Analyflca Chlmrca Acta, 241 (1991) 79-82
Elsevter Sctence Pubhshers B.V., Amsterdam
19
Conductimetric assay of elastase in the supernatants of cultures of Pseudomonas aeruginosa strains
J.M. Saulnier and J.M. Wallach *
Laboratotre de Btochlmte Analytlque, ZCBMC, Bat 303, 69622 Vdieurbanne Cedex (France)
(Received 2nd January 1991)
Abstract
A conducttmetric assay of Pseudomonas aerugmosa elastase actrvtty was developed, usmg etther a synthettc peptrde
(tetraalamne) or unmochfted msotuble etastm as substrate Using punned etastase for cahbratrons, the actrvrttes of 45
cuhure supematants of strams from patients wtth cystic fibrosis were determmed. A hnear relatronsfnp was obtamed
between the two assays, with a slope of 0.98 These results clearly Indicate that measurement of the productton of
elastase IS possible by using a conductrmetric method. Whereas elastm represents a more selective substrate (not
cleavable by P aerugmosa alkaline protemase), tetraalanme 1s a very convement substrate tf routme and/or fast assays
are requtred
Keyworcis Conductrmetry, Blologtcal samples, Elastase, Enzyme acttvtty, Pseudomonas aerugrnosa
Pseudomonas aerugznosa is an opportunistic pathogen which may cause pulmonary infections [1,2] and cornea1 ulcers in man [3,4] and various other infections (for a review, see [5]). Several metabolites related to its virulence have been iden- tified and characterized. Of these, together with exotoxin A, those studied the most are two pro- teinases, an alkaline type and a metalloproteinase which is an elastase. Both have been purified and well studied by Morihara and co-workers [6-81. Substrate specificity is not well defined but it resembles that of thermolysin, and peptides which contain a Pr’ phenylalanyl, tyrosyl or leucyl res- idue (in the nomenclature of Schechter and Berger [9]) are cleaved more rapidly than others. It was recently demonstrated that, in contrast to pan- creatic elastase, P. aeruginosa elastase may hydro- lyse tetrapeptides such as tetraalanine [lo].
Protease production by P. aerugmosa isolates has been investigated by various methods, but they were qualitative rather than quantitative. Ra-
dioimmunoassays were used in two studies to compare elastase production by different Pseudo- monas strains [11,12]. Different elastase assays have been developed, using dye- or radiolabelled- elastin [12,13]; measurements were made after a long incubation of the elastin-supematant mix- ture and no initial rate assays could be obtained.
For a long time in this laboratory the develop- ment of devices and procedures for metabolite and enzyme activities assays has been under inves- tigation [14-171. The methodology has been suc- cessfully adapted for elastolysis monitoring, using native insoluble elastin as a substrate [l&20]. The method is precise and easy to perform but it is quite lengthy. Development of a now commer- cially available conductimetric cell allows reliable and sensitive measurements and recently a partly automated device designed for monitoring enzyme activities was proposed [21]. For routine assays of P. aeruginosa elastase, it would be helpful to use specific synthetic substrates. In this paper, results
0003-2670/91/$03.50 0 1991 - Elsevrer Scrence Publishers B V
80
on conductimetric measurements of P. aeruginosa
elastase activity using either tetraalanine or insolu- ble elastin as a substrate are presented.
EXPERIMENTAL
Materials
Elastin, extracted from bovine neck ligament and purified by alkaline extractton, was obtained from Elastin Products (Pacific, MO, U.S.A.). P.
aerugwzosa elastase and alkaline proteinase were
purchased from Nagase (Osaka, Japan). Their pur- ities were checked by polyacrylamide gel electro- phoresis according to Laemmli [22] and their con- centrations were determined spectrophotometri- tally [8]. Tetraalanine was from Bachem (Buben- dorf, Switzerland) and its purity was checked by liquid chromatography. All other products (salts and buffers) were of analytical-reagent grade. The cultures of P. aeruginosa strains were grown on trypticase soy broth (TSB) as described previously
v31.
