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New methods for the determination of elastase
CLINICA
NEW
CHIMICA
327
ACTA
METHODS
FOR
THE
DETERMINATION
c. GEOKAS,P.SILVERMAN,Y.LILLAKD
H. RINDERKNECHT,M. B.J.HAVERBACK Department of Medicine, Hospital,
Los Angeles,
(Received November
OF ELASTASE”
University of Southern Calif. 90033 (U.S.A.)
Califwnia
and the Los
.~ND Angeles
Couuty
Geneva1
Ist,1967)
SUMMARY
Highly sensitive fluorimetric and calorimetric methods for estimation of pancreatic elastase have been developed. Two covalently labeled substrates, Fluorescein-elastin and Remazolbrilliant Blue-elastin, were prepared for this purpose. Pancreatic elastase has been assayed selectively in the presence of trypsin and chymotrypsin. The potentiating effect of these enzymes on the activity of elastase is described. The influence of this synergism on elastase determinations and its significance in acute pancreatic disease is discussed.
INTRODUCTION
Recent work suggesting a key role for elastase in the aging process1 and acute pancreatiti9 has drawn considerable attention to this enzyme. Methods for the determination of elastase in pancreatic extracts, juice and in blood have been the subject of numerous reports which have been reviewed by MandP, Ha114, and others. In an earlier paper5 we have commented on the lack of sensitivity of older techniques and pointed out that current more sensitive methods which utilize elastin substrates dyed with Congo Red or Orcein, give erroneous results in the presence of albumin and other plasma proteins. Moreover, the influence of other pancreatic enzymes, notably trypsin and chymotrypsin, on the action of elastase and their possible contribution to elastase readings in various assay systems have never been investigated. The need for a sensitive, selective method for determining pancreatic elastase thus remained. In this communication we describe new, highly sensitive and specific methods for assaying elastolytic activity based on substrates covalently labeled with chromophores or fluorophores. Using these techniques, we have selectively assayed elastase in pancreatic extracts and juice in the presence of trypsin and chymotrypsin.
* This work was supported by USPHS
Grants No. AM
04683-07 and AM
08293-04.
Clin. Chim. Acta,
19 (1968)
327-339
323 METHODS
RINDERKNECHT
AND
et d.
MATERIALS
Pre$aratio?z of Fluorescei+elastin Elastin powder (Worthington Biochem. Corp., Freehold, N.J.) (rg g) was suspended in 500 ml I M sodium bicarbonate solution in a 2-1 Erlenmeyer flask by means of a Vibromix. A filtered solution of 140 mg fluorescein isothiocyanate (Calbiochem, Los Angeles, Calif.) in 500 ml of acetone was added at once to the vigorously stirred suspension. Stirring was continued at room temperature for 30 min and the reaction mixture was centrifuged. The supernatant was discarded and the yellow precipitate washed with five 75o-ml portions of a mixture of acetone and 0.1 M sodium bicarbonate solution (I :I), two 75o-ml portions of 0.1 M sodium bicarbonate, five 75o-ml portions of water and two 75o-ml portions of methanol. The product was dried over phosphorus pentoxide in a vacuum desiccator protected from light, ground to a fine powder and stored in the cold with exclusion of moisture and light. Yield: rg g. Pre$aratiolz of Remazolbrilliant Blade-elastilz (RBB-elastin) A 1-1 Erlenmeyer flask containing a suspension of 20 g elastin powder in 200 ml of water was placed into a constant temperature water bath at 50’ and the contents were stirred with a magnetic stirrer or Vibromix. Remazolbrilliant Blue (Farbwerke Hoechst A.G., Frankfurt (M))Hoechst, Germany) (0.8 g) dissolved in zoo ml of water was added to the suspension and stirring continued. During the following 20 min sodium sulfate (40 g) was added in several portions. At the end of this period 2 g of trisodium phosphate in 50 ml of water was added in order to adjust the pH of the reaction mixture to II. Stirring at 50’ was continued for a further 30 min. The product was then centrifuged and the supernatant discarded. The RBB-elastin was resuspended in water, separated by centrifuging and this procedure was repeated until the supernatant was quite colorless. After 3 washes with 500 ml of methanol the material was dried in a vacuum desiccator over phosphorus pentoxide. The brilliant blue RBB--elastin (IS g) was then ground to a fine powder and stored under refrigeration and exclusion of moisture. Pre$aration of N&o-elastin Concentrated nitric acid (go:/o), 200 ml, was added to 20 g elastin powder in a 4-l beaker with stirring and external cooling with tap water. Stirring was continued for 5 min. Ice-water (2000 ml) was then added to the mixture with continued stirring and the suspended yellow product centrifuged off. The supernatant was discarded and the precipitate washed with five 5oo-ml portions of water, five 5oo-ml portions of 0.1 M sodium bicarbonate, four zoo-ml portions of water and finally with three 5oo-ml portions of methanol. The yellow material was dried in a vacuum desiccator. The dry product (IS g) was ground to a fine powder in a mortar and stored with refrigeration and exclusion of moisture. General assay conditions The following procedure was employed for the determination of elastolytic activity of authentic enzyme solutions, pancreatic extracts or juice. Variables as indicated below were : type and concentration of modified elastin, addition of inhibitors or additional enzymes or combinations thereof. All experiments were carried out Clin. Chinz. Acta,
rg (1968) 3~7-339
ELASTASE
DETERMINATION
329
in duplicate. Test tubes (0.5 x 15 cm) were charged with substrate (x mg); solution or test samples in Bicine buffer (N,N-bis-(z-hydroxyethyl)glycine,
enzyme Calbio-
them, Los Angeles, Calif.) 0.05 M, pH 8.8 were added and the final volume adjusted to 5 ml with the same buffer. The tubes were stoppered, placed in an incubator at 37’ protected from incident light and inverted every 5 min for an hour. The tubes were then removed from the incubator and placed in ice-water. The contents were filtered through Whatman No. 50 (No. I for RBB-elastin) filter paper and the filtrate assayed immediately in a spectrophotometer or fluorimeter. Blanks were obtained similarly (unless indicated otherwise) by adding authentic enzymes or elastase-containing test samples at the end of the incubation period, immediately before filtration of the mixture. Values obtained from blanks were subtracted from the readings of the test samples. Assay
with Fluorescein-elastin Elastase determinations were performed with 20 mg of substrate/tube. The incubation mixture was protected from incident light during incubation. After cooling in ice-water and filtration it was diluted immediately with g volumes of Bicine buffer, 0.05 M, pH 8.8, or more as required in order to obtain a fluorescence intensity reading preferably not exceeding 35 units. Fluorescence intensity (= o/otransmission scale on the photomultiplier) was measured in an Aminco-Kiers spectrophotofluorimeter (excitation wave length 500 nm, emission wave length 525 nm) with the photomultiplier scale set at 0.3 and the slit arrangement A,B,C,D,E, = 5,3,4,4,3. Test samples which furnished fluorescence intensity in excess of 200 units when recalculated for a I : IO dilution were rejected and reassayed at a lower concentration. The relationship of fluorescence intensity of I : 20 dilutions of incubation mixtures to elastase (porcine, electrophoretically purified, 8o-1oo~/~ pure, Worthington Biochem. Corp., Freehold, N.J.) concentration is illustrated in Fig. I,A. Determination of total elastase in pancreatic extracts and juice. Total elastolytic activity in pancreatic extracts and juice was assayed in the presence of excess trypsin (5000 pg) which liberates elastase and other proteolytic enzymes (chymotrypsin, endogenous trypsin, etc.) from their inactive zymogens. A set of standard curves (Fig. 4) was prepared which include corrections for the potentiating effect of trypsin (2 x tryst., salt free, Worthington Biochem. Corp.) (5000 pg) and varying amounts of a-chymotrypsin (3 x tryst., salt free, Mann Research Laboratories, New York) (o-1000 ,ug). Fluorescence intensity was plotted against chymotrypsin concentration for each level of elastase (Fig. 4). Total elastase concentration of test samples (incubated with 5000 ,ug trypsin) was determined with the aid of Fig. 4 from observed Auorescence intensity of the filtrates and the chymotrypsin concentration of test samples determined separately 6p7.Experience showed that blanks could be omitted in routine assays: blank readings, i.e., fluorescence readings obtained following incubation of substrate with 5000 ,I_LQ trypsin and addition of pancreatic juice or extract after incubation, were virtually unaffected by the presence or absence of such extracts or juice. There is, however, a measurable contribution to fluorescence arising from the elastolytic action of trypsin on the substrate (separate from the potentiating effect of this enzyme on elastase!), but this is the same in assays of test samples and authentic elastase solutions. Blanks are therefore redundant if standard curves are set up to include this constant “trypsin reading” for every point and if trypsin of the same Clin. Chim. Acta, 19 (1968)
327-339
RINDERKNECHT et al.
