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Susceptibility of various kinds of elastin to elastolytic enzymes, trypsin and chymotrypsin
CLINICA CHIMICA ACTA
52I
SUSCEPTIBILITY OF VARIOUS KINDS OF ELASTIN TO ELASTOLYTIC ENZYMES, TRYPSIN AND CHYMOTRYPSIN* WlM A. LOEVEN
Gerontology Research Center, National Institute of Child Health and Human Development, National Institutes of Health, Department of Health, Education and Welfare, Baltimore City Hospital% Baltimore, Maryland 2z2z4 (U.S.A.) (Received September z8, x969)
SUMMARY
When measuring the elastolytic activity of elastoproteinase with various elastin preparations and the synergistic effect of the elastomu~ ases Em-I and Em-S on this activity, the following observations were made: x. Staining alkali-treated elastin with Congo Red and orcein largely decreases the susceptibility of elastin to elastoproteinase, while it increases the activities'ot the elastomucases, especially when using Congo Red as a dye. 2. Whereas orcein staining of acid-treated elastin does not affect tile susceptibility of elastoproteinase and the enhancement of this enzyme by Era-I, the synergistic effect of the elastomucase Em-S is completely suppressed. 3. Chymotrypsin and trypsin, which both have no elastolytic activity of themselves, show synergistic effects on unstained acid-treated elastin and elastin stained with DNS (5-dimethylamine-t-naphthalene sulfonyl chloride). Trypsin shows only a slight effect when measuring the activation activity against orcein- and Congo Red-stained acid-treated elastin. 4- As a result of this investigation, a procedure is proposed to measure the concentration of elastoproteinase and elastomucase Em-I in crude enzyme preparation and in pancreatic extracts and to estimate the amount of elastomucase Em-S. Some results of experiments with rats using the proposed assay are given.
INTRODUCTION
Elastin is rapidly dissolved by elastoproteinase (elastase, E~, pancreatopeptidase E, E.C. 3.4.4.7) and the determination of elastolytic activity of pancreatic extracts is based on the solubilization of unstained or stained elastin, mostly prepared from bovine nuchal ligament or aorta. The methods for assaying this solubilization of elastin are based on measuring the loss of weight of the undissolved clastin, the * Part of the experiments described in this paper were performed in Leiden, The Netherlands (Netherlands Institute for Preventive Medicine, TNO). Clin. Chim. Acta, 27 (I97O) 521-533
522
LOEVEN
protein content in the elastolyzate (Biuret method or Folin-Ciocalteau reaction) or the release of dye in the case of stained elastin (Congo Red- and orcein-stained elastin being frequently used). Some of these methods have been used to study the role of elastase in acute pancreatitis in man~, ~. However, the estimates of elastolytic activity in pancreatic extracts are higher than their elastoproteinase content would warrant, since not only the mucolytic elastase components (elastomucases or elastomucoproteinases) but also trypsin and chymotrypsin are able to act synergistically on the activity of elastoproteinase ~-6. Loeven 7 has already demonstrated that small amounts of elastomucase can be isolated from some commercial trypsin and chymotrypsin preparations and that these contaminating agents are in part responsible for the activation activity of these proteolytic enzymes. On the other hand, the measurements of the activity of the elastomucases present in crude elastase preparations give too high values, not only due to the contaminating effect of trypsin and chymotrypsin but also to the fact that the combined activity of the two elastomucases so far identified in pancreas (called Em-I and Era-S) is higher than the summation of the activity of the individual elastomucases 5. Although column chromatography on DEAE-Sephadex is extremely suitable for a complete separation of all the components of the elastase complex*-L this purification procedure is too time-consuming for routine determinations of the enzyme concentrations especially when only small amounts of pancreatic extracts or crude enzyme preparations are available*. Several years ago, the author observed that with the orcein method (alkalitreated elastin stained with orcein) it was very hard to find an activation of the elastoproteinase activity by elastomucase (at that time the presence of only o n e elastomucase was known). This finding, confirmed by Banga (personal communication, April i963), was used by Banga, Loeven and Romh~nyi 8 in their histochemical study of the two-component system in elastin. Whereas a synergistic effect of elastomucase Em-I could be observed when using orcein-elastin, the other elastomucase Em-S was completely inactive. A systematic study of the activity of purified components of the elastase complex, trypsin, and chymotrypsin on various kinds of e!~tin preparations was carried out. As a result of this study, presented in this paper, a procedure is proposed which allows the measuring of the activity of elastoproteinase and of elastomucase Em-I in crude enzyme preparations without the contaminating effects of elastomucase Era-S, trypsin, and chymotrypsin, and at the same time gives an estimated value for the concentration of elastomucase Em-S.
* It is sometimes p~.ssible to separate the enzymes present in crude pancreas extracts into t w o fractions: a. the monomer form of elastoproteinase and elastomucase Em-I present in a precipitate formed during exhaustive dialysis against distilled water for several days of an acetic acid extract of pancreas powder; b. the dimer form of elastoproteinase and elastomucase Em-S both present in the supernatant after removing this dialysis precipitate. This procedure not only requires a large amount of crude material, but it was also observed that with some pancreas extracts (e.g. from rat and rabbit) no precipitate was formed.
Clin. Chim. Acta, 27 (x97o) 521-533
SUSCEPTIBILITY OF ELASTIN TO ENZYMES
523
MATERIAL AND METHODS
I. Elastin preparations Bovine nuchal ligament obtained fresh from the slaughterhouse was used for the preparation of elastin. Both alkali-treated and acid-treated elastin prepared as described in details elsewhere 9 were used as: x. unstained elastin preparations: 2. stained with Congo Red (Fisher certified reagent) according to HallOo; 3- stained with orcein (Fisher certified reagent) according to Banga~'; 4. stained with DNS (5-dimethylamino-x-naphthalene sulfonyl chloride; Eastman Organic Chemicals) according to Rinderknecht et al. z. Since the elastolytic activity increases as the size of the particles decreases ~z, all elastin samples were shaken in an electric Laboratory Test Sieve Vibrator (Derrick Co., Buffalo, N.Y.). Only elastin powder passing the sieve with openings of 44 microns (Tyler equivalent mesh: 325) was used in all experiments. I I . Enzyme preparations Elastoproteinase, elastomucase Em-I and elastomucase Em-S were purified from crude pancreas powder (Organon N.V., Oss, The Netherlands) using DEAESephadex column chromatographyL Chymotrypsin (3 × salt free from ethanol) and trypsin (z × cryst, salt free) were purchased from Nutritional Biochemicals Corp. The chymotrypsin used in the experiments was a mixture of equal amounts of ~-, /~-, F-, and A-chymotrypsin. I I I . Methods used with purified enzyme preparations All elastin preparations were used as suspensions of 5 mg per ml of buffer. Since the velocity of elastolysis varies largely with the ionic strength of the buffer solutions used, partly due to inhibitory effects of certain buffer salts and partly to the decrease of pH values during elastolysis when using buffer solutions with low buffer capacity ~s, a sodium-carbonate-HCl buffer of o.x M and pH 8.8 was used throughout. Only in the case of DNS-elastin, tile original bicine buffer of Rinderknecht et al. s was used. Although the elastomucase activity is higher at lower pH values, a pH of 8.8 was chosen in the experiments because the activity of a fourth component of the elastase complex (the elastolipoproteinase, primarily releasing lipid-like material from elastin) is extremely low at pH 8.85. The reaction mixture consists of : 5 ml of the elastin suspension (z5 mg elastin) pipetted in wide test tubes; x ml of an elastoproteinase solution; y ml of an elastomucase (or trypsin or chymotrypsin) solution. The total volume was made up to 6 ml with buffer. All enzyme solutions were in the carbonate buffer. The test tubes with the substrate were placed for I5 min in an Eberbach water bath shaker at 37 ° before adding the enzymes. The incubation time was always 30 min (except DNS-elastin: x h). The residual elastin was centrifuged off and the amount of solubilized elastin in the supernatant determined; unstained elastin by means of the Biuret method °, stained elastin by measuring the extinction of the dyes at 59° nm (orcein) or at 485 nm (Congo Red) in a Gilford Spectrophotometer, DNS-elastin by measuring tlle fluorescence intensity of the supernatant as described by Rinderknecht et al. ~ in an Aminco Spectrophotofluorometer (excitation wave length: 32o rim, emission wave length: Clin. Chim. Acta, 27 (x97 o) 52x-533
524
LOEVE~
49° nm). For all kinds of elastin preparations investigated, standard curves were made, allowing us to calculate the amount of solubilized elastin from the extinction or fluorescence intensity values measured. PROPOSED PROCEDURE