Methods
A conductimetric method was used to de- termine the enzymatic activity of supematants, with either tetraalanine or unmodified insoluble elastin as a substrate. The device used for these experiments was described previously [14-171. All
its components are now commercially available. In a typical experiment, 4 ml of the substrate solu- tion in 5 mM Tris-HCl (pH 8.6) were added to a 4-ml temperature-regulated conductimetric cell (Type MCCD, Solea-Tacussel, Villeurbanne, France). The enzymatic reaction was initiated by addition of small amounts (lo-40 ~1) of culture
supematants. Conductance changes were meas- ured over 15-20 min at 30 f 0.01” C with a B-640 Wayne Kerr bridge or alternatively with a con- ductimeter (Solea-Tacussel, Type CD-810). Con- ductimetric data were analysed as described previ- ously [18] and activities are expressed in pS_’ h-i. When necessary, the curves were calibrated
J M SAULNIER AND J M WALLACH
with standard hydrochloric acid, and the rate of hydrolysis is expressed in pM[H+] h-‘.
RESULTS AND DISCUSSION
When elastin is hydrolysed at pH > 8, the spht- ting of peptide bonds liberates protons, which, when associated with convenient buffers, lead to a significant conductance change. As an example, the use of Tris buffer leads to an increase m conductance, as Tris, unprotonated in its basic
form, becomes positively charged. Hydrolysis of
synthetic substrates may also be accompanied by conductance changes [24]. Hydrolysis of tetra- alanine by P. aeruginosa elastase can be consid- ered to follow the reaction shown in Scheme 1.
Prelimmary experiments were conducted to de-
termine the substrate concentrations to be used m th experiments. For tetraalanme, as K, (the Michaelis constant) is 4 mM [lo], it was impossi- ble, owing to its solubility, to use concentrations above K,, as is usually done. Further, to avoid the buffer capacity of dialanme resulting from tetraalanine hydrolysis, it was necessary to use a Tris buffer concentration in the range 5-10 mM. Considering the experimental sensitivity required for the measurements, 5 mM Tris-HCl buffer (pH 8.6) was used and it was demonstrated that a concentration of about 0.5-l mM tetraalanine was convenient. In this range, a linear change in conductance with enzymatic hydrolysis was found for up to 10 min, with a pH change less than 0.1.
In subsequent experiments 0.75 mM was used. For insoluble elastin, the choice of concentration was defined by taking into account the expenmen- tal sensitivity of the device in the presence of a suspension of msoluble elastin; a 2 g 1-l elastin
suspension was considered as optimum. Another set of experiments was done to check
the linearity of the initial rate of elastolysis, mea- sured by conductimetry, with increasing volume of supernatant. For the standardization of the method, the strain IFO-3455 was used. With the
NH,CH(CH,)CONHCH(CH,)CONHCH(CH,)CONHCHoH3)COO- pH 8.6
2 NH,CH(CH,)CONHCH(CH,)COO-+ H+
Scheme 1.
CONDUCTIMETRIC ASSAY OF P AERUGINOSA ELASTASE ACTIVITY 81
TABLE 1
P. at-rug~~sa elastase actlvlty (V), determmed by conductlme-
try, usmg tetraalamne or elastm as a substrate, for different
enzyme concentrations
Elastase
concentration
(mg 1-l)
Y (pS h-l)
Tetraalamne a Elastm a
0 0
0.185 8 OkO.1
0 31 18.4k1.3
0 56 22.4+ 1.3
0 74 31.4*1 1
0.93 38.2*32
1.11
1.48
1 85 _
a MeanfS.D. (n = 3)
1.22 f 0.02
1 81 f 0.26
2.80 f 0 24
4 14*0 15
5 10*0.15
$ 13 5
0 -4 0 1 2 3
PsE (mg/l)
supematants of its cultures in TSB medium, a linear relationship from 10 to 100 ~1 of super- natant was demonstrated.
To measure the elastase production of the cul-
tures, calibration graphs were prepared with P. aerugmosa elastase, purified from strain IFO-3455.
