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Inhibition of bovine trypsin with human plasma inhibitors
THROMBOSIS
RESEARCH
@Pergamon
hwss
14; 413-422 Printed
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INHIBITION OF BOVINE TRYPSIN WITH HUMAN PLASMA INHIBITORS
Akikazu Takada, Shigeji Fukuda and Yumiko Takada Department of Physiology, Hamamatsu University, School of Medicine, Hamamatsu-shi, Shizuoka-ken, Japan, 431-31
(Received
19.6.1978; hcepted by
in revised sorm 21.11.1978. Edi..tor C.M. Jackson)
ABSTRACT
’
ester) by trypsin Hydrolysis of TAMe .._ (tosyl arginine methyl ._._. . was not inhibited by undiluted plasma or Its pseudoglobulin fraction. Dilution of plasma resulted in inhibition of trypsin activity. Inhibition of tryptic hydrolysis of TAMe by urinary trypsin inhibitor (UTI) was prevented by macroglobulin fractions of pseudoglobulin. When casein was used as the substrate a similar phenomenon was not observed because the trypsin- Qnacroglobulin complex does not hydrolyze casein. Highly purified ¯oglobulin (ocJM) protected trypsin against other proteinase inhibitors (d,antitrypsin or UTI). Mixture of 1.1 )rM ofOc,an itrypsin (cx,AT) and 9.1 x lo-2pM of ClzM did not inhibit 8.3 x 10' of trypsin sign'ficantly, but one tenth amounts ofd,AT (1.1 x 10' MM) and 9.1 10' pM of &M showed a GM _$ 1 large extent of inhibition of 8.3 x 10 PM of trypsin. These results may suggest that &M rather protected trypsin from its inactivation by other proteinase inhibitors. Such&M-trypsin complex still hydrolyzes molecules of certain molecular weights such as tripepti'depNA (H-D-Val-Leu-Lys-pNA, M.W. 552).
INTRODUCTION There are seven established proteinase inhibitors in human plasma;a,AT, $antichymotrypsin, inter-0c-trypsin inhibitor (IdI), antithrombin III,(AT III), Cl-inactivator (Cl-INA), &.M and&plasmin inhibitor (&PI). Among these, s,AT, Id I, AT lII,OGM,&PI may be related to the regulation of hemostasis. Cl-INA may also be related to hemostasis since Cl-MA inactivates not only Cl (first component of the complement) but also plasmin (l), and since tranexamic acid, potent inhibitor of fibrinolysis, is shown to be effective in treatment of hereditary angioneurotic edema (congenital deficiency of Cl-INA) (2, 3). Plasma inhibitors may also be important in protecting plasma enzymes against pancreatic proteinases which are liberated during pancreatitis. 413
414
INHIBITION
OF TRYPSIN
Vol.lk,No.2/3
Although many important contributions were done on chemical properties of trypsin inhibitor and its interaction with trypsin (4, 5, 6), . . not _ much is known about in vivo interaction of trypsin and proteinase inhibitors. It is suggested thatdaM forms a complex with proteinase and the complex is eliminated from the circulation, thus&M serving to protect our body against destruction by proteinases (7). Recently we have recognized that tryptic hydrolysis of TAMe is not inhibited by undiluted serum, but is inhibited when serum is 100 times diluted. The similar observation was reported by Bieth et al which indicates that increase in the amount of serum did not fully inhibit trypsin (8). In the present paper we report some results which may indicate different roles of&M and &AT in inhibition of trypsin in vivo. MATERIALS AND METHODS Plasma
Plasma was obtained from out-dated blood.
Pseudoglobulin Plasma was dialyzed against 20 times volume of water, then pH was adjusted to 5.2 by acetic acid. After centrifugation, the supernatant was frozen as pseudoglobulin till use. One ml of pseudoglobulin contained 0.63 mg (11 PM) of oi,AT and 3.0 mg (4.1 PM) of oclM. Trypsin
Bovine trypsin. type 1, from Sigma Co., Ltd. was used.
&-macroglobulin
and d,-antitrypsin
Highly purified&M andd/AT were kindly provided by Drs. T. Ikenaka, M. Morii and Y. Ohta, 2nd Department of Biochemistry, Niigata, Japan (9). Crude d,AT was prepared as indicated by Bieth et al: Gel filtration of pseudo lobulin through Sephadex G-200 and concentration of fractions having was used in the present experiments unless otherwise o(tiAT98). Crude&AT stated. Hydrolysis of TAMe Trypsin (0.1 ml containing 2 )J ), 0.1 ml of inhibitor (Y,AT, &M or UTI), and 0.1 ml of pseudoglobulin 9 buffer was added to adjust the volume to 0.5 ml) were incubated for 3 min at 37"C, and 0.5 ml of TAMe (10 u moles/O.5 ml) was added. The mixture was incubated for 15 min at 37"C, and Hesterin reagents were added as reported previously (10, 11). The optical density of the solution was measured at 530 nm. Caseinolysis Four percent casein solution was prepared. 0.2 ml of trypsin (4 ug), 0.1 ml of inhibitor and 0.1 ml of pseudoglobulin and 0.1 ml of buffer were incubated for 3 min, and 0.5 ml of 4 % casein solution was added. After 60 min incubation, 3 ml of 15 % solution of trichloroacetic acid was added. The mixture was centrifuged for 10 min at 4°C. After the supernatant was filtered through Whatman No. 1 filter paper, the optical density of the filtrate was measured at 280 nm.