The following procedure is proposed for the determination of the activity of elastoproteinase and of elastomucase Em-I and the estimation of the activity of elastomucase Em-S in crude elastase preparations and pancreatic extracts. Unstained and orcein-stained acid-treated elastin prepared as discussed on page 523 are homogenized in a sodium-carbonate-HCl buffer of o.I M and pH 8.8. Wide test tubes are filled with 5 ml of this suspension (25 mg of elastin) and preheated for I5 rain at 37 ° in a water bath shaker. To a series of test tubes are added x mg of a highly purified elastoproteinase preparation, y ml of a crude enzyme sample or pancreatic extract to be tested, both dissolved in the carbonate buffer. The amount of enzymes added is chosen so that no more than 5o% and not less than 20% of the initial substrate is hydrolyzed. Under these conditions the enzyme activity is essentially linear. Maximally o.2 mg of the elastoproteinasp is added. The value for y varies with the total elastolytic activity present in the unknown sample and has to be determined in a separate assay (using only the substrate and the crude enzyme with omission of the purified elastoproteinase). For rat pancreatic extract, y appears to be maximally 1. 5 rag. The incubation time is exactly 3o min. After centrifugation, the supernatant is used for determining the amount of elastin solubilized as outlined in the preceding section. The following enzyme units will be used: (i) One elastoproteinase unit (E.U.) of activity is such that I m g of elastin is solubilized under the conditions of the assay: (if) The synergistic effect of the extract is expressed in percents of enhancement of the elastoproteinase (El) activity present in the system: mg elastin dissolved by [x mg El + y mg extract]-I00 X
mg elastin dissolved by x mg El mg elastin dissolved by x mg El
(iii) One elastomucase unit (Em. U.) is the activity of an elastomucase preparation resulting in an enhancement of the activity of a highly purified elastoproteinase preparation by 50% under the experimental conditions of the assay. By using this method and the calculations mentioned above, we obtain for each crude enzyme sample or pancreatic extract: a. values for the total elastolytic activity (a E.U./mg prep.) and the synergistic effect (b Em. U./mg prep.) when using unstained acid-treated elastin; b. values for the same activities with orcein-stained acid-treated -~l-~-"n 1~ E.U./mg prep. and d Em. U./mg prep., respectively). The values for the total elastolytic activity include also the synergistic effect of the elastomucase present in the sample and must be corrected for this enhancement Clin. Chim. Acta, 27 (x97 o) 52Jt-533
SUSCEPTIBILITY OF ELASTIN TO ENZYMES
525
of the real concentration of elastoproteinase. Since the synergistic effect is independent of the low elastoproteinase concentration present in the reaction mixture and is also proportional to the elastomucase concentration, the correction can be made as follows: a. for unstained elastin: E.U./mg corrected = a E.U./mg I + r/2 b Em.U./mg b. for orcein elastin: E.U./mg corrected -c E.U./mg I + I/2 d Em. U./mg The d Em.U./mg prep gives the amount of elastomucase Em-! in units (with an extremely small contribution of trypsin as mentioned in the I)ISCUSSION); the b Em.U./mg prep gives the total amount of elastomucases in units; (b--d) Em.U./mg prep. is the rough estimate for the units of elastomucase Em-S (contaminated with trypsin and chymotrypsin activities). RESULTS
I. P_,i~,ding of dye to the elastic fibers Gotte et al. as have shown that mild hydrolytic damage to autoclaved elastin (comparable with our acid-treated elastin) by the action of 0.5 N NaOH at 25 ° results in a progressively lower uptake of orcein. Boiling of elastin with o.I N NaOH for 45 min at 980 (Lansing method as used for the preparation of alkali-treated elastin) seems to result in only small damage. When measuring the anaount of orcein bound to elastin, we found in the case of acid-treated elastin a mean value for the extinction of orcein at 59 ° nm of o.15o per nag solubiiized elastin and for alkalitreated elastin of O.ll 7 per mg elastin (ratio (extinction acid-treated elastin) :(extinction alkali-treated elastin)-- 1.28). About the same ratio (1.18) was found when comparing the fluorescence intensity of DNS acid-treated and DNS alkali-treated elastin (19. 5 and 16.5 fluorescence units per mg solubilized elastin, respectively, with the photomultiplier scale set at I.O). While in the case of orcein and Congo Red ionic and hydrogen bonds are involved, DNS reacts primarily with free amino groups (covalent linkages). In this connection, it is very surprising that there appears to be a large difference in staining capacity of Congo Red for these two kinds of elastin. Acid-treated elastin binds about five times more of the dye than alkali-treated elastin (mean values for the extinction per mg solubilized elastin at 485 nm: o.o67 and o.oi2, respectively).
II. Elastolytic activity of elastoproteinase and the other enzymes studied on various kinds of elastin preparations Fig. x shows the elastolysis of the elastin preparations by elastoproteinase. The effect of this enzyme on all three types of acid-treated elastin is a zero order reaction up to about 50% solubilized substrate. This effect was already observed for orcein-stained elastin by Banga 1~ and Crepaldi et al. ~*. The higher susceptibility Clin. Chim..4eta, 27 (197 o) 521-533
526
LOEVEN ALKALI-TREATED ELASTIN pH 8.8 I
I
:" 1
i
.........
i
ACID-TREATED ELASTINpH 8.8 =|
w
'/
"~'-'
4
EO
0
0.2
0.4
'
i. . . .
I
"
T
~" 12
CONGO I:O4;T
'"u)~'
I
l
0.6
0.8
.
ORCEIH-STAINEO
4
II
1.0
0
0.1
11.2
0.3
0.4
0.5
mg ELASTOPROTEINASE
m 9 ELASTOPROTEINASE Fig.
'
I. Elastolysis-elastoproteinase concentration curve of various kinds of elastin preparations.
of Congo Red-elastin for elastoproteinase was explained by Hall t° as being a result of the increased negative charge of the substrate after binding the dye. However, this explanation ,:..'!~es not hold for the staining of alkali-treated elastin. When ~:omparing the susceptibility of the alkali-treated elastin preparations to elastoproteinase, only the unstained elastin behaves in the same way as the acid-treated elastins as was found by Lamy et al. ~5 and in our own experiments performed under other experimental conditions6, °. The elastolysis-enzyme concentration curves for the two stained alkaLLtreated elastins are nearly superimposed on each other. They both show the very slow elastin dissolving part up to about 0. 5 mg enzyme, followed by a very accelerated dissolving process at higher enzyme concentrations as also found by Rancati et al. ~6 for orcein-stained elastin. During the first part of the curve only 12 o,;o / of the total amount of elastin present is solubilized. We must, however, mention that the susceptibility of unstained alkali-treated elastin to the enzyme is less than that of the three acid-treated elastin preparations studied. Table I gives data on the susceptibility of the elastin preparations to the elastomucases, trypsin, and chymotrypsin without any addition of elastoproteinase. These data again confirm the earlier observations that pure trypsin and chymotrypsin TABLE
I
ELASTOLYTIC ACTIVITY OF ELASTOMUCASES, T R Y P S I N AND CH'VMOTRYPS!N
K i n d of elastin
preparation
mg of
Elastin solubilized by
Era- 1
Unstained-alkali Congo Red-alkali Orcein-alkali Unstained-acid Congo Red-acid Orcein-aeid DNS-alkali
Trypsin
Em-S cone. i~ mg
cone. in n~¢
Chymotrypsin
cone. in mg
conc. in mg
0.06
o.r2
o.z5
o.zz
o.z~
0.50
0.5
z.o
o.5
z.o
0.3 o.i o.2 o. 7 o.2 o.5 o. z
0. 7 0.2 0.3 1.2 o. 5 1.3 o.2
I.x o. 3 o.3 t .9 x.o 2.t o. 5
0.2 o.t o. z o.4 o.i o.2 o.o
o, 4 o.i o.2 o, 7 0.2 o. 3 o,o
0. 4 o.i o.2 x.o o. 3 o, 4 o, [
o.I 0.2 o.o o,o o.e o.2 o,o
0.2 o. 3 o.o o.o o.2 o.2 o.o
o.I o.I o.o o,o o.o o.o o.o
o.2 o. I o o.o o.I o.o
Clin. Chim. Acta, 2 7 ( z 9 7 o) 5 2 z - 5 3 3
o.z .... ....
SUSCEPTIBILITY OF ELASTIN TO ENZYMES
527
themselves cannot attack elastin. Even in relatively high ccnce_ntrat,_'e.--.s (==, chinpared to the concentration of elastoproteinase), no more than z% of the substrate is clis~ived. Whereas elastomucase Em-S can dissolve no more than about z½% of the alkali-treated elasth~ and elastomucase Em-I about 4/o, o/ the elastol)~ic activity of these enzymes on acid-treated elastin is somewhat higher (up to 4% and 8%, respectively, as a result of the release of polysaccharide-protein complexes6). Although the solubilizing effect is extremely low, here too we found that unstained alkali-treated elastin can be attacked better than the stained ones and that the susceptibility of unstained and orcein-stained acid-treated elastin to Em-I is exactiy the same.
UNSTAINED
ACID-TREATEDELASTINpH 8.8
ALKALI-TREATEDELASTINpH 8.8 20
-
-
~ I ~ l
~
I
~ I
I-
-
t
0.25 mg Em-I -.--0.12 m9 Em-t . . . . 0.06 mg Em-i
¢= 16
20
"
T - - '
"
|
|
,._, 16
•
¢,.,t,,,I
>
--"
' ~ r ~
ss e
35% }
21
" z I,*,.-
"=~'- B ~
J.};,.~.,.", BLANK
-
[
~
,...,,a
e
t
E 4
If 0
0.2
|
|
I
1
1
i
I
0.4
0.6
0.8
0.1
0.2
0.3
0.4
m 9 ELASTOPROTEINASE
mg ELASTOPROTEINASE
UNSTAINED
ACID-TREATEDELASTINpH 8.8
ALKALI-TREATEDELASTINpH 8.8 II
|
|
20
|
1
~T . . . .
'
0.50 mg EmS ----0.25 mo Eros
16
!
/
. . . . 0.12 mg Em-S 30%
B
8tA.K
NK
•~' 4
I
O
0.2
0.4
0.6
mg ELASTOPROTEINASE
0.8
. . . . . .
