synthetic peptide substrates for a conductimetric assay ofpseudomonas aeruginosaelastase

ANALYTICAL BIOCHEMISTRY 237, 216–223 (1996)ARTICLE NO. 0232

Synthetic Peptide Substrates for a Conductimetric Assayof Pseudomonas aeruginosa Elastase1

Christine Besson, Joelle Saulnier, and Jean M. Wallach2

Laboratoire de Biochimie Analytique et de Synthese Bioorganique, Institut de Chimie et de Biologie Moleculaire etCellulaire, Batiment 303, 69622 Villeurbanne Cedex, France

Received November 28, 1995

tase would prevent the enzymatic destruction of manyPseudomonas aeruginosa is a zinc metalloprotease proteins, including those of connective tissue, delaying

which may be involved in many infection processes, then the progress of the disease. For the design of suchespecially in the lung. In order to evaluate the produc- inhibitors, the knowledge of enzyme specificity is fun-tion of the enzyme in culture supernatants, we devel- damental. Previous studies from Morihara and Tsu-oped an assay using peptide derivatives; the conducti- zuki (1, 2) have demonstrated that Phe, Leu, or Tyrmetric method was used for monitoring the enzymatic residues in the P*1 position (according to the nomencla-activities. Tetrapeptide derivatives were enzymati- ture of Schechter and Berger (3)) are favoring the pep-cally synthesized by coupling Z-Ala2 and X-AlaR using tide bond splitting. In recent papers from our labora-either thermolysin or P. aeruginosa elastase itself. In tory, we reported that tetrapeptides may be cleaved bythese substrates, X could be phenylalanine, tyrosine, elastase and we confirmed Morihara et al.’s results byor leucine and C-protection was performed by either

using tetrapeptides deriving from tetraalanine by in-an amide (NH2) or a methyl (OMe) group. Z-Ala2-Phe-troducing various amino acids in the P*1 position (4, 5).AlaNH2 was found to be the best substrate, giving a

Assays of P. aeruginosa elastase activity are usuallycatalytic ratio kcat/KM of 8600 mM01.s01. The evaluationperformed by measuring the hydrolysis rate of peptideof the alkaline protease activity with this substratebonds either by UV spectrophotometry (6) or by spec-showed that the catalytic ratio is 1000-fold lower. Thetrofluorometry (7). The first method is limited, as thesensitivity of the conductimetric method was alsofurylacryloylpeptides usually employed are poor sub-demonstrated with as little as 1 nM elastase (0.13 mg),strates, with quite high KM values (over 30 mM) and lowbeing easily and accurately detected (SD, 3.8% for 10solubilities. On the other hand, being quite a sensitivemeasurements). Furthermore, the enzymatic activitymethod, use of spectrofluorometry is not suitable inwas measured in a culture supernatant from a clinical

strain. q 1996 Academic Press, Inc. complex media, such as supernatants of bacterial cul-tures.

Protease production by P. aeruginosa isolates wasinvestigated by various methods, but they were quali-

The elastase of Pseudomonas aeruginosa is a metal- tative rather than quantitative. Radioimmunoassaysloproteinase which is thought to play a causative role were used in studies to compare elastase production byin many infection processes, especially in the lung. It different Pseudomonas strains (8–10). Different elas-is therefore an attractive target for therapeutic inter- tase assays were carried out using dye or radiolabeledvention in such diseases. In chronic infections such as elastin (10, 11) but measurements were made after athose associated with cystic fibrosis, inhibitors of elas- long incubation of the elastin–supernatant mixture

and no initial rate assays could be obtained.In our laboratory, we have been promoting for many1 This work was supported by grants from ‘‘Etablissement Public

Regional Rhone-Alpes,’’ from the ‘‘Association Francaise de Lutte years a sensitive conductimetric method for monitoringcontre la Mucoviscidose’’ (AFLM), and from the University Lyon I. the activities of various enzymes, including elastases

2 To whom correspondence should be addressed at Laboratoire de (12–15). Its principle is very simple. The conductanceBiochimie Analytique et de Synthese Bioorganique, ICBMC, Bat 303,of a substrate solution is measured in a thermostated43, Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.

