a continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

ANALYTICALBIOCHEMISTRY

Analytical Biochemistry 344 (2005) 115–121

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A continuous Xuorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

Mark Shepherd, Harry A. Dailey ¤

Biomedical and Health Sciences Institute, University of Georgia, Athens, GA 30602, USA

Received 26 April 2005Available online 22 June 2005

Abstract

A continuous spectroXuorimetric assay for protoporphyrinogen oxidase (PPO, EC 1.3.3.4) activity has been developed using a 96-well plate reader. Protoporphyrinogen IX, the tetrapyrrole substrate, is a colorless nonXuorescent compound. The evolution of theXuorescent tetrapyrrole product, protoporphyrin IX, was detected using a Xuorescence plate reader. The apparent Km (Kapp) valuesfor protoporphyrinogen IX were measured as 3.8 § 0.3, 3.6 § 0.5, and 1.0 § 0.1 �M for the enzymes from human, Myxococcusxanthus, and Aquifex aeolicus, respectively. The Ki for aciXuorfen, a diphenylether herbicide, was measured as 0.53 �M for the humanenzyme. Also, the speciWc activity of mouse liver mitochondrial PPO was measured as 0.043 nmol h¡1/mg mitochondria, demonstrat-ing that this technique is useful for monitoring low-enzyme activities. This method can be used to accurately measure activities as lowas 0.5 nM min¡1, representing a 50-fold increase in sensitivity over the currently used discontinuous assay. Furthermore, this contin-uous assay may be used to monitor up to 96 samples simultaneously. These obvious advantages over the discontinuous assay will beof importance for both the kinetic characterization of recombinant PPOs and the detection of low concentrations of this enzyme inbiological samples. 2005 Elsevier Inc. All rights reserved.

Keywords: Heme biosynthesis; Protoporphyrin; Enzyme kinetics; Fluorescence; Herbicide

Protoporphyrinogen oxidase (PPO,1 EC 1.3.3.4) cata-lyzes the oxygen-dependent six-electron oxidation ofprotoporphyrinogen IX to the fully conjugated macro-cycle of protoporphyrin IX [1]. Mutations in the PPOgene may give rise to a condition known as variegateporphyria [2]. In eukaryotic organisms, PPO has beenlocalized to the cytosolic side of the inner mitochondrialmembrane and requires molecular oxygen for catalysisto proceed [3–5]. The human, Bacillus subtilis, Myxococcusxanthus, Aquifex aeolicus, and mouse enzymes have pre-

* Corresponding author. Fax: +1 706 542 7567.E-mail address: [email protected] (H.A. Dailey).

1 Abbreviations used: PPO, protoporphyrinogen oxidase; FAD, Xa-vin adenine dinucleotide; MOPS, 4-morpholinepropanesulfonic acid;Tris, Tris(hydroxymethyl)aminomethane carbonate; PMSF, phenyl-methylsulfonyl Xuoride; EDTA, ethylenediamine tetraacetic acid.

0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2005.06.012

viously been cloned, overexpressed in Escherichia coli,and puriWed to homogeneity [6–10]. A noncovalentlybound Xavin adenine dinucleotide (FAD) cofactor wasdetected in each of these enzymes, and several wereinhibited by aciXuorfen, a diphenylether herbicide. Also,a recent study reported that aciXuorfen is a competitiveinhibitor of human PPO [11].

The Wrst assays of PPO exploited the change in absor-bance elicited by the accumulation of protoporphyrin IX[12]. The enzymatic rate was calculated by measuring thediVerence between the Q-band absorption peaks of pro-toporphyrin IX (450–700 nm) before and after catalysis.This was found to be preferable to an alternate proce-dure involving the spectrophotometric detection of pro-toporphyrin IX in acid solution under anaerobicconditions that caused rapid autooxidation of theprotoporphyrinogen substrate [13].