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OF TRYPSIE;
Basic experiments on hydrolysis of TAMe and casein by trypsin 0.1 ml of trypsin (2 pg) was mixed with 0.4 ml of tris-buffer, and 0.5 ml of TAMe (10 u moles) and incubated for various time intervals. 0.2 ml of trypsin (4 pg) was mixed with 0.3 ml of tris-buffer, and 0.5 ml of 4 % casein solution and incubated for various time intervals. Hydrolysis of TAMe and casein was determined as described above. Fig. 1 shows that hydrolysis of TAMe was linear for at least 20 min, and that of casein was linear for at least 1 hr.
Hydrolysis of TAHe
p moles
Caseinolysis 0°280 0.4
. 5 0.2
. / / c
5
10
15
15
20
45
60
Incubation time (min)
Incubation time (min)
FIG.
30
1
Time course of hydrolysis of TAMe and casein by trypsin
Determination ofg,AT
andarM
The amounts of d,AT and ,r,M were determined by using radial immunodiffusion plates (Hoechst). Purification of urinary trypsin inhibitor (UTI) UT1 was prepared by adsorption to Bentnite of urine and gel-filtration through G-100 after its elution with 2 % NH4OH containing 0.2 M NaCl. One unit of UT1 was determined as the amount to inhibit one pg of trypsin (10, 11). Usually the unit was determined by multiplying by 2 the amount of UT1 to inhibit 50 % trypsin. Concentration of protein All units were expressed as p mole /L (= PM).
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RESULTS 1) Inhibition of trypsin activity by human plasma 0.1 ml of trypsin (2 vg) was incubated with 0.1 ml of serially diluted plasma for 3 min, then 0.5 ml of TAMe was added. Hydrolysis of TAMe was measured after 15 min incubation.
FIG. 2 Inhibitory pattern of trypsin activity with human plasma Abscissa indicates the amounts of plasma used in the experiments which are expressed in logarithmic scale. For example the use of ten times diluted plasma means the use of l/100 ml, thus - 10g(1/100) = 2
I 1
2 3 4 - Log (ml'of plasma)
As shown in Fig. 2, no inhibition of TAMe hydrolysis was seen by using 2 times diluted plasma (Final volume of plasma was 0.05 ml.). Rather enhancement of hydrolysis was shown. Increase in the dilution of plasma resulted in greater extent of inhibition of trypsin activity. One hundred times dilution (Final volume was 1 x 10-31131.) of plasma showed largest extent of inhibition. Further dilution resulted in expected decreased inhibition of trypsin activity. 2) Depression by pseudoglobulin of inhibitory effects of UT1 on trypsin Fig. 3 shows the results of experiments in which trypsin was mixed with UT1 and pseudoglobulin before TAMe was added. No inhibition of trypsin was shown by using undiluted or 2 times diluted pseudoglobulin solutions. Increase in dilution of pseudoglobulin to 100 to 1000 times showed no effect on the inhibition of trypsin by UTI. 3) Effect of pseudoglobulin on the inhibition by UT1 of tryptic digestion of casein
INHIBITION
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OF TRYPSIN
FIG. 3 Effects of pseudoglobulin on the inhibition of trypsin by UT1 (2 u). + trypsin -.
+ TAMe
Line indicated by (UT1 t trypsin) means % inhibition of trypsin by UT1 in the absence of pseudoglobulin. The region A is the region where trypsin is protected by&H from inactivation caused by &AT or UTI.
I 1
4 3 2 - Log (mlof pseudoglobulin)
Fig. 4 shows the results of experiments in which pseudoglobulin, UT1 and trypsin were preincubated before casein solution was added. As shown here, tryptic digestion was fully inhibited by pseudoglobulin and UTI. No suppression of inhibition of UT1 action on trypsin was seen in contrast to the use of TAMe as a substrate.
-3
UT1
+ pseudoglobulinCtizM+alAT)
t trypsin r:
-+
z 6 : Y
1
)/
3
4
2
Inhibition of tryptic caseinolysis by UT1 in the presence of pseudoglobulin.
cascin
UT1 + trypsin 4
FIG. 4
Abscissa indicates the amounts of pseudoglobulin. (UT1 + trypsin) indicates tryptic caseinolysis in the presence of UT1 (2 u). 5
- Loglo (ml of pseudoglobulin)
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4) Gel filtration of pseudoglobulin through Sephadex G-200
In order to know which fractions of pseudoglobulin were effective in prevention of UT1 activity on trypsin, 18 ml of pseudoglobulin was gel-filtered through Sephadex G-200. Effluent was mixed with UT1 and trypsin, then TAMe was added.
G-200.
3
I
50 m
As shown in Fig. 5, little inhibition of trypsin activity by UT1 was seen by adding the effluent of the macroglobulin fractions.
\
UT1* effluent + tryps,n -+TAwc
FIG. 5 Gel filtration of pseudoglobulin
IS
30
25
20
Buffer: 0.1 M Tris-HCl, 0.2 M NaCl, pH 8.0 + - - -o:tryptic hydrolysis of TAMe in the presence of UT1 and effluent Ordinate indicates % trypsin activity measured by using 10 1 moles TAMe for hydrolysis.
35
Fraction"0.
5) Effects of &M
on hydrolysis of TAMe by trypsin in the presence of UT1
-+ L ClqM + UTI
\
+
trypsin
TAMe
I-trypsin
1
2
3
4
- Loglo (ml of#&l)
FIG. 6A Effects of highly purified&M on tryptic hydrolysis of TAMe in the presence of UTI. One ml contains 0.66 mg (0.91 uM) of C@l.
,trypsin
+
UT1
v, 0 c
+
trypsin
+
UTI-P+&M
UT1
1
2
3
4
S
- Loglo (ml ofU#
FIG. 68 Effects of preincubation of UT1 and trypsin on tryptic hydrolysis of TAMe in the presence of ;X2M. Preincubation was 5 min.