0
I
:
0.I
.
0.2
I
l
0.3
0.4
~9 ELASTOPROTEIN,",~"
Fig. 2. The synergistic effect of elastomucase Em-I (upper p a r t of the figure) and of elastomucase Em-S (lower part) on the elastolytic a c t i v i t y of elastoproteinase using u n s t a i n e d elastin as substrafe.
Clin. Chim. Acta, 27 (x97 o) 5 z i - 5 3 3
528
LOEVL,'N
III. Synergistic effect of elastomucases Em-I and Em-S on the activity of elastoprotdnase It has been demonstrated that the primary effect of the elastomucases is the release of polysaccharide-protein complexes present in the elastic structure, in this way increasing the possibility of permeation of elastoproteinase into the inner parts of the elastic fibers. Fig. 2 shows that the synergistic effect ot Em-I is larger than that of Em-S and that in both cases the enhancement of the activity of elastoproteinase on alkalitreated elastin is nearly proportional with the elastomucase concentration and independent of the eiastoproteinase concentration. When using acid-treated elastin
CONGO RED-STAINED ALKALI-TREATEDELASTINpH 8.8 I
! -
t
~
I
..... 0.25 m9 Em I 20~-- -0.12 mg Em ' | .... 0.06 mg Eml
1
ACID-TREATEDELASTINpH 88
z ',
1
f //
It
~o2~-~/i
~z
~3o~ ~ / / /
'F
hi..
"
/
136% ,Z •/ ,a, ' .g A A N K
0
322~.///
/229%,t
./.'"
~'Ot,NK
"""
///,//~ 0
0.2 0.4 0.6 0.8 mg ELASTOPROTEINASE
0.05 0.1 0,15 0.2 m9 ELASTOPROTEINASE
CONGO RED STAINED ALKALI-TREATEDELASTINpH 8.8 2O -
"
,"
i~ . . . . . . . .
i'
~
0.50 mgEm.S 16
] J,
---0.25 mg Em-S
.... 0.12 mg Em.S
/~
.=.
,//,
__t
~ 12
__J
E
/~/
/
. I
0.2
I
I
l
0.4
0.6
0.8
m9 ELASTOPROTEINASE
I
I
29%
'2'~9
4 :y ":'"~
E
4
0
I ....
~e
8
,oo~\
,!
'[
12
104%'~. i,f ~
ACID-TREATEDELASTINpH 8.8 20
I
~f,:~LANK
5%~/,,~y-
0
0,05
i
I
I
0.1
0.15
0.2
m9 ELASTOPROTEINASE
Fig. 3. The enhancement of the activity of elastoproteinase on Congo Red-stained elastin of elastomucase Em-I (upper part) and of elastomucase Em-S (lower part).
Ciin. Chim. Acla, 27 (x97o) 52x-533
SUSCEPTIBILITY OF ELASTIN TO E~ZYME$
529
as substrate this is only true up to about z5% dissolved elastin due to the inhibition of the elastomucase activity by the elasto!yzate (Loeven; unpubilshed results). These data confirm results obtained under other experimental conditions6, 0. When using Congo Red-stained elastin, there appears to be a distinct difference between the activity of both elastomucases on alkali- and acid-treated elastin (Fig. 3). The synergistic effect largely depends on the elastoproteinase concentration in the mixture, especially in the case of alkali-treated elastin. The acceleration of the elastolysis in the second part of the curve (above 0. 4 mg elastoproteinase) is sometimes twice that occurring at lower elastoproteinase concentrations. Even in the first part. of the curve (below 3 mg dissolved elastin), the activation activity of the elastomucases is higher than when using unstained alkali-treated elastin as substrate. In comparison
ORCEIN-STAINED ACID-TREATEDELASTINpH 8.8
ALKALI-TREATEDELASTINpH 8.8 20
,
,
1
~
0.25 mg Em I -----0.12 m9 Eml . . . . 0.06 mg Em-I /
20-
1
,
......
,
,
--F---
---
16
]
/
tat
12
/
~ 8
69%~
./
34,
.......
2
/./ : ,#
E
~ 2 1 % 0.2
=
!
l
0.4
0.6
0.8
|
!
0.1 0,2 0.3 mg ELASTOPROTEINASE
mg ELASTOPROTEINASE
_
0.4
ORCEIN-STA~NED ACID-TREATEDELASTiNpH 8.8
ALKALI-TREATEDELASTINpH 8.8 20
I
I
!
I
......0.50 mg Em-S ----0.25 m9 Em.S 16 . . . . 0.12 mg Eros
}
.j/"
:=p
~_ 12
e's
=p i,,,-,
-
E
"/'
-
4 2 1 ~ ~
~. .. ~ , 7%
0.2
0.4
0.6
mg ELASTOPROTEINASE
0.8
0
0.1
n_.?
0.3
0.4
mg ELASTOPROTEINASE
Fig. 4. The activation activity of elastomucase Em-I (upper part) and of elastomucase E m - S (lower part) on the activity of elastoproteinase when usiag orcein-stained elastin as substrate.
Clin. Chin~. Acta. z 7 (z97 o) 5 e z - 5 3 3
530
LO~VE~
with the effect on this kind of substrate, the presence of Em-I in a reaction mixture containing Congo Red-acid-treated elastin results in an extremely high synergistic effect at low elastoproteinase concentrations. The activation activity of elastonmcase Era-S, however, is not only maximally the same as for the Congo Red-stained alkah-treated elastin but decreases rapidly at higiler concentrations of ~:!a~toproteinase. This is caused by an inhibition of the enzyme activity by the releas~:d polysaccharidecontaining substances in the elastolyzate as mentioned above. As a result of this, the curves run almost parallel with the blank. Since the synergistic effect of both elastomucases is so largely dependent on the elastoproteinase concentration, this kind of substrate cannot be used at all for exact measurements of elastomucase activities. The elastolysis-enzyme concentration curves for Em-I and Em-S with orceinstained alkali-treated elastin (Fig. 4) are mainly the same as obtained for Congo Red-stained elastin. The only differences are the lower percentages of activation activity, an indication that orcein-staiaJ~ elastin is less susceptible to elastomucase, especially to Em-S. That orcein staining indeed inhibits the activity of Em-S is clearly demonstrated when acid-treated elastin is used. Over the whole range of elastoproteinase concentrations investigated with this stained substrate, the enhancement of the enzyme activity by Em-S is completely absent. The small effect present at low elastoproteinase concentration is not real since the same amount of solubilized elastinprotein was measured without and in the presence of Em-S. The small amount of orcein released is in any case not protein-bound. The activity of Em-I is still present, independent of the concentration of elastoproteinase and at low concentration of Em-I approximately the same as measured for unstained acidtreated elastin. The synergistic effect of the elastomucases on DNS-alkali-treated elastin was only measured in the low concentration range of elastoproteinase (o-5o/~g) used by Rinderknecht et al. ~. Table II shows that although the synergistic effect is independent of the elastoproteinase concentration, low concentrations of elastomucase give a rapid increase in activation activity leveling off at higher concentrations. Further experiments with this substrate are in progress.
IV. Synergistic effect of trypsin and chymotrypsin on various kinds of dastin preparations In tile same way as elastoproteinase complements the action of trypsin and TABLE II SYNERGISTIC EFFECT OF TIIE ELASTOMUCASES E M - I AND E M - S ON TIIE ACTIVITY O F E L A S T O P R O T E I N A S E U S I N G AS S U B S T R A T E D N ~ A L K A L I - T R E A T E D ELASTIN
Elastomucase
Enzyme conc. in mg
.'~ynergistic efj~ci in o/ /O
Em-I
0.05 o.i 0.25 0.5 o.i2 0.25 0.5
4° 67 78 90 27 47 73 92
Em-S
x.o
Clin. Chim. Acta, 27 (I97o) 521-533
SUSCEPTIBILITY' OF ELASTIN TO ENZYMES
531
chymotrypsin by splitting bonds not in the specificity range of the two other proteases, these proteolytic enzymes without any elastolytic activity of themselves (see Table I) are able to attack the elastic fibers already partially digested by elastoproteinase, in this way enhancing the activity of this enzyme3, *. The present author has shown that to some extent the synergistic effect of tr3~)sin and chymotrypsin is due to a contamination of these enzyme preparations with elastomucase 7. Table IIl shows TABLE
III
S Y N E R G I S T I C E F F E C T O F T R Y P S I N A N D C H Y M O T R Y P S I N ON T H E A C T I V I T Y O F E L A S T O P R O T E I N A S E U S I N G V A R I O U S K I N D S O F E L A S T I N P R E P A R A T I O N S AS S U B S T R A T E
Kind of dastin
Unstained-alkali* Congo Red-alkali Orcein-alkali Unstained-acid* Congo Red-acid Orcein-acid DNS-alkali**
Buyer and pH
Na--carbonateHC1 buffer. o.I M a n d p H 8.8
B i c i n e buffer p H 8.8
Synergistic effect (in %) of Trypsin Chymotrypsin in mg in mg 0.5
l.o
0.5
,,.o
o o 5 35 9 7 31
o o 9 6I 16 15 44
o o o 32 o o 4 t)
o 6 2 53 o 3 7°
* E l a s t o p r o t e i n a s e r a n g e i n v e s t i g a t e d : 0 - 0 . 6 m g ( a l k a l i - t r e a t e d elastin) a n d 0-0.3 mg ( , c i d t r e a t e d elastin). ** 2o m g e l a s t i n i n s t e a d of 25 m g p e r 5 ml; i n c u b a t i o n t i m e I h i n s t e a d of 3 ° rain; e l a s t o p r o t e i n a s e r a n g e o - 5 o / ~ g i n s t e a d of o - o . 6 mg.