330
batch is used throughout. In a series of experiments one or two blanks were usually included to ascertain proper assay conditions. ~ete~~~~~t~5n of frea ~~~st~se 6~ ~u~~~ea~~c e&acts and juice. Samples of pancreatic extracts or juice were mixed with 5000 units of “Trasylol” (Protease inhibitor, Farbenfabriken Bayer A.C., Leverkusen, Germany, Preparation A 12S, I ml = 5000 KIU) and assayed with Fluorescein-elastin under standard conditions. Fluorescence .220 210 ,200
100
.I 90 .I 80
90
.I 70 .I 60
80
E c
.I 50
:
.I 40 :
-50
:
Q80
0 !i 0
.060
-40
,070 -30
5 fi =: &! 0 3 tL
- 20
-
0
2 )lg
1,
4
ELASTASE
I
6
I
,
I
8
,/5ml
I
I
10
I 12
/
INCUBATION
I
IO
I, 14
MIXTURE
Fig. I. Relationship of optical density (fluorescence intensity) to elastase concentration. h: Fluerescein-elastin (20 mg substrate/incubation tube), fluorescence readings represent I : .m dilutions of filtered incubation mixtures. B: RBB-elastin (20 mg substrate/incubation tube) read at 595 nm, C: Pu’itro-elastin (60 mg snbstrate-sorbitol mixtnre~~ilcubation tube) read at 446 nm.
readings observed were used to determine elastase concentrations from a standard curve which relates fluorescence intensity to increasing concentrations of elastase assayed in the presence of a constant amount of “Trasylol” (5000 units)?. As in the determination of total elastase, blanks were used only to determine proper assay conditions. Assay with RBB-elastiqt Assays were carried out with Clin. Chim.
A&,
19 (x968)
327-339
30
mg substrate/tube
and optical
density
of the
ELASTASE DETERMINATION
filtered incubation density to elastase
331
mixture was determined at 595 nm. The relationship concentration for this substrate is illustrated in Fig.
of optical Assays
I, B.
of pancreatic extracts and juice were performed in the presence of 5000 ,~g trypsin as described above for the Fluorescein-elastin substrate. Standard curves with corrections for the potentiating effects of trypsin and chymotrypsin were obtained analogously (Fig. 5). However, blanks obtained in the usual fashion were used with this substrate. In contrast to assays with Fluorescein-elastin in which variations in “native” fluorescence of extracts (not due to enzymatic action) can be neglected since such readings are very small, and in addition are taken of I :IO to I: IOO diiutions, variations in light absorption by individual extracts assayed with RBB-elastin are much more likely to constitute a measurable part of the total absorption observed after incubation, and experimental readings therefore require the correction of a blank. Assay
with Nitro- elastin To facilitate uniform dispersion Nitro-elastin was mixed with an equal part of sorbitol and the mixture ground to a fine powder. Assays were performed with 60 mg of substrate mixture/tube. Optical density of the filtered incubation mixture was measured at 446 nm. A standard curve for this substrate was obtained by plotting optical density vs. elastase concentration (Fig. I,C). Total solubilization of elastin substrate Graded amounts of each substrate
were incubated
in Bicine
0450-
buffer
0.05
M,
, ,/'X
0425 /'
0.4000.3750.3500325 2 0.300 2 0.273-
-200 -I50 0.125-
$ z u" L1: 2 5 ,'
--I00
0.1
0.2
0.3
0.4 0.5 0.6 mg SUBSTRATE/ml
0.7
0.6
09
1.0
Fig. 2. Total solubilisation of elastin substrates. Relationship of amount of substrate dissolved to optical density (fluorescence intensity). D : Fluorescein-elastin (fluorescence readings represent I : IO dilutions of the incubation mixture). E : RBB-elastin, read at 595 nm. F: Nitro-elastin, read at 446 nm. C&?. Chim. 4ctn. TO (1968) 327--119
332
et ad.
RINDERKNECHT
pH 8.8 with an excess elastase (300 pug/ml)until complete solubilization was obtained. The relationship of fluorescence intensity or optical density to amount of substrate solubilized is illustrated in Fig. z,D,E and F. Pancreatic
mtracts
frozen pancreatic tissue was homogenized with rg parts of o.og M Bicine buffer pH 8.8 in the cold room. The homogenate was centrifuged for 30 min in a refrigerated centrifuge at 78500 g. The clear supernatant (I ml/assay tube) or dilutions thereof were used for elastase assays. Fre;sh
RESULTS
Fig. r,A,B, and C, shows the correlation of optical density or fluorescence intensity and increasing amounts of enzyme in our assay with each of three different substrates and indicates the respective range of elastase concentrations within which zero order kinetics can be observed. The relationship between optical density or fluorescence and the amount of each of the three substrates dissolved by elastase is represented by Fig. z,D,E and F. With the aid of the two sets of plots (Figs. I and 2) elastase readings in micrograms can be converted to elastase units. An elastase unit is defined as the amount of elastase capable of digesting I mg of substrate by incubation at 37’ for I h under standard conditions. The number of micrograms of elastasej unit is dependent upon the susceptibility of the substrate to the action of elastase and varies for the three elastin derivatives described in this paper (Table I). I
TABLE
sUSCE&‘TIBlLTTY OF ELrisTIN SUBSTRATES -
--------
Substrate
___~. Coilgo
Red--elastin
TO
ELASTASE,
TRYPSIN
AND
CHYMOTRYPSIN
-----
pg Pwe elastase equiv!alent to I unit (assayed with:)
rq
A’o. of molrcu.& of waavkw per
1000 alnino
Relative sapsceptibility to elastase
acid residues
7
Fluorescein-elastin z3 0.9 4.5 RBB-elastin 3.4 3 33 DNS-elastin 2.1 2.5 4o ^. Nitro-elastin I8*’ 2 50 -._-. __ -_.._ ~__.. ~~_~_~ ~ * Values obtained with trypsin of different sources. ** Value obtained by spectrophotometric assay of hydrolysate, based of 3.5.dinitrotyrosine.
Susceptibility to ~000 &Lgof trypsin. Equivalent to peg rlastasP _
0
I.1 0.75, 1.0* o, 1.5*, 6.0* 3.0 ~~~ -...
on E,,,:
6.3x
1r.s 103,
A,,,
: 342
Although our new substrates possess a high order of specificity for elastase they are susceptible to a very minor degree to high concentrations of chymotrypsin and trypsin. Table I lists the susceptibility of five elastase substrates to trypsin and chymotrypsin in microgram equivalents of elastase. Variations in the results obtained with trypsin are due to the use of different batches of trypsin and probably reflect contamination of trypsin with traces of elastase. Since our investigations are primarily concerned with human elastase whose occurrence in pancreatic tissue is still controversial*, we directed most of our efforts toward the development of the method with the highest sensitivity, i.e., the assay based on Fluorescein-elastin. In a series CEin. Chivn. Acta,
rg (1968) 327-339
nm
ELASTASE DETERMINATION
333
of experiments we incubated increasing concentrations of trypsin or chymotrypsin with Fluorescein-elastin under standard conditions, with and without the addition of z ,ug of elastase/s ml incubation mixture. The results, presented in Fig. 3, show that both trypsin and chymotrypsin potentiate the action of elastase. Since all three enzymes are present in pancreatic extracts or juice, their combined synergistic action will be measured in any assay utilizing an elastin substrate. Accordingly, we undertook a study designed to provide a basis for the determination of elastase in the pres-
,a--* ,3’;2
~0
_@--
_--0
C
ELASTASE
.--4
-I
5000
~4
CHYMOTRYPSIN
IR_Y_tzfj
PER
5ml
INCUBATION
MIXTURE
Fig. 3. Potentiation of elastase activity by trypsin and chymotrypsin. Determination with Fluorescein-e&tin. Fluorescence readings represent r : IO dilutions of filtered incubation mixtures. A: Elastolytic action of trypsin alone. B: Elastolytic action of chvmotrvpsin alone. C: Potentiating effect of trypsin on ;*IJ~ elastase. D : Potentiating elfect of chymotripsin on 2 pg elastase.