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Since macroglobulin fractions prevented the inhibition of trypsin by UTI, we mixed highly purified&AM with UT1 and then inmtediatelytrypsin was added. After mixing these three components, 0.5 ml of TAMe solution was added. As shown in Fig. 6A, no inhibition of tryptic hydrolysis of TAt was seen when 66 pg (9.1 x lO'*BM) of 3GM was mixed with 2 pg (8.3 x 10'9 PM) of trypsin and 2 unit of UTI. UT1 fully inhibited trypsin in the presence of 0.66 ug (9.1 x 10'8FM) of c&i. We incubated UT1 with trypsin for 5 min at 37Y, then &M was added. Three minutes later, TAMe was added. Fig. 68 shows that&M had no effect on the inhibition of trypsin by UT1 when LITYI and trypsin reacted for 5 min. 6) Effects of &antitrypsin
on hydrolysis of TAMe by trypsin
Crude &AT solution was mixed with trypsin, and AMe was added. 61 pg 8.3 x 10'b pM) of trypsin, but no (1.1 PM) of D'IAT completely inhibited 2 of or,AT was mixed with inhibition was seen when 0.61 pg (1.1 x trypsin (Fig. 7).
FIG. -
trypsin
??
&AT-
??
Effects of3oAT lysis of TAMe.
TAM
7
’ on tryptic hydro-
One ml contained 0.61 mg (11 PM) of &AT. Abscissa indicates the amounts of&AT solution.
/ 1
2
3
4
5
6
- LCSlo (ml ofeAT)
7) Effects of the mixture of&,AT
and&M
on tryptic hydrolysis of TAMe
We incubated 61 c(g (1.1 PM) of highly pu:jfiedtlrATand 66 ug (9.1 x lo-* uM) of highly purified&M with 2 ug (8.3 x 10 FM) of trypsin, then TAk was added. Fig. 8 shows that 49 % of trypsin a_Stivitywas seen. When 6,l ug (1.1 x 10 PM) of QAT and 6.6 ug (9.1 x 10 PM) of&M were incubated with trypsin, 23 % of trypsin aQivity was shown. When 0.61 c(g (1.1 x lO'*pM) of alrAT and 0.66 ug (9.1 x 10 PM) of I&M were used, little inhibition was seen. In order to compare inhibitory patterns between pseudoglobulin and the mixture of&AT and&M, 0.1 m of serially diluted pseudoglobulin solution was mixed with 2 ug (8.3 x 10'4FM) of trypsin. Fig. 8 also shows that undiluted pseudoglobulin_Tather enhanced try sin activity. Ten times diluted solution (ti,AT; 1.1 x 10 FM, &M; 4.1 x lo-BFM) showed a largest extent of inhibition.
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The solution ofd#AT and ULEl and that of pseudo lobulin had different&M concentration (0.91 uM and 4.1 JJM, respectively P , but almost same concentration ofUrAT(10.7 uM and 10.9 uM, respectively). This is the reason why 100 times dilution (- log = 3) resulted in the return to almost 100 % recovery to trypsin activity in both solutions.
FIG.
8
Effects of the mixture ofollAT andUzM on tryptic hydrolysis of TAMe One ml contained 0.66 mg(9.1 x lO"uM) of highly purified cf,aMand 0.61 mg (11 PM) of highly purified r&AT. One ml of pseudoglobulin contained 11 )tMof c&AT and 4.1 JIM of&M. 0
-.
-:
0:
Effects of pseudoglobulin on tryptic hydrolysis of TAMe. Effects of drM and &AT on tryptic hydrolysis of TAMe.
DISCUSSION Trypsin is inhibited by proteinase inhibitors of human plasma. These are 3(,AT, Id1 and &M. When trypsin is liberated during acute pancreatitis, many plasma proteins are eligible for its attack. For example, C3 is known to :,esplit by trypsin (12). It is known that cleavage of C3 by trypsin takes place during acute pancreatitis (13). Unfortunately c&AT which is an effective inhibitor of human trypsin reacts slowly with human trypsin (8). Bieth et al showed that 75 % of the proteinase bind to&M within less than 2 sec. (8) &M forms complex with trypsin, which still hydrolyzes small molecular weight substances. Recently we studied inhibition of trypsin by serum inhibitors. To our surprise, undiluted plasma did not inhibit tryptic hydrolysis of TAMe (Fig.2). Since plasma contains trypsin inhbitors, the present result indicates that these inhibitors are ineffective in inhibition of trypsin. Diluted plasma rather inhibited trypsin. . We then wanted to know what fraction of pseudoglobulin had activity to protect trypsin against UTI. As shown in Fig. 5, the macroglobulin fraction on G-200 gel filtration had such activity, but when casein was used as a substrate, trypsin activity was totally inhibited. Interaction of trypsin and UT1 before the addition of G&M resulted in no prevention of inhibitory effect andO(,AT were mixed with trypsin, the inhibitory Pattern was of UTI. When&M
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OF TRYPSIN
421