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116 Continuous Xuorimetric assay for protoporphyrinogen oxidase / M. Shepherd, H.A. Dailey / Anal. Biochem. 344 (2005) 115–121

The discontinuous spectrophotometric technique [12]was later modiWed to monitor the evolution of protopor-phyrin IX Xuorescence [14]. Although this improved sensi-tivity, the assay remained discontinuous. The reactionconditions of this discontinuous Xuorescence assay wereidentical to those used in the discontinuous spectrophoto-metric assay except that Tween was included to enhanceprotoporphyrin IX Xuorescence. The inclusion of glutathi-one was intended to help reduce the autooxidation of theprotoporphyrinogen IX substrate, although this had beenshown to inhibit PPO activity from certain microbialsources [15]. Also, chelating agents were used to preventthe insertion of divalent cations into the tetrapyrrole ringbecause ferrochelatases in the cell extracts may incorpo-rate zinc or ferrous iron. This is no longer necessary whenpuriWed recombinant PPOs are assayed.

Both of these assay procedures were subsequentlyassessed [16]. Poulson’s discontinuous spectrophotomet-ric assay [12] was found to have a limit of sensitivity of56 nM min¡1, whereas Brenner’s discontinuous Xuores-cence assay [14] was slightly more sensitive, exhibiting alower limit of 28 nM min¡1. The major shortcomings ofthese assays are a lack of sensitivity for the detection oflow-enzyme activities in biological samples and the auto-oxidation of substrate. In this article, we describe a con-tinuous Xuorescence assay that detects PPO activities aslow as 0.5 nM min¡1. Multiple samples and controls maybe monitored simultaneously, facilitating the subtractionof spontaneous protoporphyrinogen IX oxidation. Also,by increasing the wavelength and reducing the intensityof excitation light, we have managed to greatly reducethis autooxidation. We compare data obtained using thecontinuous and discontinuous methods and highlightthe beneWts of the continuous assay for use in clinicaldiagnosis and enzyme kinetics alike.

Materials and methods

Materials

Protoporphyrin IX was purchased from PorphyrinProducts (Logan, UT, USA). Tween 20 was obtainedfrom EM Science (Gibbstown, NJ, USA). 4-Morphol-inepropanesulfonic acid (MOPS) was purchased fromFisher ScientiWc (Pittsburgh, PA, USA). Tris(hydroxy-methyl)aminomethane carbonate (Tris), potassium chlo-ride, sodium dihydrophosphate, and sodium metal werepurchased from J.T. Baker. The remaining chemicalswere purchased from Sigma–Aldrich (St. Louis, MO,USA) unless otherwise speciWed.

Protein expression and puriWcation

Recombinant PPOs were overexpressed and puriWedas reported previously [7,9,10]. E. coli JM109 cells were

transformed, and the cells were grown overnight at30 °C. The growth media were supplemented with ribo-Xavin at a Wnal concentration of 0.75 �g ml¡1 2 h prior toharvesting. The cells from 1 L of cell culture were col-lected by centrifugation and resuspended in buVer(50 mM Tris/MOPS (pH 8.0), 100 mM KCl, 0.5% Tween20 (v/v)) containing 1 mg ml¡1 phenylmethylsulfonylXuoride (PMSF), sonicated on ice for 6 £ 30 s, and cen-trifuged at 100,000g for 30 min to remove membranesand insoluble proteins. The supernatant was applied toTalon (Clontech, Palo Alto, CA, USA) chelate columns(bed volume 3 ml), which were subsequently washed with20 column volumes of buVer and a further 20 volumes ofbuVer containing 15 mM imidazole. The puriWed pro-teins were eluted with buVer containing 250 mMimidazole. The imidazole was subsequently removedusing a Biogel (Bio-Rad, Hercules, CA, USA) desaltingcolumn with a bed volume of 30 ml. Protein preparationswere analyzed using UV–visible spectroscopy to conWrmthe presence of the noncovalently bound FAD cofactor.Protein concentrations were determined spectrophoto-metrically using calculated extinction coeYcients basedon the amino acid composition. These �270 values were48.0, 21.9, and 34.6 mM¡1 cm¡1 for enzymes fromhuman, M. xanthus, and A. aeolicus, respectively.

Preparation of mouse liver mitochondria

A 1.6-g liver from a C57BL/6x inbred mouse washomogenized in 8 ml of ice-cold homogenization buVer(10 mM Tris–HCl (pH 8.0), 0.25 M sucrose, 1 mM ethy-lenediamine tetraacetic acid (EDTA), 0.2 mg ml¡1

PMSF). The homogenized sample was aliquotted into1.5-ml Eppendorf tubes on ice. The samples were centri-fuged at 10,000g for 10 at 4 °C. The supernatants weredecanted, and the pellets were weighed. The pellets wereresuspended in ice-cold PPO assay buVer (50 mMNaH2PO4 (pH 8.0), 0.2% Tween 20 (v/v)) to give a con-centration of 273 mg mitochondria/ml, of which 8 to25 �l was used in the kinetic assays.