Fax: (33) 72 43 15 00. E-mail: [email protected]. cell with a high-precision conductimeter, provided with

216 0003-2697/96 $18.00Copyright q 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

AID AB 9576 / 6m16$$$$41 05-05-96 23:15:55 abas AP: Anal Bio

CONDUCTIMETRIC ASSAY OF P. aeruginosa ELASTASE 217

alternative current to avoid electrode polarization. ml H2O. The pH was adjusted to 6.5–7 and the mixturewas incubated at room temperature with 10 mg of d-When the enzyme is added into the cell, if the enzy-

matic reaction occurs with changes in charge distribu- chymotrypsin under gentle stirring. The precipitatewas collected by centrifugation and the product crystal-tion, conductance is also varying and it is possible to

correlate the enzymatic rate with this change. It is this lized from methanol (MeOH) (final yield 60%). Z-Tyr-AlaNH2 was hydrogenolyzed at normal pressure overcase for peptide hydrolysis, as when peptide bonds are

broken, new charged groups appear. The method is ac- 10% Pd/C overnight to give Tyr-AlaNH2. Ala2-Phe-AlaNH2 was obtained from Z-Ala2-Phe-AlaNH2 usingcurate and easy to perform. Development of a now com-

mercially available conductimetric cell has allowed re- the same procedure.Phenylalanylalanine methylester was prepared ac-liable and sensitive measurements and recently a

partly automated device has been proposed (12). Con- cording to Rachele and Ichakawa (19). The product waspurified on a silica column (eluent:dichloromethaneductimetry has been successfully adapted for elasto-

lysis monitoring, using native insoluble elastin as a (CH2Cl2)/MeOH (9:1)) (final yield 40%).Ala3-Phe-Ala was chemically synthesized using 9-substrate, but the method, although precise, is quite

lengthy. For routine measurements of elastase activi- fluorenyl-methyloxycarbonyl (Fmoc) strategy. Fmoc-Ala or Fmoc-Phe (Bachem) dissolved in dimethylform-ties in cultures of P. aeruginosa strains from cystic fi-

brosis patients, it would be better to develop a specific amide with diisopropyl-carbodiimide and hydroxy-1benzotriazole (Milligen) were successively added toand sensitive assay, independent of the other proteases

found in the supernatant, mainly an alkaline protease Fmoc-Ala-KA resin (Bachem). Each step of deprotec-tion was performed by 20% piperidine and 95% trifluo-and another protein fragment, named LasA, which pos-

sesses proteolytic and elastolytic activity. Considering roacetic acid (TFA)3 was used for the final cleavagefrom the resin.our knowledge concerning the specificity of Pseudomo-

nas elastase, we have designed and prepared various All other products (salts, buffers, and solvents) wereof the highest commercially available analytical grade.substrates, by either chemical or enzymatic proce-

dures. In this paper, we report the synthesis of thesesubstrates and their validation for a continuous con- Methodsductimetric assay of P. aeruginosa elastase. Melting points were determined on a Leitz Wetzlar

apparatus and were uncorrected. Purity of peptidesMATERIALS AND METHODS and of their derivatives was checked by mass spectrom-

etry (positive fast atom bombardment) and by high-Materialsperformance liquid chromatography (HPLC), using

Purified P. aeruginosa elastase (74.2 mPU/mg pro- Pharmacia-LKB equipment fitted with a LKB RP 18tein) was obtained from Nagase Biochemicals Ltd. (Fu- Spherisorb ODS-2 column (4 1 250 mm, particle sizekuchiyama City, Kyoto, Japan), as a crystallized sus- 5 mm). Elution was carried out with H2O/acetonitrile/pension in 3.0 M ammonium sulfate, 10 mM sodium TFA (generally 70/30/0.05) and monitored at 214 nm.acetate, 2 mM calcium chloride, and 0.05 mM zinc chlo-