very similar to that of plasma or pseudoglobulin. Dieth et al indicated that the association rate constant of human trypsin is higher for&M than &AT (8). This will explain the present results well. Trypsin quickly reacted witharM andcc+M-trypsin complex is still active for hydrolysis of TAMe. Also this will explain why the mixture of 2 times diluted plasma and trypsin (Fig. 2) or pseudoglobulin and trypsin (Fig. 8) resulted in trypsin activity higher than 100 %. Trypsin loses activities during incubation due to autodigestion, but trypsin entrapped by&M is protected from autodigestion, and shows higher activities than activities of trypsin withoutdrM. Higher Q+&psinactivities in diluted region (Fig. 4 or Fig. 7) may be due to prevention by diluted pseudoglobulin solution or&AT solution of adsorption of trypsin to glass surface, thus prevntion of inactivation of trypsin. lichen plasma was diluted, trypsin reacted withd,AT more effectively. The concentration of&AT in plasma is 200-400 mg/dl (34-68 uM) and that of&M in plasma is 150-350 mg/dl (2-4.8 PM). Even if the concentration of d/AT is more than 10 times in a molar basis, trypsin reacts with&M quicker. Uhen plasma or pseudoglobulin was diluted to 10 to 100 times, trypsin reacted with &AT since the molar concentration of&M is 0.02 to 0.002 JJH (use of 0.1 ml of 10 to 100 times dil$ed plasma in 1 ml assay system) which is too small to react with 2 pg (8.3 x 10 @) "5 trypsin. Fig. 8 shows thaltwhen 61 Pg (1-i pM) of d,AT and 66 pg (9.1 x 10 PM) ofoQl were mixed with 2 yg (8.3 x 10 pM) of trypsin, trypsin reacted with&M. 'Ten times dilution of3(,AT and &fl resulted in interaction of& AT with trypsin. In the case of ten times dilution, the molar ratio ofjCAT to trypsin was 0.11/0.083 = 1.3, and that ofc&M to trypsin was 0.11. Barrett and Starkey (14) put forth the theory that trypsin enters&M, and that trypsin cleaves a peptide bond inside&H molecule. Trypsin is then entrapped inNAM so that only molecules of a definite size can be hydrolyzed by entrapped trypsin. Our results seem to support the theory. Trypsin which is entrapped in&M can not be inhibited by other inhibitors (o(,ATor UTI). Once trypsin is I:iixed with UT1 previously, then trypsin-UT1 complex does not react with &M anyislore(Fig. 6). The present research indicates that trypsin is capable of hydrolyzing substances of certain molecular weights even if it is entrapped by&M. Peptides are easily attacked by&M-trypsin complex. This indicates that&M rather protects trypsin against other proteinase inhibitors. Physiological role of&M is still uncertain. It may be possible that ;G M keeps proteinase from inactivation by antiproteinase, this proteinase being still effective in breaking down physiologically active substances such as peptide hormones. j(Jl-trypsin complex is capable to hydrolyze synthetic chromogenic substrate (S-2251: H-D-Val-LeuLYs-PNA) which will be reported elsewhere. In this view, af.1 may be regarded not asaninhibitor of proteinase but as their protector. ACKNOULEDGMENTS We are grateful to Dr. T. Ikenaka, 2nd Dept of Biochemistry, Niigata Univ., Sch. of Med., for providing us highly purified&M and +AT preparations. &tl was purified by Dr. Y. Ohta andd,AT was purified by Dr. M. Morii of the same dept. We are also grateful to Dr. Ikenaka for his critical review of the manuscript and valuable suggestions.
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REFERENCES 1. HARPEL, P.C. 568, 1970.
Cl inactivator inhibition by plasmin.
J. Clin. Invest.
49,
2. SHEFFER, A.L., AUSTEN, K.F., and ROSEN, F.S. Treatment of hereditary angioneurotic edema with trans-AMCHA. J. Allerw 49, 133, 1972. 3. SHEFFER, A.L., FEARON, K.T., AUSTEN. K.F., and ROSEN, F.S. Tranexamic acid; Preoperative prophylactic therapy for patients with hereditary angioneurotic edema. J. Allergy 60, 38, 1977. 4. BUNDY, H.F.,and MEHL, J.W. Chem. 234, 1124. 1959.
Trypsin inhibitors of human serum.
J. Biol.
5. KRESS, L.F., and LASKOWSKI, M. SR. Purification, properties, and composition of,z+trypsin inhibitor from human plasma. In Proteinase Inhibitors H. Fritz, H. Tschesche, L.J. Greene and E. Truscheit (Eds) SpringerVerlag, Berlin-Heidelberg, New York 1974, p.23. 6. SKLATVALA, J., WOOD, G.C., and WHITE, 0.0. Isolation and characterization of human plasma proteinase inhibitor and a conformational study of its interaction with proteinase. Biochem. J. 157, 339, 1976. 7. OHLSON. K. Elimination of 1251-trypsin o(,-macroglobulin complexes from blood by reticuloendothelial cells in dog. Acta Physiol.Scand. 81,269,1971. 8. BIETH,J., AUBRY, M.,and TRAVIS, J. The interaction of human cationic trypsin and chymotrypsin II with human serum inhibitors. In Proteinase Inhibitors H. Fritz, H. Tschesche, L.J. Greene and E. Truscm Springer-Verlag, Berlin-Heidelberg, New York 1974, p. 53. 9. MORII, M., ODANI, S., KOIDE, T., and IKENAKA, T. Human a,-antitrypsin. Characterization and N- and C-terminal sequences. J. Biochem. 83.269,1978. 10. SUMI, H., TAKADA, Y.,and TAKADA, A. Studies on human urinary trypsin inhibitor. 1. Its modification on treatment of urine with acid. Thrombosis Res. 11, 747, 1977. 11. SUMI, H., TOKI, N., TAKADA, Y.,and TAKADA, A. Studies on human urinary enzymes and inhibitors. Concentration method and characterization. J. Biochem. 83, 141, 1978. 12. SEELIG, R.,and SEELIG, H.P. The possible role of serum complement system in the formal pathogenesis of acute pancreatitis. Acta Hepato-Gastroenterol. 22, 263, 1975. 13. MINTA, J.O., NAN, D., and MOVAT, H.Z. Kinetic studies on the fragmentation of the third component of complement (C3) by trypsin. J. Immunol. 118, 398, 1977. 14. BARRETT, A.J., and STARKEY, P.M. The interaction of &-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem. 5.133, 709, 1973.