that under the conditions used in our experiments trypsin and chymotrypsin up to a concentration of I mg have only a pronounced synergistic effect on DNS alkalitreated elastin and on unstained acid-treated elastin. Chymotrypsin is without any effect on the stained acid-treated elastin, while the activation activity of trypsin on orcein- and Congo Red-stained acid-treated elastin is less as compared with its effect o n the unstained one. DISCUSSION
Hall and Czerkawski t~ have demonstrated that three reactions take place during elastolysis: (i) adsorption of enzyme to the surface of the solid substrate and initial loosening of the surface structure; (if) ensuing permeation of the enzyme into the solid resulting in solubilization of elastin and formation of high-molecular weight ~-elastin; (iii) hydrolytic activity of the enzyme by converting ~-elastin into fl-ela~tin of low-molecular weight. The differences in elastolysis-enzyme concentration curves for alkali- and acid-treated elastin might very well be caused by either a reduced adsorption of elastoproteinase to the surface of elastin after alkali treatment or a reduced penetration capacity or both. The elastic structure seems to become more condensed during alkali treatment. The same explanation can be given for the even lower susceptibility of this kind of elastin to the enzyme after binding dye molecules to the structure. The high activity o zhe elastomucases on the stained alkali-treated elastin can be explained by the low susceptibility of these kinds of elas[in to elastoproteinase. Clin. Cltim. Acta, 27 (x97 o) 5 2 1 - 5 3 3
532
LOEVEN
Since the binding of dye molecules largely abolishes the permeation of the enzyme, any attack of the substrate that opens its structure (e.g. release of polysaccharide compotmds by elastomucase) results directly in a higher elastoprotelnase activity. Although it is assumed that Congo Red and orcein bind to the elastinprotein, it is not known as yet how the attachment occurs. Banga tt demonstrated that the same elastin preparation stained differently with orcein produced by different firms. That orcein is attached to elastin by another mechanism than Congo Red can be concluded if one compares the enzyme activity on these two substrates (see Figs. I, 3 and 4). In contrast with Congo Red, binding of orcein to acid-treated elastin has no effect at all on the activity of elastoproteinase and the enhancement of this activity by elastomucase Em-I. The phenomenon of the complete inhibition of Em-S activity when this kind of substrate is used cannot be expkdned at the moment but must be certainly a consequence of the different reaction mechanisms of the two elastomucases6,18-~0. Whereas the polysaccharide-protein complex present in alkali-treated elastin can be attacked by both elastomuca~es, the binding of orcein molecules to acidtreated elastin seems to block completely the groups susceptible to Em-S. In any case, this phenomenon makes it possible to determine the activity of Em-I in crude elastase preparations without tile interfering activation activity of Em-S and chymotrypsin and with a minimal effect of trypsin (see Table III). Since r mg of trypsin gives a synergistic effect of I5% and in the proposed assay procedure (p. 524) only I-2 mg of a crude enzyme preparation or pancreas extract will be used, the contribution of trypsin to the total synergistic effect of Em-I is very small indeed. Furthermore, the curves for unstained and orcein-stained acid-treated elastin are almost superimposed on each other at low concentration of elastoproteinase in the system (up to o.2 mg; see Fig, I). The values for the synergistic effect of Em-I on elastoproteinase when using these two substrates lie very close together at low Em-I concentrations (compare Figs. 2 and 4). In both cases, this effect is independent of the amount of elastoproteinase and proportional with the elastomucase concentration. A subtraction of the activity of Em-I in a crude enzyme preparation calculated for orcein-stained acid-treated elastin from the total elastomucase activity measured with unstained acid-treated elastin gives a rough estimate for the concentration of elastomucase Em-S. This estimate is too high for two reasons" (i) the phenomenon observed in previous work that the elastomucases Em-I and Em-S also activated each others activity0; and (ii) the small synergistic effect of chymotrypsin and trypsin. The contaminating effect mentioned under (ii) needs further research. A Trasylol® level sufficient to inhibit the activity of trypsin and chymotrypsin was not adequate to suppress the synergistic effect of these enzymes on elastoproteinase 2 (Trasylol ® only slightly i~hibiting the activity of elastoproteinase). The applicability of the assay procedure proposed in MATERIALAND ~ETHODS has been shown in the author's laboratory using rat pancreata. Table iV shows drastic changes in the concentration of pancreatic elastolytic enzymes of hypophysectomized female rats (r9-months-old and hypophysectomized at an age of 9 months) as compared with x9-months-old control rats al. The same assay procedure was also used measuring the age relationship of elastolytic enzymes in male and female rat pancreata (Loeven, in manuscript). Geokas et al. ~ and Rinderknecht a al. ~ have mentioned that there are indicaClin. Chim. Acta, 27 (x97o) 52x-533
SUSCEPTIBILITY OF ELASTIN TO ENZYMES
52]3
T A B L E IV CONCENTRATIONS OF PANCREATIC ELASTOLYTIC ENZYMES OF HYPOPHYSECTOMIZED AND UNTREATED FEMALE RATS
(I9 m o n t h s old" h y p o p h y s e c t o m i z e d a t an age of 9 months) E n z y m e units per g acetone-dried pancreas.
Substrate used
Enzyme measured
Enz~,,me activity in pancreas of hypophysect, rats control rats
Unstained acidt r e a t e d elastin
Elastoproteinase E m - I + Em-S e s t i m a t e Em-S Eiastoproteinase Em-I
45-5 i28.2 46.6 34.5 81.6
Orcein-stained acid-elastin
! 6.1 E.U. -+2 ~o.9 E m . U Em.U. ~: 4-9 E.U. ± 9.5 Era. ~.
204.9 41.z 24. 4 t88.6 i6.8
~ 24.8 E.U. ~: 6.2 Em.U. Em.U. -~: I4.6 E.U. ~ 3.3 E m . [ ' .
tions that the assay systems used by them "still lacks sufficient selectivity to exclusively measure elastoproteinase" and these authors feel that, e.g., elastomucase is a logical contender for the enhancing factor. The assay procedure proposed by us might be able to solve this problem. ACKNOWLEDGEMENT
The author wishes to acknowledge the technical assistance of Miss P. Los and MI,:. A. G. A. Sack-Janssen (N.I.P.G./TNO, Leiden) and Mrs. M. M. Baldwin (GRC, Baltimore). REFERENCI';~ t M. C. GEOKAS, S. EISENSTEIN, R. D. MCKENNA AND !. T. BECK, J. Lab. Clin. Med., 69 (1967) 530. 2 H. R I N D E R K N E C H T , M. C. GEOKAS, P. SILVERMAN, Y . L I L L A R D AND B. J. H/.VERBACK, Clin. Chim. A a a . I9 (1968) 3z7 . 3 A. AMATI AND B. CASTELL1, ltal. J. Biochem., lO (1961) 292. 4 G. N. GRANT, Biochem. J., 75 (I96o) I4P. 5 W. A. LO]gVEN, in Proceedings NATO-Conference on The Structure and Function of Connective and Skeletal Tissues, B u t t e r w o r t h , Inc., L o n d o n - E d i n b u r g h , x965, pp. xo9-116. 6 W. A. LOEVEN, Acta Physiol. Pharmacol. Neerl., x3 (1965) 278. 7 W. A. LOEVEN, Acta Physiol. Pharmacol. Neerl., x2 (1963) 57. 8 1. BANOA, W. A. LOEVEN AND GY. ROM~ANVl, Acta Morphol. Acad. Sci. Hung., 13 (I965) 385 • 9 W. A. LoEvz~;, Acta Physiol. Pharmacol. Need., 9 (I96o) 473Io D. A. HALL, Biochem. J., xol (I966) 29. I t I. BANGA, Acta Physiol. Acad. Sci. Hung., 24 (1963) x. tz W. A. LOEVEN, in D. A. HALL (Ed.), International Review Connective Tissue Research, Vol. I, Academic Press, New York, 1963, pp. 183-24 o. 13 L. GOTTE, P. STEnN, D. F. ELSDEN AND S. M. PARTRIDGE, Biochem. J., 87 (I963) 344. t 4 G. CREPALDI, G. ZENI, R. CARLI AND P. AVOGARO, Giorn. Gerontol., Iz (I963) 97. 15 F. LAMY, C. P. CRAIG AND S. TAUBER, J. Biol. Chem., 236 (x96I) 86. t6 G. RANCATL P. MARRAMA, C. FERRARI A,~D B. BONATL Giorn. Biochem., 8 (t959) 67. t 7 D. A. HALL AND J. W. CZERKAWSKL Biochem. J., 8o (I96z) I z l , t28, -'34. I8 M. J. BARNES AND S. M. PARTRIDGE, Biochem. J., xo9 (x968) 883. 19 W. A. LOEVEN, Acta Physiol. Pharmacol. Neerl., 14 (I967) 475. 2o I. BANGA, Structure and Function of Elastin and Collagen, Akaddmiai Kiad6, Budapest, I966, pp. 63-67. 2x W. A. LOEVEN, Proceedings 8th Intern. Congr. Gerontology, W a s h i n g t o n D.C., Aug. I969, Vol. 2, No. 55, P. 16; Full-length p a p e r s u b m i t t e d to Endocrinology.