Porphyrin and inhibitor preparation

Protoporphyrin IX (3 mg) was solubilized with fourdrops of 30% ammonium hydroxide and dissolved in2.5 ml of freshly prepared 10 mM KOH containing 20%ethanol. This was then diluted with 2.5 ml of 10 mMKOH solution. Sodium amalgam was prepared, asdescribed previously [14,17], by mixing 3.5 g of sodiummetal with 75 g of mercury under a stream of nitrogen ina fume hood. It was important to ensure that all of theoil was removed from the sodium metal before mixing.The hot amalgam was cooled to room temperature, bro-ken into 1-cm3 pieces, and stored under nitrogen for upto 1 week. Immediately prior to use, 20 g of amalgamwas pulverized with a mortar and pestle and was

Continuous Xuorimetric assay for protoporphyrinogen oxidase / M. Shepherd, H.A. Dailey / Anal. Biochem. 344 (2005) 115–121 117

transferred to a Xask under nitrogen in the dark. The5-ml solution of protoporphyrin IX was transferred tothe Xask. The Xask was sealed and shaken until the solu-tion became colorless, making sure to vent any evolvedgas. The pH was adjusted to 8.0 using a solution of 2 MMOPS. The resultant protoporphyrinogen solution wasWltered (using a 0.2-�m syringe Wlter), placed in the darkunder nitrogen, and used immediately.

In assays where aciXuorfen was present (Chem Ser-vices), the aciXuorfen was added from a stock solution indimethyl sulfoxide. The addition of an equivalent vol-ume of dimethyl sulfoxide alone had a negligible eVecton PPO catalysis. All assay procedures were carried outin a darkened room. Concentrations of protoporphyrinIX were determined in 2.7 N HCl using �557 D 20.7mM¡1 cm¡1 [13].

PPO assays

PPO activity was monitored using a continuous assayvia the detection of porphyrin Xuorescence. This wasperformed on a Synergy HTI plate reader using528 § 20-nm and 545 § 40-nm bandpass Wlters and a550-nm highpass Wlter for the excitation light. A635 § 32-nm bandpass Wlter was used on the emissionside. The excitation Soret band for protoporphyrin IX at406 nm was not used because this caused saturation ofthe photomultiplier. Three Wlters were used for the exci-tation light because a reduction in light intensity greatlyreduced the autooxidation of protoporphyrinogen IX.

The instrument was calibrated with known concen-trations of protoporphyrin IX. A linear relationshipbetween Xuorescence and [protoporphyrin IX] wasachieved up to 2.5 �M porphyrin. An opaque, clear-bot-tom, 96-well, full-diameter plate (Corning, Corning, NY)was used for the assays. The reaction mixtures were pre-incubated at 37 °C in the dark for 3 min. The reactionswere started by the addition of PPO, which was also pre-incubated at 37 °C, and the evolution of protoporphyrinIX was monitored for 30 min. The reaction mixtures forthe A. aeolicus enzyme were preincubated and moni-tored at 50 °C, the maximum temperature for the platereader. The nonenzymatic rates were determined con-comitantly and were subsequently subtracted. Thesebackground rates were typically less than 5% of theenzyme-catalyzed rates. Wells containing heat-inacti-vated samples were used as controls for the mouse livermitochondria assays. The mitochondrial extract washeated at 85 °C for 20 min and was assayed for PPOactivity in parallel with the other samples. On the addi-tion of the mitochondrial extract, the reaction mixturebecame slightly turbid. However, this did not seem toaVect the detection of protoporphyrin IX Xuorescence.Reaction conditions and concentrations of reactants/inhibitors are quoted in the Wgure legends. The totalvolume of the reaction mixture was 100 �l and contained

50 mM NaH2PO4 (pH 8.0), 0.2% Tween 20 (v/v), and 2.5glutathione. The mouse liver mitochondria were assayedin the presence of 2.5 mM EDTA. The concentration ofprotoporphyrinogen IX was determined retrospectivelyafter the complete autooxidation of the stock solution toprotoporphyrin IX, which was quantiWed spectrophoto-metrically in 2.7 N HCl [13].