RESEARCH
@Pergamon
hwss
14; 413-422 Printed
Ltd.1979.
in Great Britain 004g-3848/7g/o3o1-0413
$02.00/O
INHIBITION OF BOVINE TRYPSIN WITH HUMAN PLASMA INHIBITORS
Akikazu Takada, Shigeji Fukuda and Yumiko Takada Department of Physiology, Hamamatsu University, School of Medicine, Hamamatsu-shi, Shizuoka-ken, Japan, 431-31
(Received
19.6.1978; hcepted by
in revised sorm 21.11.1978. Edi..tor C.M. Jackson)
ABSTRACT
’
ester) by trypsin Hydrolysis of TAMe .._ (tosyl arginine methyl ._._. . was not inhibited by undiluted plasma or Its pseudoglobulin fraction. Dilution of plasma resulted in inhibition of trypsin activity. Inhibition of tryptic hydrolysis of TAMe by urinary trypsin inhibitor (UTI) was prevented by macroglobulin fractions of pseudoglobulin. When casein was used as the substrate a similar phenomenon was not observed because the trypsin- Qnacroglobulin complex does not hydrolyze casein. Highly purified ¯oglobulin (ocJM) protected trypsin against other proteinase inhibitors (d,antitrypsin or UTI). Mixture of 1.1 )rM ofOc,an itrypsin (cx,AT) and 9.1 x lo-2pM of ClzM did not inhibit 8.3 x 10' of trypsin sign'ficantly, but one tenth amounts ofd,AT (1.1 x 10' MM) and 9.1 10' pM of &M showed a GM _$ 1 large extent of inhibition of 8.3 x 10 PM of trypsin. These results may suggest that &M rather protected trypsin from its inactivation by other proteinase inhibitors. Such&M-trypsin complex still hydrolyzes molecules of certain molecular weights such as tripepti'depNA (H-D-Val-Leu-Lys-pNA, M.W. 552).
INTRODUCTION There are seven established proteinase inhibitors in human plasma;a,AT, $antichymotrypsin, inter-0c-trypsin inhibitor (IdI), antithrombin III,(AT III), Cl-inactivator (Cl-INA), &.M and&plasmin inhibitor (&PI). Among these, s,AT, Id I, AT lII,OGM,&PI may be related to the regulation of hemostasis. Cl-INA may also be related to hemostasis since Cl-MA inactivates not only Cl (first component of the complement) but also plasmin (l), and since tranexamic acid, potent inhibitor of fibrinolysis, is shown to be effective in treatment of hereditary angioneurotic edema (congenital deficiency of Cl-INA) (2, 3). Plasma inhibitors may also be important in protecting plasma enzymes against pancreatic proteinases which are liberated during pancreatitis. 413
414
INHIBITION
OF TRYPSIN
Vol.lk,No.2/3
Although many important contributions were done on chemical properties of trypsin inhibitor and its interaction with trypsin (4, 5, 6), . . not _ much is known about in vivo interaction of trypsin and proteinase inhibitors. It is suggested thatdaM forms a complex with proteinase and the complex is eliminated from the circulation, thus&M serving to protect our body against destruction by proteinases (7). Recently we have recognized that tryptic hydrolysis of TAMe is not inhibited by undiluted serum, but is inhibited when serum is 100 times diluted. The similar observation was reported by Bieth et al which indicates that increase in the amount of serum did not fully inhibit trypsin (8). In the present paper we report some results which may indicate different roles of&M and &AT in inhibition of trypsin in vivo. MATERIALS AND METHODS Plasma
Plasma was obtained from out-dated blood.
Pseudoglobulin Plasma was dialyzed against 20 times volume of water, then pH was adjusted to 5.2 by acetic acid. After centrifugation, the supernatant was frozen as pseudoglobulin till use. One ml of pseudoglobulin contained 0.63 mg (11 PM) of oi,AT and 3.0 mg (4.1 PM) of oclM. Trypsin
Bovine trypsin. type 1, from Sigma Co., Ltd. was used.
&-macroglobulin
and d,-antitrypsin
Highly purified&M andd/AT were kindly provided by Drs. T. Ikenaka, M. Morii and Y. Ohta, 2nd Department of Biochemistry, Niigata, Japan (9). Crude d,AT was prepared as indicated by Bieth et al: Gel filtration of pseudo lobulin through Sephadex G-200 and concentration of fractions having was used in the present experiments unless otherwise o(tiAT98). Crude&AT stated. Hydrolysis of TAMe Trypsin (0.1 ml containing 2 )J ), 0.1 ml of inhibitor (Y,AT, &M or UTI), and 0.1 ml of pseudoglobulin 9 buffer was added to adjust the volume to 0.5 ml) were incubated for 3 min at 37"C, and 0.5 ml of TAMe (10 u moles/O.5 ml) was added. The mixture was incubated for 15 min at 37"C, and Hesterin reagents were added as reported previously (10, 11). The optical density of the solution was measured at 530 nm. Caseinolysis Four percent casein solution was prepared. 0.2 ml of trypsin (4 ug), 0.1 ml of inhibitor and 0.1 ml of pseudoglobulin and 0.1 ml of buffer were incubated for 3 min, and 0.5 ml of 4 % casein solution was added. After 60 min incubation, 3 ml of 15 % solution of trichloroacetic acid was added. The mixture was centrifuged for 10 min at 4°C. After the supernatant was filtered through Whatman No. 1 filter paper, the optical density of the filtrate was measured at 280 nm.
vo1.14,~o.2/3
INHIBITIOIC
415
OF TRYPSIE;
Basic experiments on hydrolysis of TAMe and casein by trypsin 0.1 ml of trypsin (2 pg) was mixed with 0.4 ml of tris-buffer, and 0.5 ml of TAMe (10 u moles) and incubated for various time intervals. 0.2 ml of trypsin (4 pg) was mixed with 0.3 ml of tris-buffer, and 0.5 ml of 4 % casein solution and incubated for various time intervals. Hydrolysis of TAMe and casein was determined as described above. Fig. 1 shows that hydrolysis of TAMe was linear for at least 20 min, and that of casein was linear for at least 1 hr.