ence of chymotrypsin and trypsin. Chymotrypsin, trypsin and elastase normally occur in pancreatic tissue and juice in the form of enzymatically inactive zymogens which can be activated by exogenous trypsin. Pancreatic tissue and juice also contain several inhibitors of these enzymes8-12. In order to minimize their effect and to complete the activation process the addition of a large excess of trypsin to the incubation mixture is necessary. Thus, five different concentrations of elastase (o-3 ,~~g/3ml incubation mixture) were assayed in the presence of a constant amount of trypsin (5000 pug) and varying concentrations of chymotrypsin (o-1000 ,ug). The results of these experiments which were carried out with two different substrates, Fluoresceinelastin and RBB-elastin, are summarized in Figs. 4 and 5. Each curve represents the relationship of fluorescence intensity or optical density to a given elastase concentration, a constant amount of trypsin and varying concentrations of chymotrypsin. The intercept of the chymotrypsin concentration of a sample of pancreatic juice or extract (determined as indicated under METHODS) and the fluorescence or optical Clin. Chim. Acta, rg (1968) 327-339
334
RIh-DERKNECHT
et Cd.
density reading obtained by the elastase assay in the presence of 5000 pug of trypsin furnishes, by interpolation, the true elastase value, corrected for potentiation by trypsin and chymotrypsin, all present in the pancreatic extract or juice. It should be noted that the only parameters that need to be measured are elastase and chymotrypsin. Endogenous trypsin is completely “blotted out” in this system by the addition of a very large excess of exogenous trypsin. Variations of a few hundred micrograms in the amount of endogenous trypsin likely to be present in the sample of extract or juice to be tested, are of no consequence as the increase in fluorescence due to the presence of trypsin is only minor between the IOOO and 5000 pg level of trypsin, (cf. Fig. 3, curves A and C). 5 cg
230
5000
ELASTASE pg TRYPSIN
220 210
41~9 EL 5000~~
.21rg
TRY
EL
5OOOvgTRY
IIrg EL 5000
__a---
--
~9
5006~pg
owaEL
5OOOvg
0 pg
200 CHYMOTRYPSIN
400
600 /
5ml
600 INCUBATION
TRY
TRY
TRY
1000 MIXTURE
Fig. 4. Standard curves for the determination of total elastase with Fluorescein-elastin, corrected for the potentiating effect of trypsin and chymotrypsin. Fluorescence readings represent I: IO dilutions of filtered incubation mixture. “Trypsin blanks” (cf. p. 329) are included in the readings. Bars represent readings obtained with elastase alone.
In a study of the reproducibility of our new method we assayed a I : 5 dilution of a sample of human pancreatic extract IO times. The chymotrypsin content of the I : 5 dilution was 36 ,ug/ml. The average fluorescence reading of the incubation mixtures (I :IO dilutions) was 34.8 fluorescence units with a standard deviation of 5 1.7. The elastase content obtained from Fig. 4 was 0.26 & 0.05 ,ug elastase/ml diluted Clin. Chim. Acta,
19 (1968) 327-339
ELASTASE
335
DETERMINATION
or 5 x (0.26 & 0.05) = 1.3 * 0.25 pg elastase/ml original extract. We checked the reliability and sensitivity of our method by assaying several samples of human pancreatic juice or extract with and without the addition of a small amount of authentic elastase. Using data obtained from assays of pancreatic juice or extracts we also reconstituted such samples by mixing appropriate arnounts of authentic enzymes and reassaying these test mixtures. Table II lists the results of these experiments. 5 pg
5000~9
ELASTASE TRYPSIN
4vg EL 5000 ~(1 TRY
3vg EL 50OOl~g
TRY
21~9 EL 5OOOpg
TRY
lpg EL 5000 pg TRY
Opg EL 5OOOpg
pg CHYMOTRYPSIN
,&I
INCUBATION
TRY
MIXTURE
Fig. 5. Standard curves for the determination of total elastase with RBB-elastin, corrected for the potentiating effect of trypsin and chymotrypsin. “Substrate blanks” are subtracted from all readings. Bars represent readings obtained with elastase alone.
Although proteolytic enzymes in pancreatic tissue and juice normally occur in the form of their inactive proenzymes, free proteolytic activity in pancreas and pancreatic juice of patients suffering from acute pancreatitis has recently been found by Geokas et a1.7913. The technique for the estimation of total elastase described above obviously cannot be employed for such samples since the addition of trypsin would liberate further quantities of proteolytic enzymes still present in the form of inactive zymogens. We therefore attempted to inbibit selectively chymotrypsin and trypsin, the enzymes known to interfere with the determination of elastase, by addition of the specific trypsin and chymotrypsin inhibitor “TrasylolJJ7 to the incubation mixture. In a series of experiments designed to determine the amount of inhibitor needed to cancel the potentiating action of trypsin and chymotrypsin on elastase we made C&n. Chim.
Acta,
19 (1968)
327-339
RINDERKXECHT
336 TABLE
et al.
II
RECOVERYOF ELASTASE;ADDED Material
TO
PANCREATIC
Chymotrypsin
fOundP&T
SAMPLES
(added) /ml
-.
AND
IN -_.
Elastase found pug/ml
ARTIFICIAL
MIXTURES
Elastase added fig/ml
Total elastasp recovered /q/ml
_
Pancreatic extract # PEH H-66
‘5
0.15
I.0
I.0
Pancreatic extract # PEB H-66
18
0.15
0.5 I .o
0.65 1.1
Pancreatic extract # PET H-66
20
0.35
0.5
0.75 7.4
Pancreatic juice # PJ M-22
26
Pancreatic juice # PJ M-5-24
252
0.5
Pancreatic juice # PJ P-8-18
450
13.0
Mixture #I Mixture #r Mixture #3 ~~~ ______. * Determination ** Determination *** Determination
I.0
15 33 4o
I.0
IO.0
5 .o
rq.o*
6.0**
IO.0
22.0***
20.0
36.0
0.4 I.3 2.3
__-. carried out with a I : IO dilution of pancreatic juice. carried out with a I : 5 dilution of pancreatic juice. carried out with a I : 20 dilution of pancreatic extract.
0.4 1.4 2.3
several interesting observations: Elastase was only slightly inhibited by “Trasylol”; a z5oo-fold (w/w) excess of inhibitor was needed to reduce enzyme activity to 500,/o. A “Trasylol” level sufficient to reduce the activity of a mixture of chymotrypsin and trypsin to a base line value (near zero) was not adequate to suppress the potentiating effect of these enzymes on elastase activity. An overwhelming excess (5000 units) of “Trasylol” was necessary to minimize the interaction of the three enzymes. A reasonable estimate of the free elastase content of pancreatic extracts or juice may be obtained if such samples are assayed in the presence of 5000 units of “Trasylol” and elastase values are read off a standard curve in which fluorescence intensity is plotted against increasing concentrations of elastase assayed in the presence of aconstant amount of “Trasylol” (5000 units). Details of these findings are being published elsewhere’. DISCUSSION
The phenomenal success of covalently labeled fluorescent antibodies in immunology and microbiology prompted us to employ similar techniques in the construction of substrates for analytical enzymology. We have recently reported on the assay of elastolytic activity in mammalian plasma with the aid of a fluorescent elastin substrate labeled with dimethylaminonaphthalenesulfonyl chlorideI and on a new calorimetric amylase determination based on a starch substrate possessing the covalently attached marker Remazolbrilliant Bluers. The present study summarizes our specific substrates for the estimation of work on the design of highly sensitive, elastase in pancreatic extracts and juice. Unlike the staining of elastin with dyes such as Congo Red or Orcein which involves ionic and hydrogen bonds only, covalent Clin.
Chim.