Enzymatic assays. Initial rates of peptide hydroly-ride. Its purity was checked by polyacrylamide gel elec-sis were conductimetrically measured, as previouslytrophoresis according to Laemmli (16). Its concentra-described (5, 20). In a typical experiment, 4 ml of ation was determined spectrophotometrically, aftersubstrate solution in 5 mM Tris–HCl, pH 8.6, 2.5% N-dilution in the appropriate buffer, using an absorptionmethylpyrrolidone was added to a temperature-regu-coefficient value of 14.52 (17). Lyophilized P. aerugi-lated conductimetric cell (type MCCD, Radiometer).nosa alkaline protease was also obtained from NagaseConductance was recorded over 5–10 min (T Å 30 {Biochemical Ltd. Thermolysin from Bacillus thermo-0.017C) with a CD-810 conductimeter (Radiometer).proteolyticus rokko and d-chymotrypsin were pur-

At regular intervals, 10 ml of the reacting mediumchased from Peptide Institute (Osaka, Japan) andwas removed for calibration of the hydrolysis rate; eachSigma, respectively. Carbobenzoxydialanine (Z-Ala2),sample was diluted in 30 ml of the HPLC eluent, pHcarbobenzoxytyrosine (Z-Tyr), alaninamide (AlaNH2),2.2, to stop the reaction; both substrate and productsphenylalanylalaninamide (Phe-AlaNH2), leucylalani-were further analyzed by HPLC, and the concentrationnamide (Leu-AlaNH2), phenylalanylalanine (Phe-Ala),of the products was directly correlated to the experi-and tetra- and pentaalanine were obtained frommental conductance change.Bachem (Bubendorf, Switzerland).

Enzymatic parameters (Vm and KM) were derivedTyrosylalaninamide (Tyr-AlaNH2) was synthesizedfrom the Michaelis and Menten relationship using theenzymatically according to Bizzozero et al. (18). Nine

hundred milligrams of Z-Tyr was dissolved in 4 ml of0.67 M NaOH and mixed with 1.1 g of AlaNH2 in 2.4 3 Abbreviation used: TFA, trifluoroacetic acid.


BESSON, SAULNIER, AND WALLACH218

TABLE 1

Conditions for Z-Ala2-X-AlaR Peptide Derivative Synthesis

Substrate Z-Ala2-Phe-AlaNH2 Z-Ala2-Tyr-AlaNH2 Z-Ala2-Leu-AlaNH2 Z-Ala2-Phe-AlaOMe

Z-Ala2 (M) 0.15 0.15 0.15 0.05X-AlaR (M) 0.20 0.35 0.30 0.15Enzyme Thermolysin P. aeruginosa elastase Thermolysin Thermolysin

added per milliliter 1.7 mg 5.6 mg 2.2 mg 0.4 mgof media

Time for 5–10 min 24 h 15 min 2–3 hprecipitation

Solvent for Methanol Methanol/water Methanol Methanol/acetonecrystallization

Yield (%) 85 60 70 70–90

widest concentration range and calculated from a non- Relative Velocities of Hydrolysis of the SynthesizedPeptideslinear curve-fitting program (K. cat, Macintosh).

Preliminary studies, using RP-HPLC, have demon-RESULTS strated that the Z-Ala2-X-AlaR substrates were split in

a single position, leading to Z-Ala2 and X-AlaR. ThisEnzymatic Synthesis of the N- and C-Protectedis consistent with our previous results concerning theTetrapeptides and Their Characterizationhydrolysis of tetraalanine derivatives (5). Further-The synthesis of four Z-Ala2-X-AlaR tetrapeptide de-more, the presence of Z and R in P3 and P*3 , respectively,rivatives by enzymatic coupling of Z-Ala-Ala to the cor-does not induce any other site of cleavage, which wasresponding nucleophile was performed in the presencethe case when hexaalanine was used as substrate (21).of a metalloproteinase (thermolysin or P. aeruginosa

