17, 22). The vortexed. For a particular w 0 value, the required aque-ous volume injected was calculated as described else-physiological role and the natural substrate of PAPhave remained unknown to date (16, 17). Reverse mi- where (24).celles may form the microenvironment for the investi- Continuous enzyme assay in aqueous solution. Thegation of enzymatic and structural properties of pros-incubation mixture contained phenyl phosphate solu-tatic phosphatase and may allow the employment of tion of the appropriate concentration in 0.95 ml of oneapolar or amphipatic substrates.of 50 m M buffers. The reaction was started by addingTo study the catalytic properties of human prostatic0.05 ml of phosphatase solution (18.5 1 10 0 6 M concen-acid phosphatase included in reverse micelles, thetrated enzyme stock solution, in 10 m M Tris–HClpreparation of a convenient method for continuous ki-buffer, pH 7.4, containing 100 m M NaCl, diluted to thenetic recording of acid phosphatase activity in acidicrequired concentration using one of the 50 m M acetateconditions was necessary. Phenyl phosphate is hydro-buffers) and stirring. In the blank sample, the enzymelyzable by PAP entrapped in AOT–isooctane–water re-was omitted. The assay was conducted at 20  C. Theverse micelles. Because no data on the spectral proper-enzyme stock solution and substrate stock solutionties of both phenyl phosphate and phenol in reversewere kept on ice during the course of the experimentmicelles existed, the author had to establish them.and under the conditions employed the spontaneousA new continuous assay for acid phosphatase activitysubstrate hydrolysis can be neglected. Acid phospha-determination in an acidic environment (at pH 3.8 totase-catalyzed hydrolysis of phenyl phosphate in acidic8.8) using a well-known substrate, phenyl phosphate,solution was monitored continuously by measuring theis proposed. The method is based on the direct spectro-increase of phenol absorbance at the 278-nm wave-photometric determination of phenol, the product of thelength. The measurement can also be performed at theenzymatic hydrolysis, by the measurement of ab-wavelength range of 274 to 278 nm. Initial rates weresorbance at wavelength 274 nm (or between 274 anddetermined by the Gillford Spectrophotometer pro-278 nm) in aqueous environment and at a 278-nmgram.wavelength in AOT–isooctane–water reverse micelles. Continuous enzymatic hydrolysis of phenyl phos- phate in AOT –isooctane–water reverse micelles. En-zyme activity determination in reverse micelles was MATERIALS AND METHODS performed by combining 0.5 ml of the substrate micel-  Materials. Disodium salt of phenyl phosphate andlar solution with 0.5 ml of the enzyme micellar solutiondisodium salt of succinate acid bis(2-ethylhexyl)esterunder the same conditions (buffer, pH, w 0 ) and stirring(AOT) were purchased from Sigma Chemical Co. Isooc-using a Waring blender. The assay was conducted im-tane for uv spectroscopy, sodium acetate, acetic acid,mediately at 20  C by continuous monitoring of phenolimidazole, and Tris (tris(hydroxymethyl)aminometh-absorbance at 278 nm. The initial velocity was calcu-ane) come from Fluka. Phenol was produced by POCHlated by Gillford Spectrophotometer software. The con-(Poland) and redistilled. All other chemicals were of centrations in reverse micelles were expressed as over-the highest purity available and were used withoutall concentrations.further purification. Calibration curves. In water solution the standard  Enzyme preparation. Homogeneous human pros-curves for phenol were performed at the wavelengthtatic acid phosphatase was purified from human sem-range of 274 to 278 nm. In reverse micelles the stan-inal plasma according to the method of Van Ettendard curves were performed by measuring the phenoland Saini (23). The concentration of phosphatase wasabsorbance at 278 nm. The molar absorption coeffi-determined by the measurement of absorbance at 280cients were calculated by Gillford Spectrophotometernm (16).software. Spectrophotometricassays. Absorbance spectra and  Determination of kinetic constants for PAP in aque- velocity measurements were recorded at 20  C on Gill- ous solution. The values