Hydrolysis of TAHe
p moles
Caseinolysis 0°280 0.4
. 5 0.2
. / / c
5
10
15
15
20
45
60
Incubation time (min)
Incubation time (min)
FIG.
30
1
Time course of hydrolysis of TAMe and casein by trypsin
Determination ofg,AT
andarM
The amounts of d,AT and ,r,M were determined by using radial immunodiffusion plates (Hoechst). Purification of urinary trypsin inhibitor (UTI) UT1 was prepared by adsorption to Bentnite of urine and gel-filtration through G-100 after its elution with 2 % NH4OH containing 0.2 M NaCl. One unit of UT1 was determined as the amount to inhibit one pg of trypsin (10, 11). Usually the unit was determined by multiplying by 2 the amount of UT1 to inhibit 50 % trypsin. Concentration of protein All units were expressed as p mole /L (= PM).
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INHIBITION
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OF TRYPSIN
RESULTS 1) Inhibition of trypsin activity by human plasma 0.1 ml of trypsin (2 vg) was incubated with 0.1 ml of serially diluted plasma for 3 min, then 0.5 ml of TAMe was added. Hydrolysis of TAMe was measured after 15 min incubation.
FIG. 2 Inhibitory pattern of trypsin activity with human plasma Abscissa indicates the amounts of plasma used in the experiments which are expressed in logarithmic scale. For example the use of ten times diluted plasma means the use of l/100 ml, thus - 10g(1/100) = 2
I 1
2 3 4 - Log (ml'of plasma)
As shown in Fig. 2, no inhibition of TAMe hydrolysis was seen by using 2 times diluted plasma (Final volume of plasma was 0.05 ml.). Rather enhancement of hydrolysis was shown. Increase in the dilution of plasma resulted in greater extent of inhibition of trypsin activity. One hundred times dilution (Final volume was 1 x 10-31131.) of plasma showed largest extent of inhibition. Further dilution resulted in expected decreased inhibition of trypsin activity. 2) Depression by pseudoglobulin of inhibitory effects of UT1 on trypsin Fig. 3 shows the results of experiments in which trypsin was mixed with UT1 and pseudoglobulin before TAMe was added. No inhibition of trypsin was shown by using undiluted or 2 times diluted pseudoglobulin solutions. Increase in dilution of pseudoglobulin to 100 to 1000 times showed no effect on the inhibition of trypsin by UTI. 3) Effect of pseudoglobulin on the inhibition by UT1 of tryptic digestion of casein
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FIG. 3 Effects of pseudoglobulin on the inhibition of trypsin by UT1 (2 u). + trypsin -.
+ TAMe
Line indicated by (UT1 t trypsin) means % inhibition of trypsin by UT1 in the absence of pseudoglobulin. The region A is the region where trypsin is protected by&H from inactivation caused by &AT or UTI.
I 1
4 3 2 - Log (mlof pseudoglobulin)
Fig. 4 shows the results of experiments in which pseudoglobulin, UT1 and trypsin were preincubated before casein solution was added. As shown here, tryptic digestion was fully inhibited by pseudoglobulin and UTI. No suppression of inhibition of UT1 action on trypsin was seen in contrast to the use of TAMe as a substrate.
-3
UT1
+ pseudoglobulinCtizM+alAT)
t trypsin r:
-+
z 6 : Y
1
)/
3
4
2
Inhibition of tryptic caseinolysis by UT1 in the presence of pseudoglobulin.
cascin
UT1 + trypsin 4
FIG. 4
Abscissa indicates the amounts of pseudoglobulin. (UT1 + trypsin) indicates tryptic caseinolysis in the presence of UT1 (2 u). 5
- Loglo (ml of pseudoglobulin)
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4) Gel filtration of pseudoglobulin through Sephadex G-200
In order to know which fractions of pseudoglobulin were effective in prevention of UT1 activity on trypsin, 18 ml of pseudoglobulin was gel-filtered through Sephadex G-200. Effluent was mixed with UT1 and trypsin, then TAMe was added.
G-200.
3
I
50 m
As shown in Fig. 5, little inhibition of trypsin activity by UT1 was seen by adding the effluent of the macroglobulin fractions.
\
UT1* effluent + tryps,n -+TAwc
FIG. 5 Gel filtration of pseudoglobulin
IS
30
25
20
Buffer: 0.1 M Tris-HCl, 0.2 M NaCl, pH 8.0 + - - -o:tryptic hydrolysis of TAMe in the presence of UT1 and effluent Ordinate indicates % trypsin activity measured by using 10 1 moles TAMe for hydrolysis.
35
Fraction"0.
5) Effects of &M
on hydrolysis of TAMe by trypsin in the presence of UT1
-+ L ClqM + UTI
\
+
trypsin
TAMe
I-trypsin
1
2
3
4
- Loglo (ml of#&l)
FIG. 6A Effects of highly purified&M on tryptic hydrolysis of TAMe in the presence of UTI. One ml contains 0.66 mg (0.91 uM) of C@l.