Analysis of kinetic data

Steady-state rates were estimated by Wtting the timecourses to straight lines using linear regression. Sometraces exhibited small burst phases (data not shown) orlag phases. The steady-state rate was taken as the maxi-mum rate that followed these burst/lag phases. Steady-state rate data (� vs. [S]) were Wtted to Eq. (1) (usingunweighted nonlinear regression in Sigma Plot 8 ofSPSS)

(1)

where � is the rate, Vapp is the apparent maximum rate,Kapp is the apparent Michaelis constant, and [S] is thesubstrate concentration. Error bars were not includedbecause the estimates of the steady-state rates wereobtained from linear regression analysis, where r2 valueswere greater than 0.95 (data not shown).

IC50 values were calculated by Wtting � versus [inhibitor]data to a single binding site model described by Eq. (2)

(2)

where y is the percentage of maximal rate, max and minare the y values at which the curve levels oV, x is the log-arithm of inhibitor concentration, and IC50 is the inhibi-tor concentration that elicits 50% of the total inhibition.

Calculated Ki values were obtained by applying thefollowing relationship, which exists for competitive inhi-bition among Ki, Km, and IC50 at any saturating sub-strate concentration (S)

(3)

Absorbance and Xuorescence spectroscopy

Absorbance spectra were recorded on a Cary 50 (Var-ian) spectrophotometer in 50 mM NaH2PO4 (pH 8.0),0.2% Tween 20 (v/v), and 2.5 mM glutathione. Fluores-cence excitation and emission spectra were recorded at25 °C with a Shimadzu RF-5301PC spectroXuorimeter.All PPO samples were in 50 mM NaH2PO4 (pH 8.0),0.2% Tween 20 (v/v), and 2.5 mM glutathione. Samples(3 ml) were incubated for at least 3 min at 25 °C before


spectra were recorded. BuVer-only spectra were recordedand subsequently subtracted using Datamax. For emis-sion spectra, an excitation wavelength of 406 nm wasused, with excitation and emission slit widthscorresponding to 1.5 and 10 nm, respectively. For excita-tion spectra, an emission wavelength of 632 nm wasused, with excitation and emission slit widths corre-sponding to 10 and 1.5 nm, respectively. In addition, a345-nm highpass Wlter was placed at the emission side ofthe cuvette holder to reduce the detection of scatteredexcitation light and to eliminate harmonic eVects.

Results and discussion

The continuous assay in this study exploits the Xuo-rescent properties of protoporphyrin IX and the lackof absorption and Xuorescence of the tetrapyrrole sub-strate, protoporphyrinogen IX. Under the assay condi-tions described, and using an excitation wavelength of406 nm, the emission maximum for protoporphyrin IXwas 632 nm (Fig. 1B). The Xuorescence excitation

spectrum for protoporphyrin IX was obtained using anemission wavelength of 632 nm (Fig. 1A). The Soretregion around 400 nm was not used for the kineticassay because the resultant high Xuorescence intensitycaused saturation of the photomultiplier tube. Also,this lower wavelength, higher energy radiation causedrapid autooxidation of the protoporphyrinogen IXsubstrate. To combat these problems, three Wlters wereused to generate a low-intensity excitation beam atapproximately 550 nm. Fluorescence intensity exhib-ited a linear relationship between 0 and 2.5 �M proto-porphyrin IX (data not shown). Furthermore, when50 nM human PPO was assayed with 3 �M protopor-phyrinogen IX, the nonenzymatic rate was found to benegligible in comparison (Fig. 1C). However, higherconcentrations of protoporphyrinogen IX exhibithigher autooxidation rates, so nonenzymatic ratesmust be monitored concomitantly. This is easilyachieved using the plate reader because one can moni-tor 32 assays simultaneously, sampling the Xuorescenceintensity for each well every 16 s. The minimum inter-val between time points increases if more wells are