Direct comparison of the hydrolysis rates of theseelastase), as indicated in Table 1. After precipitationsubstrates was realized at a concentration of 0.75 mMof the product, pellets were centrifuged and washedusing conductimetry (Table 3). The obtained values in-with 50% methanol and then with diethyl ether. Thedicate that the highest velocity is observed with Z-Ala2-peptides were finally recrystallized. Their purities werePhe-AlaNH2. Furthermore, rates are higher when thechecked by reverse-phase HPLC. All were demon-substrate is amidated rather then esterified and alsostrated to have a purity higher than 95%, except Z-enhanced when peptides are N-protected.Ala2-Phe-AlaOMe (92%), which was slightly contami-

nated by its free carboxylic acid. Table 2 presents someKinetic Parameters for Cleavage of Syntheticcharacteristics of the synthesized products (Rf , HPLC

Substratesretention time; tR , melting temperature, molecularweight, partition constant in 1-octanol/5 mM Tris–HCl Figure 1 shows the variation of conductance with

time when Z-Ala2-Phe-AlaNH2 is hydrolyzed by threebuffer, pH 8.6).

TABLE 2

Characteristics of the Synthesized Products

Melting point m/z found PartitionSubstrate Rf tR (7C) (Mr) constant

Z-Ala2-Phe-AlaNH2 0.77 (a) 15.1 (d) 250–252 512.3 (511.6) 14.9Z-Ala2-Phe-AlaOMe 0.73 (a) 37.0 (d) ND 527.2 (526.6) NDZ-Ala2-Leu-AlaNH2 0.81 (a) 13.2 (d) 236–237 478.3 (477.5) 6.57Z-Ala2-Tyr-AlaNH2 0.66 (a) 5.8 (d) 250–252 528.2 (527.6) 1.63Ala2-Phe-AlaNH2 0.68 (b) 9.8 (e) ND 378.2 (377.4) NDZ-Tyr-AlaNH2 0.70 (a) 6.0 (d) 199–200 386.1 (385.4) NDZ-Phe-AlaNH2 0.83 (a) 13.9 (d) ND 370.1 (369.4) NDPhe-AlaOMe 0.40 (c) 2.9 (d) ND 251.1 (250.3) ND

Note. Thin-layer chromatography solvents: (a) CH2Cl2/MeOH (4:1), (b) EtOH/H2O/AcOH (4:1:1), (c) CH2Cl2/MeOH (9:1). HPLC eluents:(d) H2O/acetonitrile/TFA (70:30:0.05), (e) H2O/acetonitrile/TFA (85:15:0.05).



TABLE 3 indicating a high KM value. The kinetic parameterswere directly calculated from the rate versus substrateRelative Rate of Hydrolysis of Some Peptidesconcentration curves (Table 4). Their values clearly in-by P. aeruginosa Elastasedicate that Z-Ala2-Phe-AlaNH2 is the best substrate,

Relative rate of with a kcat/KM of 8600 mM01.s01. These ratios confirmSubstrate hydrolysis (%) that the P*1 specificity of P. aeruginosa elastase is in

the decreasing order Phe ú Leu ú Tyr. Furthermore,Z-Ala2-Phe-AlaNH2 100Z-Ala2-Phe-AlaOMe 58 all these substrates have higher kcat/KM values thanZ-Ala2-Tyr-AlaNH2 19 the alanine oligomers which were previously used inZ-Ala2-Leu-AlaNH2 64 our laboratory (4).Ala2-Phe-AlaNH2 45Ala4 0.6

Linear Relationship between Rates of Hydrolysis andNote. [Elastase] Å 1 nM. 5 mM Tris–HCl buffer, pH 8.6. T Å 307C. Enzyme Concentration

Figures 3A and 3B show that the reaction rates areproportional to the concentration of the elastase added,elastase concentrations (0.3, 1, and 2 nM). Within the

first 2 min, a linear relationship may be demonstrated, in the experimental range, Z-Ala2-Phe-AlaNH2 leadingto the highest sensitivity (Fig. 3A). Once again, the Pheallowing an easy determination of initial conductance

change. residue in the P*1 position dramatically increased thesensitivity of the assay, as demonstrated when Ala4 orKinetic parameters (KM , Vm , and kcat/KM) were de-

rived from initial rate measurements of peptide hydro- Ala5 is compared to Ala2-Phe-AlaNH2 or Ala3-Phe-Ala(Fig. 3B).lysis. Figure 2 gives the Michaelian curve for Z-Ala2-