,trypsin
+
UT1
v, 0 c
+
trypsin
+
UTI-P+&M
UT1
1
2
3
4
S
- Loglo (ml ofU#
FIG. 68 Effects of preincubation of UT1 and trypsin on tryptic hydrolysis of TAMe in the presence of ;X2M. Preincubation was 5 min.
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Since macroglobulin fractions prevented the inhibition of trypsin by UTI, we mixed highly purified&AM with UT1 and then inmtediatelytrypsin was added. After mixing these three components, 0.5 ml of TAMe solution was added. As shown in Fig. 6A, no inhibition of tryptic hydrolysis of TAt was seen when 66 pg (9.1 x lO'*BM) of 3GM was mixed with 2 pg (8.3 x 10'9 PM) of trypsin and 2 unit of UTI. UT1 fully inhibited trypsin in the presence of 0.66 ug (9.1 x 10'8FM) of c&i. We incubated UT1 with trypsin for 5 min at 37Y, then &M was added. Three minutes later, TAMe was added. Fig. 68 shows that&M had no effect on the inhibition of trypsin by UT1 when LITYI and trypsin reacted for 5 min. 6) Effects of &antitrypsin
on hydrolysis of TAMe by trypsin
Crude &AT solution was mixed with trypsin, and AMe was added. 61 pg 8.3 x 10'b pM) of trypsin, but no (1.1 PM) of D'IAT completely inhibited 2 of or,AT was mixed with inhibition was seen when 0.61 pg (1.1 x trypsin (Fig. 7).
FIG. -
trypsin
??
&AT-
??
Effects of3oAT lysis of TAMe.
TAM
7
’ on tryptic hydro-
One ml contained 0.61 mg (11 PM) of &AT. Abscissa indicates the amounts of&AT solution.
/ 1
2
3
4
5
6
- LCSlo (ml ofeAT)
7) Effects of the mixture of&,AT
and&M
on tryptic hydrolysis of TAMe
We incubated 61 c(g (1.1 PM) of highly pu:jfiedtlrATand 66 ug (9.1 x lo-* uM) of highly purified&M with 2 ug (8.3 x 10 FM) of trypsin, then TAk was added. Fig. 8 shows that 49 % of trypsin a_Stivitywas seen. When 6,l ug (1.1 x 10 PM) of QAT and 6.6 ug (9.1 x 10 PM) of&M were incubated with trypsin, 23 % of trypsin aQivity was shown. When 0.61 c(g (1.1 x lO'*pM) of alrAT and 0.66 ug (9.1 x 10 PM) of I&M were used, little inhibition was seen. In order to compare inhibitory patterns between pseudoglobulin and the mixture of&AT and&M, 0.1 m of serially diluted pseudoglobulin solution was mixed with 2 ug (8.3 x 10'4FM) of trypsin. Fig. 8 also shows that undiluted pseudoglobulin_Tather enhanced try sin activity. Ten times diluted solution (ti,AT; 1.1 x 10 FM, &M; 4.1 x lo-BFM) showed a largest extent of inhibition.
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The solution ofd#AT and ULEl and that of pseudo lobulin had different&M concentration (0.91 uM and 4.1 JJM, respectively P , but almost same concentration ofUrAT(10.7 uM and 10.9 uM, respectively). This is the reason why 100 times dilution (- log = 3) resulted in the return to almost 100 % recovery to trypsin activity in both solutions.
FIG.
8
Effects of the mixture ofollAT andUzM on tryptic hydrolysis of TAMe One ml contained 0.66 mg(9.1 x lO"uM) of highly purified cf,aMand 0.61 mg (11 PM) of highly purified r&AT. One ml of pseudoglobulin contained 11 )tMof c&AT and 4.1 JIM of&M. 0
-.
-:
0:
Effects of pseudoglobulin on tryptic hydrolysis of TAMe. Effects of drM and &AT on tryptic hydrolysis of TAMe.
DISCUSSION Trypsin is inhibited by proteinase inhibitors of human plasma. These are 3(,AT, Id1 and &M. When trypsin is liberated during acute pancreatitis, many plasma proteins are eligible for its attack. For example, C3 is known to :,esplit by trypsin (12). It is known that cleavage of C3 by trypsin takes place during acute pancreatitis (13). Unfortunately c&AT which is an effective inhibitor of human trypsin reacts slowly with human trypsin (8). Bieth et al showed that 75 % of the proteinase bind to&M within less than 2 sec. (8) &M forms complex with trypsin, which still hydrolyzes small molecular weight substances. Recently we studied inhibition of trypsin by serum inhibitors. To our surprise, undiluted plasma did not inhibit tryptic hydrolysis of TAMe (Fig.2). Since plasma contains trypsin inhbitors, the present result indicates that these inhibitors are ineffective in inhibition of trypsin. Diluted plasma rather inhibited trypsin. . We then wanted to know what fraction of pseudoglobulin had activity to protect trypsin against UTI. As shown in Fig. 5, the macroglobulin fraction on G-200 gel filtration had such activity, but when casein was used as a substrate, trypsin activity was totally inhibited. Interaction of trypsin and UT1 before the addition of G&M resulted in no prevention of inhibitory effect andO(,AT were mixed with trypsin, the inhibitory Pattern was of UTI. When&M