Fig. 1. Fluorescence spectra of protoporphyrin IX and a typical kinetic assay: Excitation (A) and emission (B) spectra of protoporphyrin IX underassay conditions. Excitation and emission wavelengths were set to 406 and 632 nm, respectively. Protoporphyrin IX (1 �M) was prepared in assaybuVer as described in Materials and methods. (C) Typical assays in the presence (�) and absence (�) of 50 nM human PPO. Enzymatic rate D 5880FU min¡1 (which translates to 155 nM min¡1). Nonenzymatic rate D 0.54 nM min¡1. These rates were estimated using linear regression, with r2 valuesfor the enzymatic and nonenzymatic Wts being 0.999 and 0.911, respectively.


used, so usually only one-third of the plate is used. Thedata points in Fig. 1C were taken at 16-s intervals. Theoxidation rate may be estimated far more accurately byusing this technique than by estimating a rate fromfour time points as was necessary previously with thediscontinuous assays. The autooxidation rate inFig. 1C (0.5 nM min¡1) was Wtted to a straight lineusing linear regression (Sigma Plot), and the resultantline of best Wt had an r2 value of 0.91. This oxidationrate represents the limit of sensitivity for the continu-ous Xuorescence assay. This technique is far more sen-sitive than the previous discontinuous Xuorescencemethod, which could be used to measure rates onlyas low as 28 nM min¡1 (5 nmol h¡1 in a 3-ml cuvette)[16].

At low concentrations of protoporphyrinogen IX, thereaction rate slows down as the substrate is exhausted.This is easily observed using the continuous Xuorescencemethod, although such observations might not be imme-diately apparent using the discontinuous assays, result-ing in an underestimation of catalytic rate.

Having established the reaction conditions, we testedthe range of enzyme concentrations that could be usedwith this assay. The evolution of protoporphyrin IXXuorescence was measured for several concentrations ofhuman PPO, and the enzymatic rate was linearlydependent on the enzyme concentration over the rangeof 25–250 nM (Fig. 2). This demonstrates that the con-centration of enzyme typically used (50 nM) is within thelinear range and that one can use protein concentrationsmore than two orders of magnitude below the Kapp forsubstrate.

The current assay was assessed by determining thesteady-state parameters of puriWed recombinant PPOsfrom human, M. xanthus, and A. aeolicus sources.

Fig. 2. Dependence of the initial rate of protoporphyrin IX evolutionon PPO concentration. Initial rates were determined using 5 �M pro-toporphyrinogen IX, and PPO concentrations were 25 nM (�), 50 nM(�), 100 nM (�), 150 nM (�), and 250 nM (�). The inset depicts theenzymatic rate versus the protein concentration (�).

These data are shown in Fig. 3, and the curves were Wttedto the Michaelis equation (Eq. 1)) using nonlinearregression. The kinetic parameters, along with thoseobtained previously for these three enzymes [7,9,10],are summarized in Table 1. The Vapp and Kapp valuesare similar to those obtained previously, and the aver-age standard errors for the three plots were 4.1 and10.6% of the Vapp and Kapp values, respectively. Thisdemonstrates that one can quickly and accurately esti-mate the kinetic parameters for this enzyme withoutthe need for excessive repetition and averaging. How-ever, if averaging is required for enzymes with loweractivities, � versus [S] curves may be obtained in tripli-cate in a single 30-min assay.

The applicability for this continuous Xuorescenceassay for inhibition studies with human PPO was testedwith aciXuorfen, a competitive inhibitor with respect tothe tetrapyrrole substrate [11]. Human PPO wasassayed with varying concentrations of aciXuorfen, andthe data were Wtted to Eq. (2) using nonlinear regres-sion (Fig. 4). The concentration of aciXuorfen that elic-ited a 50% reduction in catalytic rate was1.48 § 0.20 �M. This compares with 4.2 § 0.39 �M, asreported in a previous study [11]. Using Eq. (3), theapparent Ki was calculated as 0.53 �M aciXuorfen,which compares with 0.23 �M, as determined previ-ously [11].