Acta,
19 (1968)
327-339
ELASTASE
DETERMINATION
337
linkage of a chromophore or fluorophore to a protein substrate can be expected to result in more or less profound conformational changes and loss of specificity. This has been demonstrated amply in the fluorescent labeling of antibody where overlabeling causes drastic reduction or abolition of antibody titer. Following these guide lines we prepared the elastin substrates listed in Table I. Comparison of column z (degree of substitution) with column 3 (susceptibility to elastase) shows that susceptibility to elastase decreases with increasing substitution. The specificity of the substrate (resistance to trypsin and chymotrypsin, columns 4 and 5) is likewise affected adversely by an increase in the number of substituents. The anomalous position of RBB-elastin may be explained by the assumption that the RBB-label is attached at a different site of the substrate less likely to hinder the attack by the enzyme. In contrast to fluorescein isothiocyanate and dimethylaminonaphthalenesulfonyl chloride which react primarily with free amino groups. Hydroxyl, Remazolbrilliant Blue readily forms an ether linkage with secondary hydroxyl groups. Hydroxyl groups predominate over free amino groups in elastin by a ratio of at least IO: T. Our observations on the influence of substitution on substrate susceptibility and specificity are underscored by the finding that more extensive labeling than indicated in the experimental section with any individual reagent resulted in inferior substrates of reduced susceptibility to elastase and decreased resistance to trypsin and chymotrypsin. Although elastin-Congo Red which is the least modified substrate possesses the highest susceptibility and specificity (see Table I), Fluorescein-elastin is at least five times more sensitive in our assay system than elastin-Congo Red; indeed its sensitivity is mostly limited by its solubility, however slight, in the incubation mixture which provides enough fluorescence to render the measurement of minute differences inaccurate. The sensitivity and specificity of RBB-elastin is of the same order as that of elastin-Congo Red, even though its susceptibility to elastase is reduced still further than that of Fluorescein-elastin. It is an excellent substrate for calorimetric estimation of elastase and is, in contrast to elastin-Congo Red or Orcein, not affected by albumin or other protein9. DNSelastin, still lower on the scale of susceptibility, is a much less sensitive substrate than Fluorescein-elastin, but possesses the advantage of yielding degradation products of greater fluorescence stability than the latter. Indeed, we found that incubation of peptides derived from Fluorescein-elastin with blood plasma (0.5) ml in the presence of trypsin (4000 ,ug) under standard conditions yielded less fluorescence when compared with that of blanks from which plasma had been omitted. The fluorescence decay appears to involve an enzymatic process since addition of boiled plasma or fresh plasma at the end of the incubation with trypsin causes no reduction in fluorescence. Loss of fluorescence of fluorescein isothiocyanate due to the presence of biological materials has been reported recently by Winkelman and Grossman16. It should be emphasized here that assays with fluorescent substrates must be carried out under rigorously standardized conditions with exclusion of actinic or fluorescent light during incubation. Nitro-elastin which possesses the lowest susceptibility to elastase and the lowest specificity of our substrates, is nevertheless equal in sensitivity to RBB-elastin and elastin-Congo Red. This is evidently due to the large number of chromophore groups Clin.
Chim.
Acta.
19 (1968) 327-339
338
RINDERKNECHT
et d.
(-NO,) introduced into the elastin molecule. The usefulness of this substrate is limited by its inferior specificity. It was first reported by Graham I7, that trypsin when added to elastase gave greater elastolysis than elastase alone although trypsin by itself had no elastolytic effect. W:e have confirmed these findings and extended them to chymotrypsin. It can be seen from Fig. 3 that potentiation of elastase activity by chymotrypsin is much more pronounced than that observed with trypsin. When all three enzymes are present in the incubation mixture the potentiating effects of both, trypsin and chymotrypsin, are approximately additive to the action of elastase as illustrated by Figs. 4 and 5. For example: the fluorescence reading for 2 ,ug elastase potentiated by 5000 pg of trypsin alone is 80 (Fig. 4). The potentiating effect of 600 ,ug of chymotrypsin on 2 pg of elastase is 78-40 = 38 (Fig. 3,D). The total fluorescence reading for 2 ,ug of elastase in the presence of 5000 ,ug trypsin and 600 pg of chymotrypsin therefore should be: 80+38 = 118 fluorescence units. The actual fluorescence intensity found was 125 (Fig. 4). It follows from these observations that the data reported in the literature on measurements of elastolytic activity in pancreatic extracts and juice do not represent elastase, but the combined synergistic action of elastase and other proteolytic enzymes. A glance at Fig. 4 shows that previously reported levels of pancreatic “elastase” are likely to be more than one hundred percent too high. Moreover, there are indications that our assay system, although corrected for the synergistic action of trypsin and chymotrypsin, still lacks sufficient selectivity to exclusively measure elastoproteinase (EJ. In the course of an investigation of pancreatic juice with an unusually high “elastase” content we observed that addition of authentic elastase (I ,ug) to the juice led to a recovery of over ZOO~!~in our assay. However, when we added authentic elastase to dilutions of the original juice, made to contain less than I ,ug elastase/5 ml incubation mixture, we recovered the correct amount of enzyme (ca. I pg). We interpret these findings as indicating the presence in pancreatic juice of an enhancing factor other than trypsin or chymotrypsin, which is diluted out in our assay when dilutions of juice are used to give readings of less than I pg elastase/s ml incubation mixture. We have no information regarding the nature or identity of this enhancing factor, but feel that elastomucase (E,)l* and collagenase are logical contenders for this function. The possible influence of pancreatic inhibitors on elastase readings obtained in our assav system cannot be assessed at present. Further refinement of our technique to include such parameters must await availability of these factors and a considerable amount of additional work. Although we have no experimental data as yet which might explain the nature of the potentiating effect of trypsin and chymotrypsin on elastase activity, we feel tempted to speculate that chymotrypsin and trypsin may cause conformational changes of the elastin substrate rendering certain sites more susceptible to the action of elastase. Alternatively, chymotrypsin and trypsin may interact with elastase. Although the mechanism of enzyme potentiation remains to be clarified, our findings should contribute to the understanding not only of normal digestion, but also of the pathological process of autodigestion so strikingly exhibited in acute hemorrhagic pancreatitis. Indeed, our studies on the synergistic effect of trypsin and chymotrypsin on elastase seem to support views recently expressed which assign a central role in the pathogenesis of this disease to elastase 13, the enzyme for which this assay was developed.
ELASTASE
DETERMINATION
339
REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14
D. A. HALL, Elastolysis and Ageing, Charles C. Thomas, Springfield, Ill., 1964. M. C. GEOKAS, R. D. MCKENNA .%ND I. T. BECK, Gastvoentevology, 50 (1966) 882. I. MANDL, Advan. Enzymol., 23 (1961) 163. D. A. HALL, Biochem. J., IOI (1966) 29. M. C. GEOK~S, P. WILDING, H. RINDERKNECHT AND B. J. HAVERBACK, Clin. Biochem., (1968). J. KALLOS, E. L. ARTHUR, D. RIZOCK AND D. K~Hx, Can. J. Biochem., 43 (1965) 135. M. C. GEOKAS, H. RINDERKNECHT, I’. WILDING, \-. LILLARD, C. J. BERNE .&ND B. J. HAVERBACK, Gastroenterology. in press (1967). 1~. LAMY AND S. TAUBER, J. Biol. Chcm., 238 (1963) 939. W. A. LOEVEX, Acta PhJjs,siol.Phwmucol. h’eevl., IO (1962) 2~8. M. KUNITZ AND J. H. NORTHROP, J. &%z. Physiol., 19 (1936) 991. L. A. KAZAL, D. S. SPICER AND R. A. BRAHINSKY, J. .41x. Chem. Sot., 70 (1948) 3034. J. L. GREENE, M. RIGBI AP*‘DD. S. FACKRE, J. Biol. Ckewz., 141 (1966) 5610. M. C. GEOKAS, H. RINDERKNBCHT, V. SWANSOX ‘YND B. J. HAVERBACK, to be published. H. RINDERKNECHT, M. C. G;EOK,ZS, I’. SILVERXIN .UD B. J. HAVERB.%CK, Cbi~. Chiw. z4cta, 19
15 16 17 18
(1968) 89. H. RINDERKXECHT, P. WILDING AND B. J. H.~vERB.%cI~, Experientia, J. WINKELMAN AND J. GROSSMAN, ,4nal. Chem., 39 (1967) 8. G. N. GRAHAM, Biochem. J., 75 (1960) 15 P. W. A. LOEVEN, Intern. Review Colznective Tissue Res., I (1963) 202. Clips. Chim.