Clin. Chim. Acta, 27 (I97 o) 521-533
52I
SUSCEPTIBILITY OF VARIOUS KINDS OF ELASTIN TO ELASTOLYTIC ENZYMES, TRYPSIN AND CHYMOTRYPSIN* WlM A. LOEVEN
Gerontology Research Center, National Institute of Child Health and Human Development, National Institutes of Health, Department of Health, Education and Welfare, Baltimore City Hospital% Baltimore, Maryland 2z2z4 (U.S.A.) (Received September z8, x969)
SUMMARY
When measuring the elastolytic activity of elastoproteinase with various elastin preparations and the synergistic effect of the elastomu~ ases Em-I and Em-S on this activity, the following observations were made: x. Staining alkali-treated elastin with Congo Red and orcein largely decreases the susceptibility of elastin to elastoproteinase, while it increases the activities'ot the elastomucases, especially when using Congo Red as a dye. 2. Whereas orcein staining of acid-treated elastin does not affect tile susceptibility of elastoproteinase and the enhancement of this enzyme by Era-I, the synergistic effect of the elastomucase Em-S is completely suppressed. 3. Chymotrypsin and trypsin, which both have no elastolytic activity of themselves, show synergistic effects on unstained acid-treated elastin and elastin stained with DNS (5-dimethylamine-t-naphthalene sulfonyl chloride). Trypsin shows only a slight effect when measuring the activation activity against orcein- and Congo Red-stained acid-treated elastin. 4- As a result of this investigation, a procedure is proposed to measure the concentration of elastoproteinase and elastomucase Em-I in crude enzyme preparation and in pancreatic extracts and to estimate the amount of elastomucase Em-S. Some results of experiments with rats using the proposed assay are given.
INTRODUCTION
Elastin is rapidly dissolved by elastoproteinase (elastase, E~, pancreatopeptidase E, E.C. 3.4.4.7) and the determination of elastolytic activity of pancreatic extracts is based on the solubilization of unstained or stained elastin, mostly prepared from bovine nuchal ligament or aorta. The methods for assaying this solubilization of elastin are based on measuring the loss of weight of the undissolved clastin, the * Part of the experiments described in this paper were performed in Leiden, The Netherlands (Netherlands Institute for Preventive Medicine, TNO). Clin. Chim. Acta, 27 (I97O) 521-533
522
LOEVEN
protein content in the elastolyzate (Biuret method or Folin-Ciocalteau reaction) or the release of dye in the case of stained elastin (Congo Red- and orcein-stained elastin being frequently used). Some of these methods have been used to study the role of elastase in acute pancreatitis in man~, ~. However, the estimates of elastolytic activity in pancreatic extracts are higher than their elastoproteinase content would warrant, since not only the mucolytic elastase components (elastomucases or elastomucoproteinases) but also trypsin and chymotrypsin are able to act synergistically on the activity of elastoproteinase ~-6. Loeven 7 has already demonstrated that small amounts of elastomucase can be isolated from some commercial trypsin and chymotrypsin preparations and that these contaminating agents are in part responsible for the activation activity of these proteolytic enzymes. On the other hand, the measurements of the activity of the elastomucases present in crude elastase preparations give too high values, not only due to the contaminating effect of trypsin and chymotrypsin but also to the fact that the combined activity of the two elastomucases so far identified in pancreas (called Em-I and Era-S) is higher than the summation of the activity of the individual elastomucases 5. Although column chromatography on DEAE-Sephadex is extremely suitable for a complete separation of all the components of the elastase complex*-L this purification procedure is too time-consuming for routine determinations of the enzyme concentrations especially when only small amounts of pancreatic extracts or crude enzyme preparations are available*. Several years ago, the author observed that with the orcein method (alkalitreated elastin stained with orcein) it was very hard to find an activation of the elastoproteinase activity by elastomucase (at that time the presence of only o n e elastomucase was known). This finding, confirmed by Banga (personal communication, April i963), was used by Banga, Loeven and Romh~nyi 8 in their histochemical study of the two-component system in elastin. Whereas a synergistic effect of elastomucase Em-I could be observed when using orcein-elastin, the other elastomucase Em-S was completely inactive. A systematic study of the activity of purified components of the elastase complex, trypsin, and chymotrypsin on various kinds of e!~tin preparations was carried out. As a result of this study, presented in this paper, a procedure is proposed which allows the measuring of the activity of elastoproteinase and of elastomucase Em-I in crude enzyme preparations without the contaminating effects of elastomucase Era-S, trypsin, and chymotrypsin, and at the same time gives an estimated value for the concentration of elastomucase Em-S.
* It is sometimes p~.ssible to separate the enzymes present in crude pancreas extracts into t w o fractions: a. the monomer form of elastoproteinase and elastomucase Em-I present in a precipitate formed during exhaustive dialysis against distilled water for several days of an acetic acid extract of pancreas powder; b. the dimer form of elastoproteinase and elastomucase Em-S both present in the supernatant after removing this dialysis precipitate. This procedure not only requires a large amount of crude material, but it was also observed that with some pancreas extracts (e.g. from rat and rabbit) no precipitate was formed.
Clin. Chim. Acta, 27 (x97o) 521-533
SUSCEPTIBILITY OF ELASTIN TO ENZYMES
523
MATERIAL AND METHODS
I. Elastin preparations Bovine nuchal ligament obtained fresh from the slaughterhouse was used for the preparation of elastin. Both alkali-treated and acid-treated elastin prepared as described in details elsewhere 9 were used as: x. unstained elastin preparations: 2. stained with Congo Red (Fisher certified reagent) according to HallOo; 3- stained with orcein (Fisher certified reagent) according to Banga~'; 4. stained with DNS (5-dimethylamino-x-naphthalene sulfonyl chloride; Eastman Organic Chemicals) according to Rinderknecht et al. z. Since the elastolytic activity increases as the size of the particles decreases ~z, all elastin samples were shaken in an electric Laboratory Test Sieve Vibrator (Derrick Co., Buffalo, N.Y.). Only elastin powder passing the sieve with openings of 44 microns (Tyler equivalent mesh: 325) was used in all experiments. I I . Enzyme preparations Elastoproteinase, elastomucase Em-I and elastomucase Em-S were purified from crude pancreas powder (Organon N.V., Oss, The Netherlands) using DEAESephadex column chromatographyL Chymotrypsin (3 × salt free from ethanol) and trypsin (z × cryst, salt free) were purchased from Nutritional Biochemicals Corp. The chymotrypsin used in the experiments was a mixture of equal amounts of ~-, /~-, F-, and A-chymotrypsin. I I I . Methods used with purified enzyme preparations All elastin preparations were used as suspensions of 5 mg per ml of buffer. Since the velocity of elastolysis varies largely with the ionic strength of the buffer solutions used, partly due to inhibitory effects of certain buffer salts and partly to the decrease of pH values during elastolysis when using buffer solutions with low buffer capacity ~s, a sodium-carbonate-HCl buffer of o.x M and pH 8.8 was used throughout. Only in the case of DNS-elastin, tile original bicine buffer of Rinderknecht et al. s was used. Although the elastomucase activity is higher at lower pH values, a pH of 8.8 was chosen in the experiments because the activity of a fourth component of the elastase complex (the elastolipoproteinase, primarily releasing lipid-like material from elastin) is extremely low at pH 8.85. The reaction mixture consists of : 5 ml of the elastin suspension (z5 mg elastin) pipetted in wide test tubes; x ml of an elastoproteinase solution; y ml of an elastomucase (or trypsin or chymotrypsin) solution. The total volume was made up to 6 ml with buffer. All enzyme solutions were in the carbonate buffer. The test tubes with the substrate were placed for I5 min in an Eberbach water bath shaker at 37 ° before adding the enzymes. The incubation time was always 30 min (except DNS-elastin: x h). The residual elastin was centrifuged off and the amount of solubilized elastin in the supernatant determined; unstained elastin by means of the Biuret method °, stained elastin by measuring the extinction of the dyes at 59° nm (orcein) or at 485 nm (Congo Red) in a Gilford Spectrophotometer, DNS-elastin by measuring tlle fluorescence intensity of the supernatant as described by Rinderknecht et al. ~ in an Aminco Spectrophotofluorometer (excitation wave length: 32o rim, emission wave length: Clin. Chim. Acta, 27 (x97 o) 52x-533
524
LOEVE~
49° nm). For all kinds of elastin preparations investigated, standard curves were made, allowing us to calculate the amount of solubilized elastin from the extinction or fluorescence intensity values measured. PROPOSED PROCEDURE