of  K  m and k  cat for each pHford UV–VIS Spectrophotometer Response.value were calculated from triplicate measurements of   Buffers. The following 50 m M buffers were used: v 0 for phenyl phosphate concentration in the range 0.05acetate, in pH range 3.8–5.7; imidazole–HCl, in pHto 10 m M (25). Enzyme concentration was 9.25 n M .range 6.0–7.0; Tris–HCl, in pH range 7.2–8.8. The  Determination of kinetic constants for PAP in AOT – solutions were prepared using deionized and distilled isooctane–water reverse micelles. For determinationwater.of  K  m and k  cat in AOT–isooctane–reverse micelles, theinitial velocity ( v 0 ) measurements in the micellar solu-  Reverse micellar solution. AOT reverse micelleswere formed by injection of microliter amounts of aque- tion (for each used w 0 and pH) were performed in tripli-  ACID PHOSPHATASE CONTINUOUS ASSAY 169 FIG. 2. Absorbance spectra of 0.36 m M phenol and of 0.36 m M FIG. 1. Absorbance spectra of 1 m M phenol and of 1 m M phenylphenyl phosphate in 50 m M acetate buffer, pH 4.5, entrapped in 0.1phosphate in 50 m M acetate buffer, pH 4.5: (—), phenol; (---), phenyl M AOT–isooctane–water reverse micelles, w 0 Å 20: (—), phenol;phosphate.(---), phenyl phosphate. cate. The substrate overall concentration ranged fromwater solution (nonlinearity was observed above 2 m M )0.05 to 3 m M and the enzyme overall concentrationand in reverse micelles in the studied phenol concentra-equalled 9 n M . The calculations were performed usingtion (until 0.4 m M ).program of Lutz et al. (25). Kinetic parameters of human prostatic acid phospha-tase in aqueous solution and in AOT –isooctane–water  RESULTS reverse micelles. Kinetic parameters for PAP in water  Absorbance spectra of phenyl phosphate and phenol. solution and in AOT–isooctane–water reverse mi-The typical ultraviolet absorbance spectra of phenylcelles, determined using phenyl phosphate as substratephosphate and phenol in acidic conditions (pH 4.5) inby the proposed continuous procedure, are shown inwater solution and in AOT–isooctane–water reverseTable 1. Figure 3 illustrates the catalytic activity at pHmicelles are shown in Figs. 1 and 2, respectively, as an3.8 of prostatic acid phosphatase entrapped in AOT–example. The absorbance spectra of phenyl phosphateisooctane–water reverse micelles. The reaction kinet-and phenol in water solution and in reverse micellesics in reverse micelles obeys the Michaelis–Mentendo not change in pH ranging from 3.8 to 8.8. In watersolution the wavelengths between 274 and 278 nm canbe used for direct kinetic measurement of phenol. The TABLE 1 highest sensitivity of the method is at the wavelength Kinetic Constants for Human Prostatic Acid Phosphatase 274 nm. In this paper the measurements of phenol in Using Phenyl Phosphate as the Substrate in 50 m M Acetate water solution were performed at 278 nm. In reverse Buffer, at 20  C micelles the 278-nm wavelength was chosen as themost convenient for the determination of phenol re- Water solutionpH K  m ( M ) k  cat (s 0 1 ) leased from phenyl phosphate by the action of acidphosphatase. 3.8 2.6 1 10 0 4 1200  Molar absorption coefficients for phenol in water solu- 4.5 2.7 1 10 0 4 1270 tion and in reverse micelles. The molar absorption co- 5.7 3.0 1 10 0 4 1390 efficients (pH 3.8 to 8.8) for phenol in aqueous solution Micellar solution (0.1 M AOT–isooctane–water, w o Å 20) are: 1090 M 0 1 cm 0 1 at 274 nm, 940 M 0 1 cm 0 1 at 276 nm, pH K  m,ov ( M ) k  cat (s 0 1 ) and 600 M 0 1 cm 0 1 at 278 nm. In AOT–isooctane–water 3.8 5.2 1 10 0 4 114 reverse micelles ( w 0 Å 10 to 30) the molar absorption 4.5 4.1 1 10 0 4 30 coefficient for phenol is 1500 M 0 1 cm 0 1 at 278 nm. The 5.7 2.1 1 10 0 4 25 calibration curves for phenol are linear until 2 m M in  EWA LUCHTER-WASYLEWSKA 170nol is performed by measurement of its absorbance at390 nm (31) and at 400 nm (32), as well as in the rangeof 340 to 390 nm (12). O -Carboxyphenol is measuredat 298 nm (33). Unfortunately these two methods arebased on the spectrophotometricestimation of the prod-uct at the wavelength where the substrate absorbs. Insuch a situation it is difficult to measure the concentra-tion of the product which