23 (1967)
Acta,
805.
19 (1968) 327-339
NEW
CHIMICA
327
ACTA
METHODS
FOR
THE
DETERMINATION
c. GEOKAS,P.SILVERMAN,Y.LILLAKD
H. RINDERKNECHT,M. B.J.HAVERBACK Department of Medicine, Hospital,
Los Angeles,
(Received November
OF ELASTASE”
University of Southern Calif. 90033 (U.S.A.)
Califwnia
and the Los
.~ND Angeles
Couuty
Geneva1
Ist,1967)
SUMMARY
Highly sensitive fluorimetric and calorimetric methods for estimation of pancreatic elastase have been developed. Two covalently labeled substrates, Fluorescein-elastin and Remazolbrilliant Blue-elastin, were prepared for this purpose. Pancreatic elastase has been assayed selectively in the presence of trypsin and chymotrypsin. The potentiating effect of these enzymes on the activity of elastase is described. The influence of this synergism on elastase determinations and its significance in acute pancreatic disease is discussed.
INTRODUCTION
Recent work suggesting a key role for elastase in the aging process1 and acute pancreatiti9 has drawn considerable attention to this enzyme. Methods for the determination of elastase in pancreatic extracts, juice and in blood have been the subject of numerous reports which have been reviewed by MandP, Ha114, and others. In an earlier paper5 we have commented on the lack of sensitivity of older techniques and pointed out that current more sensitive methods which utilize elastin substrates dyed with Congo Red or Orcein, give erroneous results in the presence of albumin and other plasma proteins. Moreover, the influence of other pancreatic enzymes, notably trypsin and chymotrypsin, on the action of elastase and their possible contribution to elastase readings in various assay systems have never been investigated. The need for a sensitive, selective method for determining pancreatic elastase thus remained. In this communication we describe new, highly sensitive and specific methods for assaying elastolytic activity based on substrates covalently labeled with chromophores or fluorophores. Using these techniques, we have selectively assayed elastase in pancreatic extracts and juice in the presence of trypsin and chymotrypsin.
* This work was supported by USPHS
Grants No. AM
04683-07 and AM
08293-04.
Clin. Chim. Acta,
19 (1968)
327-339
323 METHODS
RINDERKNECHT
AND
et d.
MATERIALS
Pre$aratio?z of Fluorescei+elastin Elastin powder (Worthington Biochem. Corp., Freehold, N.J.) (rg g) was suspended in 500 ml I M sodium bicarbonate solution in a 2-1 Erlenmeyer flask by means of a Vibromix. A filtered solution of 140 mg fluorescein isothiocyanate (Calbiochem, Los Angeles, Calif.) in 500 ml of acetone was added at once to the vigorously stirred suspension. Stirring was continued at room temperature for 30 min and the reaction mixture was centrifuged. The supernatant was discarded and the yellow precipitate washed with five 75o-ml portions of a mixture of acetone and 0.1 M sodium bicarbonate solution (I :I), two 75o-ml portions of 0.1 M sodium bicarbonate, five 75o-ml portions of water and two 75o-ml portions of methanol. The product was dried over phosphorus pentoxide in a vacuum desiccator protected from light, ground to a fine powder and stored in the cold with exclusion of moisture and light. Yield: rg g. Pre$aratiolz of Remazolbrilliant Blade-elastilz (RBB-elastin) A 1-1 Erlenmeyer flask containing a suspension of 20 g elastin powder in 200 ml of water was placed into a constant temperature water bath at 50’ and the contents were stirred with a magnetic stirrer or Vibromix. Remazolbrilliant Blue (Farbwerke Hoechst A.G., Frankfurt (M))Hoechst, Germany) (0.8 g) dissolved in zoo ml of water was added to the suspension and stirring continued. During the following 20 min sodium sulfate (40 g) was added in several portions. At the end of this period 2 g of trisodium phosphate in 50 ml of water was added in order to adjust the pH of the reaction mixture to II. Stirring at 50’ was continued for a further 30 min. The product was then centrifuged and the supernatant discarded. The RBB-elastin was resuspended in water, separated by centrifuging and this procedure was repeated until the supernatant was quite colorless. After 3 washes with 500 ml of methanol the material was dried in a vacuum desiccator over phosphorus pentoxide. The brilliant blue RBB--elastin (IS g) was then ground to a fine powder and stored under refrigeration and exclusion of moisture. Pre$aration of N&o-elastin Concentrated nitric acid (go:/o), 200 ml, was added to 20 g elastin powder in a 4-l beaker with stirring and external cooling with tap water. Stirring was continued for 5 min. Ice-water (2000 ml) was then added to the mixture with continued stirring and the suspended yellow product centrifuged off. The supernatant was discarded and the precipitate washed with five 5oo-ml portions of water, five 5oo-ml portions of 0.1 M sodium bicarbonate, four zoo-ml portions of water and finally with three 5oo-ml portions of methanol. The yellow material was dried in a vacuum desiccator. The dry product (IS g) was ground to a fine powder in a mortar and stored with refrigeration and exclusion of moisture. General assay conditions The following procedure was employed for the determination of elastolytic activity of authentic enzyme solutions, pancreatic extracts or juice. Variables as indicated below were : type and concentration of modified elastin, addition of inhibitors or additional enzymes or combinations thereof. All experiments were carried out Clin. Chinz. Acta,
rg (1968) 3~7-339
ELASTASE
DETERMINATION
329
in duplicate. Test tubes (0.5 x 15 cm) were charged with substrate (x mg); solution or test samples in Bicine buffer (N,N-bis-(z-hydroxyethyl)glycine,
enzyme Calbio-
them, Los Angeles, Calif.) 0.05 M, pH 8.8 were added and the final volume adjusted to 5 ml with the same buffer. The tubes were stoppered, placed in an incubator at 37’ protected from incident light and inverted every 5 min for an hour. The tubes were then removed from the incubator and placed in ice-water. The contents were filtered through Whatman No. 50 (No. I for RBB-elastin) filter paper and the filtrate assayed immediately in a spectrophotometer or fluorimeter. Blanks were obtained similarly (unless indicated otherwise) by adding authentic enzymes or elastase-containing test samples at the end of the incubation period, immediately before filtration of the mixture. Values obtained from blanks were subtracted from the readings of the test samples. Assay
with Fluorescein-elastin Elastase determinations were performed with 20 mg of substrate/tube. The incubation mixture was protected from incident light during incubation. After cooling in ice-water and filtration it was diluted immediately with g volumes of Bicine buffer, 0.05 M, pH 8.8, or more as required in order to obtain a fluorescence intensity reading preferably not exceeding 35 units. Fluorescence intensity (= o/otransmission scale on the photomultiplier) was measured in an Aminco-Kiers spectrophotofluorimeter (excitation wave length 500 nm, emission wave length 525 nm) with the photomultiplier scale set at 0.3 and the slit arrangement A,B,C,D,E, = 5,3,4,4,3. Test samples which furnished fluorescence intensity in excess of 200 units when recalculated for a I : IO dilution were rejected and reassayed at a lower concentration. The relationship of fluorescence intensity of I : 20 dilutions of incubation mixtures to elastase (porcine, electrophoretically purified, 8o-1oo~/~ pure, Worthington Biochem. Corp., Freehold, N.J.) concentration is illustrated in Fig. I,A. Determination of total elastase in pancreatic extracts and juice. Total elastolytic activity in pancreatic extracts and juice was assayed in the presence of excess trypsin (5000 pg) which liberates elastase and other proteolytic enzymes (chymotrypsin, endogenous trypsin, etc.) from their inactive zymogens. A set of standard curves (Fig. 4) was prepared which include corrections for the potentiating effect of trypsin (2 x tryst., salt free, Worthington Biochem. Corp.) (5000 pg) and varying amounts of a-chymotrypsin (3 x tryst., salt free, Mann Research Laboratories, New York) (o-1000 ,ug). Fluorescence intensity was plotted against chymotrypsin concentration for each level of elastase (Fig. 4). Total elastase concentration of test samples (incubated with 5000 ,ug trypsin) was determined with the aid of Fig. 4 from observed Auorescence intensity of the filtrates and the chymotrypsin concentration of test samples determined separately 6p7.Experience showed that blanks could be omitted in routine assays: blank readings, i.e., fluorescence readings obtained following incubation of substrate with 5000 ,I_LQ trypsin and addition of pancreatic juice or extract after incubation, were virtually unaffected by the presence or absence of such extracts or juice. There is, however, a measurable contribution to fluorescence arising from the elastolytic action of trypsin on the substrate (separate from the potentiating effect of this enzyme on elastase!), but this is the same in assays of test samples and authentic elastase solutions. Blanks are therefore redundant if standard curves are set up to include this constant “trypsin reading” for every point and if trypsin of the same Clin. Chim. Acta, 19 (1968)
327-339
RINDERKNECHT et al.