Phe-AlaNH2. Similar curves were obtained with the Reproducibility of the assay with the best substratewas estimated from 10 measurements of the activityother substrates, except Z-Ala2-Leu-AlaNH2, for which

a linear relationship between initial rate and substrate of 1 nM elastase and the standard deviation was equalto 3.8%.concentration was obtained in the 0.01–0.5 mM range,

FIG. 1. Recording of conductance changes with time for three elastase concentrations. Baseline was recorded for the substrate solutionalone, and then the increase of conductance, immediately after enzyme addition, was calculated and correlated with the hydrolysis rate.



FIG. 2. The Michaelian curve for the hydrolysis of Z-Ala2-Phe-AlaNH2. [Elastase] Å 2 nM.

Under the same experimental conditions, we have by adding 1 to 5ml of this extract directly in a substratesolution (0.3 mM Z-Ala2-Phe-AlaNH2 in 5 mM Tris–HClmeasured the hydrolysis of these N-protected substrates

by the alkaline protease, produced by P. aeruginosa to- buffer, pH 8.6). A linear relationship was demonstratedbetween hydrolysis rates and the volume of added su-gether with elastase. A similar linear relationship be-

tween rates of cleavage and protease concentration pernatant (Fig. 4). Moreover, regardless of whether aconstant volume of purified enzyme was added simulta-could be demonstrated but the hydrolysis rates were

about 1000-fold lower than with elastase. neously to the supernatant, the same slopes were calcu-lated (slopes without commercial enzyme, 0.41Preliminary experiments using crude extract super-

natants were also performed. A clinical strain was mM.h01.ml01 and with commercial enzyme, 0.39mM.h01.ml01, the correlation being 1.00 in each case).grown on complex media (casein tryptic peptone, yeast

extract, salt) for 18 h and then centrifuged to separate These results indicate that there is additivity of bothpurified elastase and supernatant activities, indicatingsupernatant from cells. Elastase assay was carried out

TABLE 4

Kinetic Parameters for Cleavage of Synthetic Substrates

Vm kcat/KM PrecisionSubstrate (mM.h01) KM (mM) (mM01.s01) (R)

Z-Ala2-Phe-AlaNH2 7.30 { 0.28 0.12 { 0.02 8600 0.98Z-Ala2-Phe-AlaOMe 2.90 { 0.20 0.09 { 0.02 4520 0.98Z-Ala2-Leu-AlaNH2 ND ND 1070 NDZ-Ala2-Tyr-AlaNH2 2.39 { 0.25 0.38 { 0.07 890 1.00Ala2-Phe-AlaNH2 3.71 { 0.90 1.08 { 0.49 500 0.92Ala3-Phe-Ala 1.30 { 0.04 0.37 { 0.04 2080 0.99Ala4

a 1.5 10.6 10 1.00Ala5

a 3.1 4.4 51 0.99

Note. [Elastase] Å 2 nM.a Catalytic constants were determined previously (4) with 4 nM elastase.



quinoleines, commonly employed in spectrophotometryor spectrofluorometry.

A few solutions have been proposed, including use offurylacryloylpeptides or of quenched fluorogenic sub-strates, but this latter approach is difficult for assaysin crude media, such as culture supernatants; further-more, the spectrophotometric assay of the hydrolysisof furylacryloylpeptides is not very specific, as the Mi-chaelis constant of these substrates is quite high. Con-ductimetry offers an interesting alternative to othertechniques as unmodified peptides may be used as sub-strates and their hydrolysis monitored with deviceswhich are now commercially available. Moreover, theuse of thermostated conductimetric cells allows a pre-cise and reliable measurement of protease activities.Numerous assays based on this methodology have beendeveloped in the past 10 years, as previously men-tioned.