increases during the courseof the reaction in the sample which also contains thesubstrate, the concentration of which decreases. Supe-riority of the phenyl phosphate method recommendedin this paper over those two methods lies especially inthe recording of phenol, the reaction product, at thewavelength where the substrate, phenyl phosphate,does not absorb.In reverse micelles two methods of acid phosphataseactivity determination have been developed to date.The first method using p -nitrophenyl phosphate as sub-strate in a continuous manner is not described in detail(26). The measurement of  p -nitrophenol is performed FIG. 3. Human prostate acid phosphatase-catalyzed hydrolysis of phenyl phosphate in 0.1 M AOT–isooctane–water reverse micelles at 320 nm in acidic micellar solution where the spectra at 20  C. The dependence of  k  cat on w 0 as obtained from initial velocity of substrate and product are not distinct enough. The measurements is plotted. second method using 1-naphthyl phosphate as sub-strate in a coupled reaction with Fast Red (27) shouldnot be applied because the precise estimation of kineticequation. Measurements were performed at least threeconstants for the enzyme-catalyzed reaction is difficulttimes and were reproducible.when the reaction studied is perturbed by a second,coupled reaction.The aim of the paper was to develop a method for DISCUSSION continuous kinetic recording of acid phosphatase activ-ity adapted both for water solution and for reverse mi-Acid phosphatase activity determination is im-portant in medical diagnostics, in biochemistry, and celles in order to study the catalytic properties of hu-man prostatic acid phosphatase entrapped in AOT–in biotechnology. Nonradioactive artificial substratesused for the determination of acid phosphatase ac- isooctane–water reverse micelles. Because the PAP in-cluded in the reverse micelles did not exhibit the activ-tivity include p -nitrophenyl phosphate, 1- and 2-naphthyl phosphate, phenyl phosphate, phosphotyro- ity for phosphotyrosine, the method developed in ourlaboratory for aqueous solution by Apostol et al. (28)sine, phosphoserine, phosphothreonine, phosphocho-line, a - and b -glycerophosphate, thymolphthalein could not be adapted for micellar studies.In the ultraviolet region of light the spectra of phenylphosphate, phenolphthalein phosphate, and 4-methyl-umbelliferyl phosphate. Discontinuous methods for phosphate and phenol are distinct not only in alkalinesolution above p K  of phenol (7, 34), which equals 9.99acid phosphatase activity measurements are based onthe colorimetric determination of products obtained in (35), but also below p K  of phenol (pH 3.8 to 8.8), bothin aqueous (Fig. 1) and in micellar solution (Fig. 2). Asenzyme catalyzed reaction (1–12). In triphasic reversemicellar solution end-point methods cannot be used. shown in Figs. 1 and 2 there is a wavelength rangewhere the absorption of phenyl phosphate is very lowContinuous acid phosphatase assays, employing aro-matic substrates, based on spectrophotometric mea- and the absorbance of phenol is high. Therefore in thiswavelength range, the direct continuous spectrophoto-surement of the reaction product in aqueous acidic so-lution have been developed to date. Phosphotyrosine metric measurement of phenol resulting from phenylphosphate in acid phosphatase-catalyzed hydrolysis re-has been used in the methods based on the measure-ment of the tyrosine absorbance at 286 nm (28) or at action is possible. In aqueous solution the measure-ment of phenol liberated from phenyl phosphate by en-280 nm (29). The method using 2,6-dichloro-4-nitrophe-nyl phosphate as the substrate is based on the mea- zyme action can be performed at any wavelength in therange between 274 and 278 nm. In reverse micelles thesurement of 2,6-dichloro-4-nitrophenol absorbance at400 nm (30). The other two substrates used are p -ni- wavelength 278 nm has been chosen as the best forcontinuous spectrophotometric measurement of phenoltrophenyl phosphate and O -carboxyphenyl phosphate.In acidic solution kinetic determination of  p -nitrophe- in the sample containing phenyl phosphate.
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