330
batch is used throughout. In a series of experiments one or two blanks were usually included to ascertain proper assay conditions. ~ete~~~~~t~5n of frea ~~~st~se 6~ ~u~~~ea~~c e&acts and juice. Samples of pancreatic extracts or juice were mixed with 5000 units of “Trasylol” (Protease inhibitor, Farbenfabriken Bayer A.C., Leverkusen, Germany, Preparation A 12S, I ml = 5000 KIU) and assayed with Fluorescein-elastin under standard conditions. Fluorescence .220 210 ,200
100
.I 90 .I 80
90
.I 70 .I 60
80
E c
.I 50
:
.I 40 :
-50
:
Q80
0 !i 0
.060
-40
,070 -30
5 fi =: &! 0 3 tL
- 20
-
0
2 )lg
1,
4
ELASTASE
I
6
I
,
I
8
,/5ml
I
I
10
I 12
/
INCUBATION
I
IO
I, 14
MIXTURE
Fig. I. Relationship of optical density (fluorescence intensity) to elastase concentration. h: Fluerescein-elastin (20 mg substrate/incubation tube), fluorescence readings represent I : .m dilutions of filtered incubation mixtures. B: RBB-elastin (20 mg substrate/incubation tube) read at 595 nm, C: Pu’itro-elastin (60 mg snbstrate-sorbitol mixtnre~~ilcubation tube) read at 446 nm.
readings observed were used to determine elastase concentrations from a standard curve which relates fluorescence intensity to increasing concentrations of elastase assayed in the presence of a constant amount of “Trasylol” (5000 units)?. As in the determination of total elastase, blanks were used only to determine proper assay conditions. Assay with RBB-elastiqt Assays were carried out with Clin. Chim.
A&,
19 (x968)
327-339
30
mg substrate/tube
and optical
density
of the
ELASTASE DETERMINATION
filtered incubation density to elastase
331
mixture was determined at 595 nm. The relationship concentration for this substrate is illustrated in Fig.
of optical Assays
I, B.
of pancreatic extracts and juice were performed in the presence of 5000 ,~g trypsin as described above for the Fluorescein-elastin substrate. Standard curves with corrections for the potentiating effects of trypsin and chymotrypsin were obtained analogously (Fig. 5). However, blanks obtained in the usual fashion were used with this substrate. In contrast to assays with Fluorescein-elastin in which variations in “native” fluorescence of extracts (not due to enzymatic action) can be neglected since such readings are very small, and in addition are taken of I :IO to I: IOO diiutions, variations in light absorption by individual extracts assayed with RBB-elastin are much more likely to constitute a measurable part of the total absorption observed after incubation, and experimental readings therefore require the correction of a blank. Assay
with Nitro- elastin To facilitate uniform dispersion Nitro-elastin was mixed with an equal part of sorbitol and the mixture ground to a fine powder. Assays were performed with 60 mg of substrate mixture/tube. Optical density of the filtered incubation mixture was measured at 446 nm. A standard curve for this substrate was obtained by plotting optical density vs. elastase concentration (Fig. I,C). Total solubilization of elastin substrate Graded amounts of each substrate
were incubated
in Bicine
0450-
buffer
0.05
M,
, ,/'X
0425 /'
0.4000.3750.3500325 2 0.300 2 0.273-
-200 -I50 0.125-
$ z u" L1: 2 5 ,'
--I00
0.1
0.2
0.3
0.4 0.5 0.6 mg SUBSTRATE/ml
0.7
0.6
09
1.0
Fig. 2. Total solubilisation of elastin substrates. Relationship of amount of substrate dissolved to optical density (fluorescence intensity). D : Fluorescein-elastin (fluorescence readings represent I : IO dilutions of the incubation mixture). E : RBB-elastin, read at 595 nm. F: Nitro-elastin, read at 446 nm. C&?. Chim. 4ctn. TO (1968) 327--119
332
et ad.
RINDERKNECHT
pH 8.8 with an excess elastase (300 pug/ml)until complete solubilization was obtained. The relationship of fluorescence intensity or optical density to amount of substrate solubilized is illustrated in Fig. z,D,E and F. Pancreatic
mtracts
frozen pancreatic tissue was homogenized with rg parts of o.og M Bicine buffer pH 8.8 in the cold room. The homogenate was centrifuged for 30 min in a refrigerated centrifuge at 78500 g. The clear supernatant (I ml/assay tube) or dilutions thereof were used for elastase assays. Fre;sh
RESULTS
Fig. r,A,B, and C, shows the correlation of optical density or fluorescence intensity and increasing amounts of enzyme in our assay with each of three different substrates and indicates the respective range of elastase concentrations within which zero order kinetics can be observed. The relationship between optical density or fluorescence and the amount of each of the three substrates dissolved by elastase is represented by Fig. z,D,E and F. With the aid of the two sets of plots (Figs. I and 2) elastase readings in micrograms can be converted to elastase units. An elastase unit is defined as the amount of elastase capable of digesting I mg of substrate by incubation at 37’ for I h under standard conditions. The number of micrograms of elastasej unit is dependent upon the susceptibility of the substrate to the action of elastase and varies for the three elastin derivatives described in this paper (Table I). I
TABLE
sUSCE&‘TIBlLTTY OF ELrisTIN SUBSTRATES -
--------
Substrate
___~. Coilgo
Red--elastin
TO
ELASTASE,
TRYPSIN
AND
CHYMOTRYPSIN
-----
pg Pwe elastase equiv!alent to I unit (assayed with:)
rq
A’o. of molrcu.& of waavkw per
1000 alnino
Relative sapsceptibility to elastase
acid residues
7
Fluorescein-elastin z3 0.9 4.5 RBB-elastin 3.4 3 33 DNS-elastin 2.1 2.5 4o ^. Nitro-elastin I8*’ 2 50 -._-. __ -_.._ ~__.. ~~_~_~ ~ * Values obtained with trypsin of different sources. ** Value obtained by spectrophotometric assay of hydrolysate, based of 3.5.dinitrotyrosine.
Susceptibility to ~000 &Lgof trypsin. Equivalent to peg rlastasP _
0
I.1 0.75, 1.0* o, 1.5*, 6.0* 3.0 ~~~ -...
on E,,,:
6.3x
1r.s 103,
A,,,
: 342
Although our new substrates possess a high order of specificity for elastase they are susceptible to a very minor degree to high concentrations of chymotrypsin and trypsin. Table I lists the susceptibility of five elastase substrates to trypsin and chymotrypsin in microgram equivalents of elastase. Variations in the results obtained with trypsin are due to the use of different batches of trypsin and probably reflect contamination of trypsin with traces of elastase. Since our investigations are primarily concerned with human elastase whose occurrence in pancreatic tissue is still controversial*, we directed most of our efforts toward the development of the method with the highest sensitivity, i.e., the assay based on Fluorescein-elastin. In a series CEin. Chivn. Acta,
rg (1968) 327-339
nm
ELASTASE DETERMINATION
333
of experiments we incubated increasing concentrations of trypsin or chymotrypsin with Fluorescein-elastin under standard conditions, with and without the addition of z ,ug of elastase/s ml incubation mixture. The results, presented in Fig. 3, show that both trypsin and chymotrypsin potentiate the action of elastase. Since all three enzymes are present in pancreatic extracts or juice, their combined synergistic action will be measured in any assay utilizing an elastin substrate. Accordingly, we undertook a study designed to provide a basis for the determination of elastase in the pres-
,a--* ,3’;2
~0
_@--
_--0
C
ELASTASE
.--4
-I
5000
~4
CHYMOTRYPSIN
IR_Y_tzfj
PER
5ml
INCUBATION
MIXTURE
Fig. 3. Potentiation of elastase activity by trypsin and chymotrypsin. Determination with Fluorescein-e&tin. Fluorescence readings represent r : IO dilutions of filtered incubation mixtures. A: Elastolytic action of trypsin alone. B: Elastolytic action of chvmotrvpsin alone. C: Potentiating effect of trypsin on ;*IJ~ elastase. D : Potentiating elfect of chymotripsin on 2 pg elastase.