Fig 1. Influence of the supernatant components on the elasto-
lytlc actlvlty of a punfled P aerugtnosn elastase. elastm (2 g
I-‘) was suspended m 5 mM Tns-HCl buffer (pH 8.6) before
addltlon of enzyme The elastase concentrations were kept
between 0 and 3 mg I-‘. Lmear relabonshps were obtamed
under the three followmg condltlons: (A) w&out supematant,
(0) m the presence of 50 pl of the supematant of the stram
IFO-3455 and (x) m presence of 50 gl of the supematant of
the stram IFO-3455, previously kept at 70 o C for 30 mm
Table 1 gives the values of the initial rates obtamed using either elastin or tetraalanine as
substrates. For almost all measurements the repro- ducibility is better than 5%. There is a linear relationship between the observed rates of hydrol- ysis and the enzyme concentration in the range 0.4-a 1.8 mg 1-l (12-60 nM) with elastin and 0.2-0.9 mg 1-l with tetraalanine.
strate at a concentration of 0.75 mM, was 25 times lower than that with elastase (these experiments were done with purified enzymes); and wrth elas- tin, hydrolysis by elastase was not modified by alkaline proteinase, even if present in a 5-fold a
60
The calibration equations and correlation coef-
ficients (r) are I’(+ h-‘) = 2.72 [PsE] (mg l-l),
r = 0.99 (n = 6), and V(pS h-‘) = 40.8 [PsE] (mg l-‘), r=0.99(n=6).
It was also demonstrated that the components of the culture medium have no influence on the calibration graph. Different cell culture super- natants were previously inactivated for 30 min at 70” C and calibration graphs were determined using purified elastase in these media. In all ex- periments, the slopes of the calibration graphs never differed from the reference by more than 10% (for an example, see Fig. 1). The measured enzyme activity was assessed to be only the elas- tase type, for two major reasons: hydrolysis by alkaline protemase, with tetraalanine as a sub-
0 20 40 60 80
PsE (mg/l) S Tetraalanme
Fig. 2. Relationslup between P aenrg~~osa elastase actlvlty, determmed by conductlmetry, using either elastm or tetra-
alamne as a substrate. Substrates were prepared m 5 mM
Tns-HCl buffer (pH 8.6) at 30°C. The slope of the graph
determined by hnear regresslon, was 0 98, the correlation coef-
f*ckent bemg 0 92. Number of different strains 45
82 .I M SAULNIER AND J M WALLACH
molar excess. It may be concluded that, for all supernatants, the measured activities are exclu- sively due to P. aerugznosa elastase.
The elastolytic activities of culture supernatants of 45 strains of P. aerugznosa isolated from pa- tients with cystic fibrosis were then determined using the above procedures. For each measure- ment, enzymatic activities were derived from the corresponding calibration graphs using purified P. aerugznosa elastase. Figure 2 shows the relation- ship between the values obtained with the two
substrates. A linear relationship was clearly dem- onstrated, with a slope of 0.98 and a correlation coefficient of 0.92.
The correlation between the two assays is high,
indicating that tetraalanine may be used as a substitute for elastin if a routme and fast measure- ment is required. The development of a partially automated device for conductimetric assays in solutions [21] allows the use of this method also for the monitoring of elastase production with time. The use of unlabelled elastin avoids the
drawbacks that may be due to non-uniform label- ling of elastin, either with dye or with radioactive atoms. Further, the measurement of activity within 30 min avoids the possible activity of alkaline
proteinase, which, inactive during the first stages of elastolysis, may hydrolyse solubilized peptides and could then interfere with elastolytic activity
after too long an incubation. The method detected elastase activities corre-
sponding to concentration of purified elastase in
the mg 1-l range (in the supernatant). In the
conductimetric cell, the concentrations are about 100 times lower. Experiments are in progress to compare the elastase activities of strains with their
elastase antigen concentrations, measured by ELISA or radioimmuno assay methods. The pre- lirnmary results indicate that a correlation exists with the present procedures but with lower corre-
lation coefficients. Such a result has been observed by Elsheik et al. [13] with strains from mink [13].
As the pathogenicity of P. aerugznosa may, at least in part, be ascribed to elastase activity, it is important to have a fast and reliable technique for
such a measurement. The conductimetnc method, using either a synthetic substrate or insoluble elastin, appears to be a useful method which may
give, for instance, rapid information on changes in
activity during the development of the disease, or on the effect of inhibitors or other chemicals on elastase production.
This work was supported by a grant from EPR Region Rhane-Alpes (programme: Mucovisci- dose). Thanks are due to G. Doring (Tiibingen) for cultures of strains from patients and for many helpful and stimulatmg discussions. Thanks are
due to C. Matray for typing the manuscript.
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