The following procedure is proposed for the determination of the activity of elastoproteinase and of elastomucase Em-I and the estimation of the activity of elastomucase Em-S in crude elastase preparations and pancreatic extracts. Unstained and orcein-stained acid-treated elastin prepared as discussed on page 523 are homogenized in a sodium-carbonate-HCl buffer of o.I M and pH 8.8. Wide test tubes are filled with 5 ml of this suspension (25 mg of elastin) and preheated for I5 rain at 37 ° in a water bath shaker. To a series of test tubes are added x mg of a highly purified elastoproteinase preparation, y ml of a crude enzyme sample or pancreatic extract to be tested, both dissolved in the carbonate buffer. The amount of enzymes added is chosen so that no more than 5o% and not less than 20% of the initial substrate is hydrolyzed. Under these conditions the enzyme activity is essentially linear. Maximally o.2 mg of the elastoproteinasp is added. The value for y varies with the total elastolytic activity present in the unknown sample and has to be determined in a separate assay (using only the substrate and the crude enzyme with omission of the purified elastoproteinase). For rat pancreatic extract, y appears to be maximally 1. 5 rag. The incubation time is exactly 3o min. After centrifugation, the supernatant is used for determining the amount of elastin solubilized as outlined in the preceding section. The following enzyme units will be used: (i) One elastoproteinase unit (E.U.) of activity is such that I m g of elastin is solubilized under the conditions of the assay: (if) The synergistic effect of the extract is expressed in percents of enhancement of the elastoproteinase (El) activity present in the system: mg elastin dissolved by [x mg El + y mg extract]-I00 X
mg elastin dissolved by x mg El mg elastin dissolved by x mg El
(iii) One elastomucase unit (Em. U.) is the activity of an elastomucase preparation resulting in an enhancement of the activity of a highly purified elastoproteinase preparation by 50% under the experimental conditions of the assay. By using this method and the calculations mentioned above, we obtain for each crude enzyme sample or pancreatic extract: a. values for the total elastolytic activity (a E.U./mg prep.) and the synergistic effect (b Em. U./mg prep.) when using unstained acid-treated elastin; b. values for the same activities with orcein-stained acid-treated -~l-~-"n 1~ E.U./mg prep. and d Em. U./mg prep., respectively). The values for the total elastolytic activity include also the synergistic effect of the elastomucase present in the sample and must be corrected for this enhancement Clin. Chim. Acta, 27 (x97 o) 52Jt-533
SUSCEPTIBILITY OF ELASTIN TO ENZYMES
525
of the real concentration of elastoproteinase. Since the synergistic effect is independent of the low elastoproteinase concentration present in the reaction mixture and is also proportional to the elastomucase concentration, the correction can be made as follows: a. for unstained elastin: E.U./mg corrected = a E.U./mg I + r/2 b Em.U./mg b. for orcein elastin: E.U./mg corrected -c E.U./mg I + I/2 d Em. U./mg The d Em.U./mg prep gives the amount of elastomucase Em-! in units (with an extremely small contribution of trypsin as mentioned in the I)ISCUSSION); the b Em.U./mg prep gives the total amount of elastomucases in units; (b--d) Em.U./mg prep. is the rough estimate for the units of elastomucase Em-S (contaminated with trypsin and chymotrypsin activities). RESULTS
I. P_,i~,ding of dye to the elastic fibers Gotte et al. as have shown that mild hydrolytic damage to autoclaved elastin (comparable with our acid-treated elastin) by the action of 0.5 N NaOH at 25 ° results in a progressively lower uptake of orcein. Boiling of elastin with o.I N NaOH for 45 min at 980 (Lansing method as used for the preparation of alkali-treated elastin) seems to result in only small damage. When measuring the anaount of orcein bound to elastin, we found in the case of acid-treated elastin a mean value for the extinction of orcein at 59 ° nm of o.15o per nag solubiiized elastin and for alkalitreated elastin of O.ll 7 per mg elastin (ratio (extinction acid-treated elastin) :(extinction alkali-treated elastin)-- 1.28). About the same ratio (1.18) was found when comparing the fluorescence intensity of DNS acid-treated and DNS alkali-treated elastin (19. 5 and 16.5 fluorescence units per mg solubilized elastin, respectively, with the photomultiplier scale set at I.O). While in the case of orcein and Congo Red ionic and hydrogen bonds are involved, DNS reacts primarily with free amino groups (covalent linkages). In this connection, it is very surprising that there appears to be a large difference in staining capacity of Congo Red for these two kinds of elastin. Acid-treated elastin binds about five times more of the dye than alkali-treated elastin (mean values for the extinction per mg solubilized elastin at 485 nm: o.o67 and o.oi2, respectively).
II. Elastolytic activity of elastoproteinase and the other enzymes studied on various kinds of elastin preparations Fig. x shows the elastolysis of the elastin preparations by elastoproteinase. The effect of this enzyme on all three types of acid-treated elastin is a zero order reaction up to about 50% solubilized substrate. This effect was already observed for orcein-stained elastin by Banga 1~ and Crepaldi et al. ~*. The higher susceptibility Clin. Chim..4eta, 27 (197 o) 521-533
526
LOEVEN ALKALI-TREATED ELASTIN pH 8.8 I
I
:" 1
i
.........
i
ACID-TREATED ELASTINpH 8.8 =|
w
'/
"~'-'
4
EO
0
0.2
0.4
'
i. . . .
I
"
T
~" 12
CONGO I:O4;T
'"u)~'
I
l
0.6
0.8
.
ORCEIH-STAINEO
4
II
1.0
0
0.1
11.2
0.3
0.4
0.5
mg ELASTOPROTEINASE
m 9 ELASTOPROTEINASE Fig.
'
I. Elastolysis-elastoproteinase concentration curve of various kinds of elastin preparations.
of Congo Red-elastin for elastoproteinase was explained by Hall t° as being a result of the increased negative charge of the substrate after binding the dye. However, this explanation ,:..'!~es not hold for the staining of alkali-treated elastin. When ~:omparing the susceptibility of the alkali-treated elastin preparations to elastoproteinase, only the unstained elastin behaves in the same way as the acid-treated elastins as was found by Lamy et al. ~5 and in our own experiments performed under other experimental conditions6, °. The elastolysis-enzyme concentration curves for the two stained alkaLLtreated elastins are nearly superimposed on each other. They both show the very slow elastin dissolving part up to about 0. 5 mg enzyme, followed by a very accelerated dissolving process at higher enzyme concentrations as also found by Rancati et al. ~6 for orcein-stained elastin. During the first part of the curve only 12 o,;o / of the total amount of elastin present is solubilized. We must, however, mention that the susceptibility of unstained alkali-treated elastin to the enzyme is less than that of the three acid-treated elastin preparations studied. Table I gives data on the susceptibility of the elastin preparations to the elastomucases, trypsin, and chymotrypsin without any addition of elastoproteinase. These data again confirm the earlier observations that pure trypsin and chymotrypsin TABLE
I
ELASTOLYTIC ACTIVITY OF ELASTOMUCASES, T R Y P S I N AND CH'VMOTRYPS!N
K i n d of elastin
preparation
mg of
Elastin solubilized by
Era- 1
Unstained-alkali Congo Red-alkali Orcein-alkali Unstained-acid Congo Red-acid Orcein-aeid DNS-alkali
Trypsin
Em-S cone. i~ mg
cone. in n~¢
Chymotrypsin
cone. in mg
conc. in mg
0.06
o.r2
o.z5
o.zz
o.z~
0.50
0.5
z.o
o.5
z.o
0.3 o.i o.2 o. 7 o.2 o.5 o. z
0. 7 0.2 0.3 1.2 o. 5 1.3 o.2
I.x o. 3 o.3 t .9 x.o 2.t o. 5
0.2 o.t o. z o.4 o.i o.2 o.o
o, 4 o.i o.2 o, 7 0.2 o. 3 o,o
0. 4 o.i o.2 x.o o. 3 o, 4 o, [
o.I 0.2 o.o o,o o.e o.2 o,o
0.2 o. 3 o.o o.o o.2 o.2 o.o
o.I o.I o.o o,o o.o o.o o.o
o.2 o. I o o.o o.I o.o
Clin. Chim. Acta, 2 7 ( z 9 7 o) 5 2 z - 5 3 3
o.z .... ....
SUSCEPTIBILITY OF ELASTIN TO ENZYMES
527
themselves cannot attack elastin. Even in relatively high ccnce_ntrat,_'e.--.s (==, chinpared to the concentration of elastoproteinase), no more than z% of the substrate is clis~ived. Whereas elastomucase Em-S can dissolve no more than about z½% of the alkali-treated elasth~ and elastomucase Em-I about 4/o, o/ the elastol)~ic activity of these enzymes on acid-treated elastin is somewhat higher (up to 4% and 8%, respectively, as a result of the release of polysaccharide-protein complexes6). Although the solubilizing effect is extremely low, here too we found that unstained alkali-treated elastin can be attacked better than the stained ones and that the susceptibility of unstained and orcein-stained acid-treated elastin to Em-I is exactiy the same.
UNSTAINED
ACID-TREATEDELASTINpH 8.8
ALKALI-TREATEDELASTINpH 8.8 20
-
-
~ I ~ l
~
I
~ I
I-
-
t
0.25 mg Em-I -.--0.12 m9 Em-t . . . . 0.06 mg Em-i
¢= 16
20
"
T - - '
"
|
|
,._, 16
•
¢,.,t,,,I
>
--"
' ~ r ~
ss e
35% }
21
" z I,*,.-
"=~'- B ~
J.};,.~.,.", BLANK
-
[
~
,...,,a
e
t
E 4
If 0
0.2
|
|
I
1
1
i
I
0.4
0.6
0.8
0.1
0.2
0.3
0.4
m 9 ELASTOPROTEINASE
mg ELASTOPROTEINASE
UNSTAINED
ACID-TREATEDELASTINpH 8.8
ALKALI-TREATEDELASTINpH 8.8 II
|
|
20
|
1
~T . . . .
'
0.50 mg EmS ----0.25 mo Eros
16
!
/
. . . . 0.12 mg Em-S 30%
B
8tA.K
NK
•~' 4
I
O
0.2
0.4
0.6
mg ELASTOPROTEINASE
0.8
. . . . . .
0
I
:
0.I
.