In the case of Pseudomonas elastase, one possibilitywould be to use native elastin as a substrate, as we hadpreviously proposed such an assay for pig pancreatic orhuman leukocyte elastases (22–24). This procedure isapplicable for pure enzyme activity measurements andfor determination of kinetic parameters, but not forassays in culture supernatants; indeed, it has recentlybeen demonstrated that an associated 22-kDa protein,named LasA, enhances the elastolytic activity of vari-ous proteases, including Pseudomonas elastase, buthas no effect on the hydrolysis of peptidic substrates(25). The use of elastin as a substrate may then leadto experimental drawbacks which could explain somestrange values obtained in our lab when assaying cul-ture supernatants, using either insoluble elastin or tet-raalanine, at a time when the possible role of LasA on

FIG. 3. Hydrolysis rate of several substrates vs concentration of elastolytic activities was not clearly defined.enzyme. (A) s, Z-Ala2-Phe-AlaNH2, v Å 2.88 [elastase] 0 0.03, R Å We had previously demonstrated that the kcat/KM val-1.00; l, Z-Ala2-Leu-AlaNH2, v Å 1.29 [elastase] / 0.08, R Å 0.99; n,

ues of alanine oligomers increased from Ala4 to Ala6,Z-Ala2-Tyr-AlaNH2, v Å 0.60 [elastase] 0 0.01, R Å 0.99; /, Z-Ala2-Phe-AlaOMe, v Å 1.02 [elastase] 0 0.08, R Å 0.99. [Substrate] Å 0.3 tetraalanine being the shortest cleavable substrate.mM. (B) s, Ala3-Phe-Ala (1.5 mM), v Å 1.85 [elastase] / 0.06, R Å However, with hexaalanine, we have observed that two1.00; l, Ala2-Phe-Ala-NH2 (1.5 mM), v Å 1.36 [elastase] / 0.02, R Å types of splitting could occur, one leading to two tri-1.00; /, Ala5 (2 mM), v Å 0.26 [elastase] / 0.04, R Å 1.00; n, Ala4 alanine molecules and the other giving rise to di- and(5 mM), v Å 0.14 [elastase] 0 0.02, R Å 1.00.

tetraalanine (4, 21). This is the reason why we havedecided to synthesize tetrapeptide derivatives, N- andC-protected, in order to avoid multiple cleavage sites,

that the supernatant compounds do not interfere with but allowing interactions with the S3 and S*3 subsiteselastase activity. We may conclude that the synthe- of the enzyme. As for many other substrates, it wassized substrates are highly specific of Pseudomonas demonstrated that C-amidated peptides were muchelastase. more easily cleaved than the corresponding esters; fur-

thermore, the yields of synthesis were significantlyDISCUSSION higher when enzymatic synthesis was performed with

amides as nucleophiles. Considering the conductimet-The development of an assay of P. aeruginosa elas-tase activity is difficult because it is a metalloprotease. ric measurements, the substrates having the structure

Z-Ala-Ala-X-AlaNH2 gave the highest experimentalFor these enzymes, specificity is directed by the aminoacid present in the P*1 position, preventing the use of conductance increase at pH 8.6, this change being

mainly due to Z-Ala-Ala carboxylate ion formation.derivatives such as C-terminal paranitroanilides or



FIG. 4. Assay of elastase in culture supernatant from a clinical strain using Z-Ala2-Phe-AlaNH2. n, crude supernatant; s, crude superna-tant / 2 ml of purified enzyme (0.5 nM). Each value is the mean of three independent measurements, while the average value is representedwith its standard deviation.

It was then possible to derive an enzymatic assay REFERENCESof Pseudomonas elastase in the nanomolar range, 1. Morihara, K., and Tsuzuki, H. (1975) Agric. Biol. Chem. Tokyowith a substrate concentration of 0.3–0.5 mM. Being 39, 1123–1128.only two to three times higher than the KM value, 2. Morihara, K., and Tsuzuki, H. (1971) Arch. Biochem. Biophys.