ence of chymotrypsin and trypsin. Chymotrypsin, trypsin and elastase normally occur in pancreatic tissue and juice in the form of enzymatically inactive zymogens which can be activated by exogenous trypsin. Pancreatic tissue and juice also contain several inhibitors of these enzymes8-12. In order to minimize their effect and to complete the activation process the addition of a large excess of trypsin to the incubation mixture is necessary. Thus, five different concentrations of elastase (o-3 ,~~g/3ml incubation mixture) were assayed in the presence of a constant amount of trypsin (5000 pug) and varying concentrations of chymotrypsin (o-1000 ,ug). The results of these experiments which were carried out with two different substrates, Fluoresceinelastin and RBB-elastin, are summarized in Figs. 4 and 5. Each curve represents the relationship of fluorescence intensity or optical density to a given elastase concentration, a constant amount of trypsin and varying concentrations of chymotrypsin. The intercept of the chymotrypsin concentration of a sample of pancreatic juice or extract (determined as indicated under METHODS) and the fluorescence or optical Clin. Chim. Acta, rg (1968) 327-339
334
RIh-DERKNECHT
et Cd.
density reading obtained by the elastase assay in the presence of 5000 pug of trypsin furnishes, by interpolation, the true elastase value, corrected for potentiation by trypsin and chymotrypsin, all present in the pancreatic extract or juice. It should be noted that the only parameters that need to be measured are elastase and chymotrypsin. Endogenous trypsin is completely “blotted out” in this system by the addition of a very large excess of exogenous trypsin. Variations of a few hundred micrograms in the amount of endogenous trypsin likely to be present in the sample of extract or juice to be tested, are of no consequence as the increase in fluorescence due to the presence of trypsin is only minor between the IOOO and 5000 pg level of trypsin, (cf. Fig. 3, curves A and C). 5 cg
230
5000
ELASTASE pg TRYPSIN
220 210
41~9 EL 5000~~
.21rg
TRY
EL
5OOOvgTRY
IIrg EL 5000
__a---
--
~9
5006~pg
owaEL
5OOOvg
0 pg
200 CHYMOTRYPSIN
400
600 /
5ml
600 INCUBATION
TRY
TRY
TRY
1000 MIXTURE
Fig. 4. Standard curves for the determination of total elastase with Fluorescein-elastin, corrected for the potentiating effect of trypsin and chymotrypsin. Fluorescence readings represent I: IO dilutions of filtered incubation mixture. “Trypsin blanks” (cf. p. 329) are included in the readings. Bars represent readings obtained with elastase alone.
In a study of the reproducibility of our new method we assayed a I : 5 dilution of a sample of human pancreatic extract IO times. The chymotrypsin content of the I : 5 dilution was 36 ,ug/ml. The average fluorescence reading of the incubation mixtures (I :IO dilutions) was 34.8 fluorescence units with a standard deviation of 5 1.7. The elastase content obtained from Fig. 4 was 0.26 & 0.05 ,ug elastase/ml diluted Clin. Chim. Acta,
19 (1968) 327-339
ELASTASE
335
DETERMINATION
or 5 x (0.26 & 0.05) = 1.3 * 0.25 pg elastase/ml original extract. We checked the reliability and sensitivity of our method by assaying several samples of human pancreatic juice or extract with and without the addition of a small amount of authentic elastase. Using data obtained from assays of pancreatic juice or extracts we also reconstituted such samples by mixing appropriate arnounts of authentic enzymes and reassaying these test mixtures. Table II lists the results of these experiments. 5 pg
5000~9
ELASTASE TRYPSIN
4vg EL 5000 ~(1 TRY
3vg EL 50OOl~g
TRY
21~9 EL 5OOOpg
TRY
lpg EL 5000 pg TRY
Opg EL 5OOOpg
pg CHYMOTRYPSIN
,&I
INCUBATION
TRY
MIXTURE
Fig. 5. Standard curves for the determination of total elastase with RBB-elastin, corrected for the potentiating effect of trypsin and chymotrypsin. “Substrate blanks” are subtracted from all readings. Bars represent readings obtained with elastase alone.
Although proteolytic enzymes in pancreatic tissue and juice normally occur in the form of their inactive proenzymes, free proteolytic activity in pancreas and pancreatic juice of patients suffering from acute pancreatitis has recently been found by Geokas et a1.7913. The technique for the estimation of total elastase described above obviously cannot be employed for such samples since the addition of trypsin would liberate further quantities of proteolytic enzymes still present in the form of inactive zymogens. We therefore attempted to inbibit selectively chymotrypsin and trypsin, the enzymes known to interfere with the determination of elastase, by addition of the specific trypsin and chymotrypsin inhibitor “TrasylolJJ7 to the incubation mixture. In a series of experiments designed to determine the amount of inhibitor needed to cancel the potentiating action of trypsin and chymotrypsin on elastase we made C&n. Chim.
Acta,
19 (1968)
327-339
RINDERKXECHT
336 TABLE
et al.
II
RECOVERYOF ELASTASE;ADDED Material
TO
PANCREATIC
Chymotrypsin
fOundP&T
SAMPLES
(added) /ml
-.
AND
IN -_.
Elastase found pug/ml
ARTIFICIAL
MIXTURES
Elastase added fig/ml
Total elastasp recovered /q/ml
_
Pancreatic extract # PEH H-66
‘5
0.15
I.0
I.0
Pancreatic extract # PEB H-66
18
0.15
0.5 I .o
0.65 1.1
Pancreatic extract # PET H-66
20
0.35
0.5
0.75 7.4
Pancreatic juice # PJ M-22
26
Pancreatic juice # PJ M-5-24
252
0.5
Pancreatic juice # PJ P-8-18
450
13.0
Mixture #I Mixture #r Mixture #3 ~~~ ______. * Determination ** Determination *** Determination
I.0
15 33 4o
I.0
IO.0
5 .o
rq.o*
6.0**
IO.0
22.0***
20.0
36.0
0.4 I.3 2.3
__-. carried out with a I : IO dilution of pancreatic juice. carried out with a I : 5 dilution of pancreatic juice. carried out with a I : 20 dilution of pancreatic extract.
0.4 1.4 2.3
several interesting observations: Elastase was only slightly inhibited by “Trasylol”; a z5oo-fold (w/w) excess of inhibitor was needed to reduce enzyme activity to 500,/o. A “Trasylol” level sufficient to reduce the activity of a mixture of chymotrypsin and trypsin to a base line value (near zero) was not adequate to suppress the potentiating effect of these enzymes on elastase activity. An overwhelming excess (5000 units) of “Trasylol” was necessary to minimize the interaction of the three enzymes. A reasonable estimate of the free elastase content of pancreatic extracts or juice may be obtained if such samples are assayed in the presence of 5000 units of “Trasylol” and elastase values are read off a standard curve in which fluorescence intensity is plotted against increasing concentrations of elastase assayed in the presence of aconstant amount of “Trasylol” (5000 units). Details of these findings are being published elsewhere’. DISCUSSION
The phenomenal success of covalently labeled fluorescent antibodies in immunology and microbiology prompted us to employ similar techniques in the construction of substrates for analytical enzymology. We have recently reported on the assay of elastolytic activity in mammalian plasma with the aid of a fluorescent elastin substrate labeled with dimethylaminonaphthalenesulfonyl chlorideI and on a new calorimetric amylase determination based on a starch substrate possessing the covalently attached marker Remazolbrilliant Bluers. The present study summarizes our specific substrates for the estimation of work on the design of highly sensitive, elastase in pancreatic extracts and juice. Unlike the staining of elastin with dyes such as Congo Red or Orcein which involves ionic and hydrogen bonds only, covalent Clin.