0.2
I
l
0.3
0.4
~9 ELASTOPROTEIN,",~"
Fig. 2. The synergistic effect of elastomucase Em-I (upper p a r t of the figure) and of elastomucase Em-S (lower part) on the elastolytic a c t i v i t y of elastoproteinase using u n s t a i n e d elastin as substrafe.
Clin. Chim. Acta, 27 (x97 o) 5 z i - 5 3 3
528
LOEVL,'N
III. Synergistic effect of elastomucases Em-I and Em-S on the activity of elastoprotdnase It has been demonstrated that the primary effect of the elastomucases is the release of polysaccharide-protein complexes present in the elastic structure, in this way increasing the possibility of permeation of elastoproteinase into the inner parts of the elastic fibers. Fig. 2 shows that the synergistic effect ot Em-I is larger than that of Em-S and that in both cases the enhancement of the activity of elastoproteinase on alkalitreated elastin is nearly proportional with the elastomucase concentration and independent of the eiastoproteinase concentration. When using acid-treated elastin
CONGO RED-STAINED ALKALI-TREATEDELASTINpH 8.8 I
! -
t
~
I
..... 0.25 m9 Em I 20~-- -0.12 mg Em ' | .... 0.06 mg Eml
1
ACID-TREATEDELASTINpH 88
z ',
1
f //
It
~o2~-~/i
~z
~3o~ ~ / / /
'F
hi..
"
/
136% ,Z •/ ,a, ' .g A A N K
0
322~.///
/229%,t
./.'"
~'Ot,NK
"""
///,//~ 0
0.2 0.4 0.6 0.8 mg ELASTOPROTEINASE
0.05 0.1 0,15 0.2 m9 ELASTOPROTEINASE
CONGO RED STAINED ALKALI-TREATEDELASTINpH 8.8 2O -
"
,"
i~ . . . . . . . .
i'
~
0.50 mgEm.S 16
] J,
---0.25 mg Em-S
.... 0.12 mg Em.S
/~
.=.
,//,
__t
~ 12
__J
E
/~/
/
. I
0.2
I
I
l
0.4
0.6
0.8
m9 ELASTOPROTEINASE
I
I
29%
'2'~9
4 :y ":'"~
E
4
0
I ....
~e
8
,oo~\
,!
'[
12
104%'~. i,f ~
ACID-TREATEDELASTINpH 8.8 20
I
~f,:~LANK
5%~/,,~y-
0
0,05
i
I
I
0.1
0.15
0.2
m9 ELASTOPROTEINASE
Fig. 3. The enhancement of the activity of elastoproteinase on Congo Red-stained elastin of elastomucase Em-I (upper part) and of elastomucase Em-S (lower part).
Ciin. Chim. Acla, 27 (x97o) 52x-533
SUSCEPTIBILITY OF ELASTIN TO E~ZYME$
529
as substrate this is only true up to about z5% dissolved elastin due to the inhibition of the elastomucase activity by the elasto!yzate (Loeven; unpubilshed results). These data confirm results obtained under other experimental conditions6, 0. When using Congo Red-stained elastin, there appears to be a distinct difference between the activity of both elastomucases on alkali- and acid-treated elastin (Fig. 3). The synergistic effect largely depends on the elastoproteinase concentration in the mixture, especially in the case of alkali-treated elastin. The acceleration of the elastolysis in the second part of the curve (above 0. 4 mg elastoproteinase) is sometimes twice that occurring at lower elastoproteinase concentrations. Even in the first part. of the curve (below 3 mg dissolved elastin), the activation activity of the elastomucases is higher than when using unstained alkali-treated elastin as substrate. In comparison
ORCEIN-STAINED ACID-TREATEDELASTINpH 8.8
ALKALI-TREATEDELASTINpH 8.8 20
,
,
1
~
0.25 mg Em I -----0.12 m9 Eml . . . . 0.06 mg Em-I /
20-
1
,
......
,
,
--F---
---
16
]
/
tat
12
/
~ 8
69%~
./
34,
.......
2
/./ : ,#
E
~ 2 1 % 0.2
=
!
l
0.4
0.6
0.8
|
!
0.1 0,2 0.3 mg ELASTOPROTEINASE
mg ELASTOPROTEINASE
_
0.4
ORCEIN-STA~NED ACID-TREATEDELASTiNpH 8.8
ALKALI-TREATEDELASTINpH 8.8 20
I
I
!
I
......0.50 mg Em-S ----0.25 m9 Em.S 16 . . . . 0.12 mg Eros
}
.j/"
:=p
~_ 12
e's
=p i,,,-,
-
E
"/'
-
4 2 1 ~ ~
~. .. ~ , 7%
0.2
0.4
0.6
mg ELASTOPROTEINASE
0.8
0
0.1
n_.?
0.3
0.4
mg ELASTOPROTEINASE
Fig. 4. The activation activity of elastomucase Em-I (upper part) and of elastomucase E m - S (lower part) on the activity of elastoproteinase when usiag orcein-stained elastin as substrate.
Clin. Chin~. Acta. z 7 (z97 o) 5 e z - 5 3 3
530
LO~VE~
with the effect on this kind of substrate, the presence of Em-I in a reaction mixture containing Congo Red-acid-treated elastin results in an extremely high synergistic effect at low elastoproteinase concentrations. The activation activity of elastonmcase Era-S, however, is not only maximally the same as for the Congo Red-stained alkah-treated elastin but decreases rapidly at higiler concentrations of ~:!a~toproteinase. This is caused by an inhibition of the enzyme activity by the releas~:d polysaccharidecontaining substances in the elastolyzate as mentioned above. As a result of this, the curves run almost parallel with the blank. Since the synergistic effect of both elastomucases is so largely dependent on the elastoproteinase concentration, this kind of substrate cannot be used at all for exact measurements of elastomucase activities. The elastolysis-enzyme concentration curves for Em-I and Em-S with orceinstained alkali-treated elastin (Fig. 4) are mainly the same as obtained for Congo Red-stained elastin. The only differences are the lower percentages of activation activity, an indication that orcein-staiaJ~ elastin is less susceptible to elastomucase, especially to Em-S. That orcein staining indeed inhibits the activity of Em-S is clearly demonstrated when acid-treated elastin is used. Over the whole range of elastoproteinase concentrations investigated with this stained substrate, the enhancement of the enzyme activity by Em-S is completely absent. The small effect present at low elastoproteinase concentration is not real since the same amount of solubilized elastinprotein was measured without and in the presence of Em-S. The small amount of orcein released is in any case not protein-bound. The activity of Em-I is still present, independent of the concentration of elastoproteinase and at low concentration of Em-I approximately the same as measured for unstained acidtreated elastin. The synergistic effect of the elastomucases on DNS-alkali-treated elastin was only measured in the low concentration range of elastoproteinase (o-5o/~g) used by Rinderknecht et al. ~. Table II shows that although the synergistic effect is independent of the elastoproteinase concentration, low concentrations of elastomucase give a rapid increase in activation activity leveling off at higher concentrations. Further experiments with this substrate are in progress.
IV. Synergistic effect of trypsin and chymotrypsin on various kinds of dastin preparations In tile same way as elastoproteinase complements the action of trypsin and TABLE II SYNERGISTIC EFFECT OF TIIE ELASTOMUCASES E M - I AND E M - S ON TIIE ACTIVITY O F E L A S T O P R O T E I N A S E U S I N G AS S U B S T R A T E D N ~ A L K A L I - T R E A T E D ELASTIN
Elastomucase
Enzyme conc. in mg
.'~ynergistic efj~ci in o/ /O
Em-I
0.05 o.i 0.25 0.5 o.i2 0.25 0.5
4° 67 78 90 27 47 73 92
Em-S
x.o
Clin. Chim. Acta, 27 (I97o) 521-533
SUSCEPTIBILITY' OF ELASTIN TO ENZYMES
531
chymotrypsin by splitting bonds not in the specificity range of the two other proteases, these proteolytic enzymes without any elastolytic activity of themselves (see Table I) are able to attack the elastic fibers already partially digested by elastoproteinase, in this way enhancing the activity of this enzyme3, *. The present author has shown that to some extent the synergistic effect of tr3~)sin and chymotrypsin is due to a contamination of these enzyme preparations with elastomucase 7. Table IIl shows TABLE
III
S Y N E R G I S T I C E F F E C T O F T R Y P S I N A N D C H Y M O T R Y P S I N ON T H E A C T I V I T Y O F E L A S T O P R O T E I N A S E U S I N G V A R I O U S K I N D S O F E L A S T I N P R E P A R A T I O N S AS S U B S T R A T E
Kind of dastin
Unstained-alkali* Congo Red-alkali Orcein-alkali Unstained-acid* Congo Red-acid Orcein-acid DNS-alkali**
Buyer and pH
Na--carbonateHC1 buffer. o.I M a n d p H 8.8
B i c i n e buffer p H 8.8
Synergistic effect (in %) of Trypsin Chymotrypsin in mg in mg 0.5
l.o
0.5
,,.o
o o 5 35 9 7 31
o o 9 6I 16 15 44
o o o 32 o o 4 t)
o 6 2 53 o 3 7°
* E l a s t o p r o t e i n a s e r a n g e i n v e s t i g a t e d : 0 - 0 . 6 m g ( a l k a l i - t r e a t e d elastin) a n d 0-0.3 mg ( , c i d t r e a t e d elastin). ** 2o m g e l a s t i n i n s t e a d of 25 m g p e r 5 ml; i n c u b a t i o n t i m e I h i n s t e a d of 3 ° rain; e l a s t o p r o t e i n a s e r a n g e o - 5 o / ~ g i n s t e a d of o - o . 6 mg.