146, 291–296.this concentration avoids the use of a too importantamount of organic solvent, generally N-methylpyrrol- 3. Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Com-

mun. 27, 157–162.idone. Moreover, the high selectivity for Pseudomo-4. Saulnier, J. M., Curtil, F. M., Duclos, M-C., and Wallach, J. M.nas elastase, when compared with alkaline protease,

(1989) Biochim. Biophys. Acta 995, 285–290.allows a precise and unambiguous assay of the en-5. Saulnier, J. M., Rayssiguie, A., Duclos, M-C., and Wallach,zyme in culture supernatants (preliminary experi-

J. M. (1990) Biochem. Soc. Trans. 18, 900–901.ments demonstrated that the assay could be used6. Feder, J. (1968) Biochem. Biophys. Res. Commun. 32, 326–332.with culture supernatants). Whereas it is usually7. Nishino, N., and Powers, J. C. (1980) J. Biol. Chem. 255, 3482–better to use a natural substrate in place of synthetic

3486.ones, this is why more reliable assays of P. aerugi-

8. Ohman, D., Cryz, S. J., and Iglewski, B. H. (1980) J. Bacteriol.nosa elastase are obtained with peptide derivatives 142, 836–842.than with insoluble elastin. Z-Ala2-Phe-AlaNH2 9. Obernesser, H. J., and Doring, G. (1981) Zentralbl. Bakteriol.might be used to assay elastase production in pulmo- Microbiol. Hyg. Abt. I Orig. A 252, 248–256.nary infections, such as those associated with cystic 10. Saulnier, J., Wallach, J. M., Doring, G., Malissard, M., Vacheron,

M. J., and Guinand, M. (1992) Eur. J. Clin. Chem. Clin. Biochem.fibrosis, even in crude culture supernatants.30, 285–290.

11. Elsheikh, L. E., Bergman, R., Cryz, S. J., and Wretlind, B. (1986)Acta Pathol. Microbiol. Immunol. Scand. Sect. B 94, 135–138.ACKNOWLEDGMENTS

12. Duffy, P., Mealet, C., Fombon, J. J., and Wallach, J. M. (1988)Anal. Chim. Acta 211, 205–211.We are greatly indebted to Sandrine Croze for typing the manu-

script. J.S. was supported by a grant from ‘‘Lyonnaise de Banque’’ 13. Duffy, P., Saad, I., and Wallach, J. M. (1988) Anal. Chim. Acta213, 267–272.and C.B. by grants from AFLM and University Lyon I.



14. Duffy, P., and Wallach, J. M. (1989) Enzyme 42, 98–102. 21. Saulnier, J. M., Thevenon, F., Besson, C., Duclos, M-C., andWallach, J. M. (1991) Biochem. Int. 23(5), 875–884.15. Saulnier, J. M., and Wallach, J. M. (1991) Anal. Chim. Acta 247,

79–82. 22. Bakala, H., Wallach, J., and Hanss, M. (1978) Biochimie 60,1205–1207.16. Laemmli, U. K. (1970) Nature 227, 680–685.

23. Manika-Saizonou, B., Bostancioglu, K., Mealet, C., Favre-Bon-17. Morihara, K., Tsuzuki, H., Oka, T., Inoue, H., and Ebata, M.vin, G., and Wallach, J. M. (1985) Anal. Lett. 18(B7), 901–913.(1965) J. Biol. Chem. 240, 3295–3304.

18. Bizzozero, S. A., Dutler, H., Franzstack, R., and Rovagnoti, 24. Saulnier, J. M., Bostancioglu, K., Favre-Bonvin, G., and Wallach,B. A. (1985) Helv. Chim. Acta 68, 981–987. J. (1988) Biol. Chem. Hoppe-Seyler 369(Suppl.), 75–78.

19. Rachele, J. R., and Ichakawa, T. (1979) Bull. Chem. 28, 3898. 25. Peters, J. E., Park, S. J., Darzins, A., Feck, L. C., Saulnier,J. M., Wallach, J. M., and Galloway, D. R. (1992) Mol. Microbiol.20. Ballot, C., Saizonou-Manika, B., Mealet, C., Favre-Bonvin, G.,

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synthetic peptide substrates for a conductimetric assay ofpseudomonas aeruginosaelastase

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