Chim.
Acta,
19 (1968)
327-339
ELASTASE
DETERMINATION
337
linkage of a chromophore or fluorophore to a protein substrate can be expected to result in more or less profound conformational changes and loss of specificity. This has been demonstrated amply in the fluorescent labeling of antibody where overlabeling causes drastic reduction or abolition of antibody titer. Following these guide lines we prepared the elastin substrates listed in Table I. Comparison of column z (degree of substitution) with column 3 (susceptibility to elastase) shows that susceptibility to elastase decreases with increasing substitution. The specificity of the substrate (resistance to trypsin and chymotrypsin, columns 4 and 5) is likewise affected adversely by an increase in the number of substituents. The anomalous position of RBB-elastin may be explained by the assumption that the RBB-label is attached at a different site of the substrate less likely to hinder the attack by the enzyme. In contrast to fluorescein isothiocyanate and dimethylaminonaphthalenesulfonyl chloride which react primarily with free amino groups. Hydroxyl, Remazolbrilliant Blue readily forms an ether linkage with secondary hydroxyl groups. Hydroxyl groups predominate over free amino groups in elastin by a ratio of at least IO: T. Our observations on the influence of substitution on substrate susceptibility and specificity are underscored by the finding that more extensive labeling than indicated in the experimental section with any individual reagent resulted in inferior substrates of reduced susceptibility to elastase and decreased resistance to trypsin and chymotrypsin. Although elastin-Congo Red which is the least modified substrate possesses the highest susceptibility and specificity (see Table I), Fluorescein-elastin is at least five times more sensitive in our assay system than elastin-Congo Red; indeed its sensitivity is mostly limited by its solubility, however slight, in the incubation mixture which provides enough fluorescence to render the measurement of minute differences inaccurate. The sensitivity and specificity of RBB-elastin is of the same order as that of elastin-Congo Red, even though its susceptibility to elastase is reduced still further than that of Fluorescein-elastin. It is an excellent substrate for calorimetric estimation of elastase and is, in contrast to elastin-Congo Red or Orcein, not affected by albumin or other protein9. DNSelastin, still lower on the scale of susceptibility, is a much less sensitive substrate than Fluorescein-elastin, but possesses the advantage of yielding degradation products of greater fluorescence stability than the latter. Indeed, we found that incubation of peptides derived from Fluorescein-elastin with blood plasma (0.5) ml in the presence of trypsin (4000 ,ug) under standard conditions yielded less fluorescence when compared with that of blanks from which plasma had been omitted. The fluorescence decay appears to involve an enzymatic process since addition of boiled plasma or fresh plasma at the end of the incubation with trypsin causes no reduction in fluorescence. Loss of fluorescence of fluorescein isothiocyanate due to the presence of biological materials has been reported recently by Winkelman and Grossman16. It should be emphasized here that assays with fluorescent substrates must be carried out under rigorously standardized conditions with exclusion of actinic or fluorescent light during incubation. Nitro-elastin which possesses the lowest susceptibility to elastase and the lowest specificity of our substrates, is nevertheless equal in sensitivity to RBB-elastin and elastin-Congo Red. This is evidently due to the large number of chromophore groups Clin.
Chim.
Acta.
19 (1968) 327-339
338
RINDERKNECHT
et d.
(-NO,) introduced into the elastin molecule. The usefulness of this substrate is limited by its inferior specificity. It was first reported by Graham I7, that trypsin when added to elastase gave greater elastolysis than elastase alone although trypsin by itself had no elastolytic effect. W:e have confirmed these findings and extended them to chymotrypsin. It can be seen from Fig. 3 that potentiation of elastase activity by chymotrypsin is much more pronounced than that observed with trypsin. When all three enzymes are present in the incubation mixture the potentiating effects of both, trypsin and chymotrypsin, are approximately additive to the action of elastase as illustrated by Figs. 4 and 5. For example: the fluorescence reading for 2 ,ug elastase potentiated by 5000 pg of trypsin alone is 80 (Fig. 4). The potentiating effect of 600 ,ug of chymotrypsin on 2 pg of elastase is 78-40 = 38 (Fig. 3,D). The total fluorescence reading for 2 ,ug of elastase in the presence of 5000 ,ug trypsin and 600 pg of chymotrypsin therefore should be: 80+38 = 118 fluorescence units. The actual fluorescence intensity found was 125 (Fig. 4). It follows from these observations that the data reported in the literature on measurements of elastolytic activity in pancreatic extracts and juice do not represent elastase, but the combined synergistic action of elastase and other proteolytic enzymes. A glance at Fig. 4 shows that previously reported levels of pancreatic “elastase” are likely to be more than one hundred percent too high. Moreover, there are indications that our assay system, although corrected for the synergistic action of trypsin and chymotrypsin, still lacks sufficient selectivity to exclusively measure elastoproteinase (EJ. In the course of an investigation of pancreatic juice with an unusually high “elastase” content we observed that addition of authentic elastase (I ,ug) to the juice led to a recovery of over ZOO~!~in our assay. However, when we added authentic elastase to dilutions of the original juice, made to contain less than I ,ug elastase/5 ml incubation mixture, we recovered the correct amount of enzyme (ca. I pg). We interpret these findings as indicating the presence in pancreatic juice of an enhancing factor other than trypsin or chymotrypsin, which is diluted out in our assay when dilutions of juice are used to give readings of less than I pg elastase/s ml incubation mixture. We have no information regarding the nature or identity of this enhancing factor, but feel that elastomucase (E,)l* and collagenase are logical contenders for this function. The possible influence of pancreatic inhibitors on elastase readings obtained in our assav system cannot be assessed at present. Further refinement of our technique to include such parameters must await availability of these factors and a considerable amount of additional work. Although we have no experimental data as yet which might explain the nature of the potentiating effect of trypsin and chymotrypsin on elastase activity, we feel tempted to speculate that chymotrypsin and trypsin may cause conformational changes of the elastin substrate rendering certain sites more susceptible to the action of elastase. Alternatively, chymotrypsin and trypsin may interact with elastase. Although the mechanism of enzyme potentiation remains to be clarified, our findings should contribute to the understanding not only of normal digestion, but also of the pathological process of autodigestion so strikingly exhibited in acute hemorrhagic pancreatitis. Indeed, our studies on the synergistic effect of trypsin and chymotrypsin on elastase seem to support views recently expressed which assign a central role in the pathogenesis of this disease to elastase 13, the enzyme for which this assay was developed.
ELASTASE
DETERMINATION
339
REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14
D. A. HALL, Elastolysis and Ageing, Charles C. Thomas, Springfield, Ill., 1964. M. C. GEOKAS, R. D. MCKENNA .%ND I. T. BECK, Gastvoentevology, 50 (1966) 882. I. MANDL, Advan. Enzymol., 23 (1961) 163. D. A. HALL, Biochem. J., IOI (1966) 29. M. C. GEOK~S, P. WILDING, H. RINDERKNECHT AND B. J. HAVERBACK, Clin. Biochem., (1968). J. KALLOS, E. L. ARTHUR, D. RIZOCK AND D. K~Hx, Can. J. Biochem., 43 (1965) 135. M. C. GEOKAS, H. RINDERKNECHT, I’. WILDING, \-. LILLARD, C. J. BERNE .&ND B. J. HAVERBACK, Gastroenterology. in press (1967). 1~. LAMY AND S. TAUBER, J. Biol. Chcm., 238 (1963) 939. W. A. LOEVEX, Acta PhJjs,siol.Phwmucol. h’eevl., IO (1962) 2~8. M. KUNITZ AND J. H. NORTHROP, J. &%z. Physiol., 19 (1936) 991. L. A. KAZAL, D. S. SPICER AND R. A. BRAHINSKY, J. .41x. Chem. Sot., 70 (1948) 3034. J. L. GREENE, M. RIGBI AP*‘DD. S. FACKRE, J. Biol. Ckewz., 141 (1966) 5610. M. C. GEOKAS, H. RINDERKNBCHT, V. SWANSOX ‘YND B. J. HAVERBACK, to be published. H. RINDERKNECHT, M. C. G;EOK,ZS, I’. SILVERXIN .UD B. J. HAVERB.%CK, Cbi~. Chiw. z4cta, 19
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