that under the conditions used in our experiments trypsin and chymotrypsin up to a concentration of I mg have only a pronounced synergistic effect on DNS alkalitreated elastin and on unstained acid-treated elastin. Chymotrypsin is without any effect on the stained acid-treated elastin, while the activation activity of trypsin on orcein- and Congo Red-stained acid-treated elastin is less as compared with its effect o n the unstained one. DISCUSSION
Hall and Czerkawski t~ have demonstrated that three reactions take place during elastolysis: (i) adsorption of enzyme to the surface of the solid substrate and initial loosening of the surface structure; (if) ensuing permeation of the enzyme into the solid resulting in solubilization of elastin and formation of high-molecular weight ~-elastin; (iii) hydrolytic activity of the enzyme by converting ~-elastin into fl-ela~tin of low-molecular weight. The differences in elastolysis-enzyme concentration curves for alkali- and acid-treated elastin might very well be caused by either a reduced adsorption of elastoproteinase to the surface of elastin after alkali treatment or a reduced penetration capacity or both. The elastic structure seems to become more condensed during alkali treatment. The same explanation can be given for the even lower susceptibility of this kind of elastin to the enzyme after binding dye molecules to the structure. The high activity o zhe elastomucases on the stained alkali-treated elastin can be explained by the low susceptibility of these kinds of elas[in to elastoproteinase. Clin. Cltim. Acta, 27 (x97 o) 5 2 1 - 5 3 3
532
LOEVEN
Since the binding of dye molecules largely abolishes the permeation of the enzyme, any attack of the substrate that opens its structure (e.g. release of polysaccharide compotmds by elastomucase) results directly in a higher elastoprotelnase activity. Although it is assumed that Congo Red and orcein bind to the elastinprotein, it is not known as yet how the attachment occurs. Banga tt demonstrated that the same elastin preparation stained differently with orcein produced by different firms. That orcein is attached to elastin by another mechanism than Congo Red can be concluded if one compares the enzyme activity on these two substrates (see Figs. I, 3 and 4). In contrast with Congo Red, binding of orcein to acid-treated elastin has no effect at all on the activity of elastoproteinase and the enhancement of this activity by elastomucase Em-I. The phenomenon of the complete inhibition of Em-S activity when this kind of substrate is used cannot be expkdned at the moment but must be certainly a consequence of the different reaction mechanisms of the two elastomucases6,18-~0. Whereas the polysaccharide-protein complex present in alkali-treated elastin can be attacked by both elastomuca~es, the binding of orcein molecules to acidtreated elastin seems to block completely the groups susceptible to Em-S. In any case, this phenomenon makes it possible to determine the activity of Em-I in crude elastase preparations without tile interfering activation activity of Em-S and chymotrypsin and with a minimal effect of trypsin (see Table III). Since r mg of trypsin gives a synergistic effect of I5% and in the proposed assay procedure (p. 524) only I-2 mg of a crude enzyme preparation or pancreas extract will be used, the contribution of trypsin to the total synergistic effect of Em-I is very small indeed. Furthermore, the curves for unstained and orcein-stained acid-treated elastin are almost superimposed on each other at low concentration of elastoproteinase in the system (up to o.2 mg; see Fig, I). The values for the synergistic effect of Em-I on elastoproteinase when using these two substrates lie very close together at low Em-I concentrations (compare Figs. 2 and 4). In both cases, this effect is independent of the amount of elastoproteinase and proportional with the elastomucase concentration. A subtraction of the activity of Em-I in a crude enzyme preparation calculated for orcein-stained acid-treated elastin from the total elastomucase activity measured with unstained acid-treated elastin gives a rough estimate for the concentration of elastomucase Em-S. This estimate is too high for two reasons" (i) the phenomenon observed in previous work that the elastomucases Em-I and Em-S also activated each others activity0; and (ii) the small synergistic effect of chymotrypsin and trypsin. The contaminating effect mentioned under (ii) needs further research. A Trasylol® level sufficient to inhibit the activity of trypsin and chymotrypsin was not adequate to suppress the synergistic effect of these enzymes on elastoproteinase 2 (Trasylol ® only slightly i~hibiting the activity of elastoproteinase). The applicability of the assay procedure proposed in MATERIALAND ~ETHODS has been shown in the author's laboratory using rat pancreata. Table iV shows drastic changes in the concentration of pancreatic elastolytic enzymes of hypophysectomized female rats (r9-months-old and hypophysectomized at an age of 9 months) as compared with x9-months-old control rats al. The same assay procedure was also used measuring the age relationship of elastolytic enzymes in male and female rat pancreata (Loeven, in manuscript). Geokas et al. ~ and Rinderknecht a al. ~ have mentioned that there are indicaClin. Chim. Acta, 27 (x97o) 52x-533
SUSCEPTIBILITY OF ELASTIN TO ENZYMES
52]3
T A B L E IV CONCENTRATIONS OF PANCREATIC ELASTOLYTIC ENZYMES OF HYPOPHYSECTOMIZED AND UNTREATED FEMALE RATS
(I9 m o n t h s old" h y p o p h y s e c t o m i z e d a t an age of 9 months) E n z y m e units per g acetone-dried pancreas.
Substrate used
Enzyme measured
Enz~,,me activity in pancreas of hypophysect, rats control rats
Unstained acidt r e a t e d elastin
Elastoproteinase E m - I + Em-S e s t i m a t e Em-S Eiastoproteinase Em-I
45-5 i28.2 46.6 34.5 81.6
Orcein-stained acid-elastin
! 6.1 E.U. -+2 ~o.9 E m . U Em.U. ~: 4-9 E.U. ± 9.5 Era. ~.
204.9 41.z 24. 4 t88.6 i6.8
~ 24.8 E.U. ~: 6.2 Em.U. Em.U. -~: I4.6 E.U. ~ 3.3 E m . [ ' .
tions that the assay systems used by them "still lacks sufficient selectivity to exclusively measure elastoproteinase" and these authors feel that, e.g., elastomucase is a logical contender for the enhancing factor. The assay procedure proposed by us might be able to solve this problem. ACKNOWLEDGEMENT
The author wishes to acknowledge the technical assistance of Miss P. Los and MI,:. A. G. A. Sack-Janssen (N.I.P.G./TNO, Leiden) and Mrs. M. M. Baldwin (GRC, Baltimore). REFERENCI';~ t M. C. GEOKAS, S. EISENSTEIN, R. D. MCKENNA AND !. T. BECK, J. Lab. Clin. Med., 69 (1967) 530. 2 H. R I N D E R K N E C H T , M. C. GEOKAS, P. SILVERMAN, Y . L I L L A R D AND B. J. H/.VERBACK, Clin. Chim. A a a . I9 (1968) 3z7 . 3 A. AMATI AND B. CASTELL1, ltal. J. Biochem., lO (1961) 292. 4 G. N. GRANT, Biochem. J., 75 (I96o) I4P. 5 W. A. LO]gVEN, in Proceedings NATO-Conference on The Structure and Function of Connective and Skeletal Tissues, B u t t e r w o r t h , Inc., L o n d o n - E d i n b u r g h , x965, pp. xo9-116. 6 W. A. LOEVEN, Acta Physiol. Pharmacol. Neerl., x3 (1965) 278. 7 W. A. LOEVEN, Acta Physiol. Pharmacol. Neerl., x2 (1963) 57. 8 1. BANOA, W. A. LOEVEN AND GY. ROM~ANVl, Acta Morphol. Acad. Sci. Hung., 13 (I965) 385 • 9 W. A. LoEvz~;, Acta Physiol. Pharmacol. Need., 9 (I96o) 473Io D. A. HALL, Biochem. J., xol (I966) 29. I t I. BANGA, Acta Physiol. Acad. Sci. Hung., 24 (1963) x. tz W. A. LOEVEN, in D. A. HALL (Ed.), International Review Connective Tissue Research, Vol. I, Academic Press, New York, 1963, pp. 183-24 o. 13 L. GOTTE, P. STEnN, D. F. ELSDEN AND S. M. PARTRIDGE, Biochem. J., 87 (I963) 344. t 4 G. CREPALDI, G. ZENI, R. CARLI AND P. AVOGARO, Giorn. Gerontol., Iz (I963) 97. 15 F. LAMY, C. P. CRAIG AND S. TAUBER, J. Biol. Chem., 236 (x96I) 86. t6 G. RANCATL P. MARRAMA, C. FERRARI A,~D B. BONATL Giorn. Biochem., 8 (t959) 67. t 7 D. A. HALL AND J. W. CZERKAWSKL Biochem. J., 8o (I96z) I z l , t28, -'34. I8 M. J. BARNES AND S. M. PARTRIDGE, Biochem. J., xo9 (x968) 883. 19 W. A. LOEVEN, Acta Physiol. Pharmacol. Neerl., 14 (I967) 475. 2o I. BANGA, Structure and Function of Elastin and Collagen, Akaddmiai Kiad6, Budapest, I966, pp. 63-67. 2x W. A. LOEVEN, Proceedings 8th Intern. Congr. Gerontology, W a s h i n g t o n D.C., Aug. I969, Vol. 2, No. 55, P. 16; Full-length p a p e r s u b m i t t e d to Endocrinology.
Clin. Chim. Acta, 27 (I97 o) 521-533