PPO from mouse liver mitochondria was assayed todemonstrate that the continuous Xuorescence assaycan be used with biological samples. Fig. 5 depicts sev-eral PPO assays using mitochondrial concentrations inthe range of 22–68 mg ml¡1. Background oxidation wasmonitored using heat-inactivated samples, and these

Fig. 3. Determination of apparent Michaelis constants for various pro-toporphyrinogen oxidases. PPO (50 nm) from human (�), M. xanthus(�), and A. aeolicus (�) was assayed using varying concentrations ofprotoporphyrinogen IX. The human and M. xanthus enzymes wereassayed at 37 °C, and the hyperthermophilic A. aeolicus enzyme wasassayed at 50 °C. The data were Wtted to single rectangular hyperbolaeusing nonlinear regression, and the kinetic parameters are shown inTable 1.


data have been subtracted. The curves possess a smalllag phase, which may be diYcult to detect if only fourtime points are used, as in the discontinuous assay. The

Fig. 4. AciXuorfen inhibition of PPO. Human PPO (50 nm) was prein-cubated for 5 min at 37 °C in the presence of varying concentrations ofaciXuorfen, a diphenylether inhibitor. Reactions were started by theaddition of 6.9 �M protoporphyrinogen IX. Reaction rates are shownas the percentages of the catalytic rate in the absence of inhibitor. Thedata were Wtted to Eq. (2) using nonlinear regression. The inhibitorconcentration that elicits 50% of the total change in activity is1.48 § 0.20 �M. The calculated apparent Ki, using Eq. (3), was 0.53 �M.

Fig. 5. PPO activity of mouse liver mitochondria. Steady-state rateswere taken as the maximum rates. Protoporphyrinogen IX (5.6 �M)was used in each assay. Background rates (of assays incorporatingthermally inactivated samples) have been subtracted. Assays were per-formed with 2.2 mg (�), 2.7 mg (�), 4.1 mg (�), 5.5 mg (�), and 6.8 mg(�) of mitochondria. The inset depicts the enzymatic rate versus themass of mitochondria (�).

steady-state rate was taken as the maximum rate thatfollows the lag phase. This highlights how useful theseprogress curves can be when monitoring a nonlinearreaction time course. The Fig. 5 inset demonstrates thatenzyme activity is directly proportional to the amountof mitochondria used. The speciWc activity(0.043 nmol h¡1/mg mitochondria) is very low, proba-bly because the samples were not sonicated. However,this is ideal for demonstrating the sensitivity of thecontinuous Xuorescence method. For example, theenzymatic rate with 2.2 mg of mitochondria was0.12 nmol h¡1 (20 nM min¡1). This activity is below thelimit of sensitivity of the discontinuous Xuorescenceassay (28 nM min¡1 [16]), demonstrating that the cur-rent method may be used to accurately measure lowenzymatic rates from biological samples.

Overall, the continuous Xuorescence assay oVersseveral signiWcant advantages over existing procedures.The steady-state rates can be determined from theslopes of progress curves, eliminating the problem ofnonlinearity in velocity calculations. The use of alower energy excitation beam around 550 nmreduces the rate of substrate autooxidation and pre-vents saturation of the photomultiplier at higher proto-porphyrin IX concentrations. Another advantage ofthis assay is that, unlike the discontinuous assays, itdoes not require one to work for extended periods inthe dark. The samples may be loaded onto the micro-plate using a multipippetter under reduced light condi-tions. In addition, all of the reactions are started at thesame time, reducing the risk of autooxidative depletionof the tetrapyrrole substrate before the addition ofenzyme.

In summary, the continuous Xuorescence assayprovides a sensitive method to measure PPO activity andcan be used for inhibition studies. Because the proceduremay be used to detect small changes in protoporphyrinIX levels in biological samples, it will be useful forassessing diminished PPO activities in variegateporphyria patient samples.

Acknowledgments

The mouse liver was kindly provided by JenniferSmith (University of Georgia, Athens). This work wasfunded by the National Institutes of Health (DK032303to H.A. Dailey).

Table 1Comparison of continuous and discontinuous assays in determination of PPO activity

PPO protein Continuous assay Discontinuous assay Reference

Kapp (�M) Vapp(�M min¡1/�M protein) Kapp (�M) Vapp (�M min¡1/�M protein)

H. sapiens 3.8 § 0.3 5.7 § 0.2 1.7 10.5 [10]M. xanthus 3.6 § 0.5 3.1 § 0.2 1.6 5.2 [9]A. aeolicus 1.0 § 0.1 0.4 § 0.009 2.8 — [7]


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a continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

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