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very similar to that of plasma or pseudoglobulin. Dieth et al indicated that the association rate constant of human trypsin is higher for&M than &AT (8). This will explain the present results well. Trypsin quickly reacted witharM andcc+M-trypsin complex is still active for hydrolysis of TAMe. Also this will explain why the mixture of 2 times diluted plasma and trypsin (Fig. 2) or pseudoglobulin and trypsin (Fig. 8) resulted in trypsin activity higher than 100 %. Trypsin loses activities during incubation due to autodigestion, but trypsin entrapped by&M is protected from autodigestion, and shows higher activities than activities of trypsin withoutdrM. Higher Q+&psinactivities in diluted region (Fig. 4 or Fig. 7) may be due to prevention by diluted pseudoglobulin solution or&AT solution of adsorption of trypsin to glass surface, thus prevntion of inactivation of trypsin. lichen plasma was diluted, trypsin reacted withd,AT more effectively. The concentration of&AT in plasma is 200-400 mg/dl (34-68 uM) and that of&M in plasma is 150-350 mg/dl (2-4.8 PM). Even if the concentration of d/AT is more than 10 times in a molar basis, trypsin reacts with&M quicker. Uhen plasma or pseudoglobulin was diluted to 10 to 100 times, trypsin reacted with &AT since the molar concentration of&M is 0.02 to 0.002 JJH (use of 0.1 ml of 10 to 100 times dil$ed plasma in 1 ml assay system) which is too small to react with 2 pg (8.3 x 10 @) "5 trypsin. Fig. 8 shows thaltwhen 61 Pg (1-i pM) of d,AT and 66 pg (9.1 x 10 PM) ofoQl were mixed with 2 yg (8.3 x 10 pM) of trypsin, trypsin reacted with&M. 'Ten times dilution of3(,AT and &fl resulted in interaction of& AT with trypsin. In the case of ten times dilution, the molar ratio ofjCAT to trypsin was 0.11/0.083 = 1.3, and that ofc&M to trypsin was 0.11. Barrett and Starkey (14) put forth the theory that trypsin enters&M, and that trypsin cleaves a peptide bond inside&H molecule. Trypsin is then entrapped inNAM so that only molecules of a definite size can be hydrolyzed by entrapped trypsin. Our results seem to support the theory. Trypsin which is entrapped in&M can not be inhibited by other inhibitors (o(,ATor UTI). Once trypsin is I:iixed with UT1 previously, then trypsin-UT1 complex does not react with &M anyislore(Fig. 6). The present research indicates that trypsin is capable of hydrolyzing substances of certain molecular weights even if it is entrapped by&M. Peptides are easily attacked by&M-trypsin complex. This indicates that&M rather protects trypsin against other proteinase inhibitors. Physiological role of&M is still uncertain. It may be possible that ;G M keeps proteinase from inactivation by antiproteinase, this proteinase being still effective in breaking down physiologically active substances such as peptide hormones. j(Jl-trypsin complex is capable to hydrolyze synthetic chromogenic substrate (S-2251: H-D-Val-LeuLYs-PNA) which will be reported elsewhere. In this view, af.1 may be regarded not asaninhibitor of proteinase but as their protector. ACKNOULEDGMENTS We are grateful to Dr. T. Ikenaka, 2nd Dept of Biochemistry, Niigata Univ., Sch. of Med., for providing us highly purified&M and +AT preparations. &tl was purified by Dr. Y. Ohta andd,AT was purified by Dr. M. Morii of the same dept. We are also grateful to Dr. Ikenaka for his critical review of the manuscript and valuable suggestions.
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REFERENCES 1. HARPEL, P.C. 568, 1970.
Cl inactivator inhibition by plasmin.
J. Clin. Invest.
49,
2. SHEFFER, A.L., AUSTEN, K.F., and ROSEN, F.S. Treatment of hereditary angioneurotic edema with trans-AMCHA. J. Allerw 49, 133, 1972. 3. SHEFFER, A.L., FEARON, K.T., AUSTEN. K.F., and ROSEN, F.S. Tranexamic acid; Preoperative prophylactic therapy for patients with hereditary angioneurotic edema. J. Allergy 60, 38, 1977. 4. BUNDY, H.F.,and MEHL, J.W. Chem. 234, 1124. 1959.
Trypsin inhibitors of human serum.
J. Biol.
5. KRESS, L.F., and LASKOWSKI, M. SR. Purification, properties, and composition of,z+trypsin inhibitor from human plasma. In Proteinase Inhibitors H. Fritz, H. Tschesche, L.J. Greene and E. Truscheit (Eds) SpringerVerlag, Berlin-Heidelberg, New York 1974, p.23. 6. SKLATVALA, J., WOOD, G.C., and WHITE, 0.0. Isolation and characterization of human plasma proteinase inhibitor and a conformational study of its interaction with proteinase. Biochem. J. 157, 339, 1976. 7. OHLSON. K. Elimination of 1251-trypsin o(,-macroglobulin complexes from blood by reticuloendothelial cells in dog. Acta Physiol.Scand. 81,269,1971. 8. BIETH,J., AUBRY, M.,and TRAVIS, J. The interaction of human cationic trypsin and chymotrypsin II with human serum inhibitors. In Proteinase Inhibitors H. Fritz, H. Tschesche, L.J. Greene and E. Truscm Springer-Verlag, Berlin-Heidelberg, New York 1974, p. 53. 9. MORII, M., ODANI, S., KOIDE, T., and IKENAKA, T. Human a,-antitrypsin. Characterization and N- and C-terminal sequences. J. Biochem. 83.269,1978. 10. SUMI, H., TAKADA, Y.,and TAKADA, A. Studies on human urinary trypsin inhibitor. 1. Its modification on treatment of urine with acid. Thrombosis Res. 11, 747, 1977. 11. SUMI, H., TOKI, N., TAKADA, Y.,and TAKADA, A. Studies on human urinary enzymes and inhibitors. Concentration method and characterization. J. Biochem. 83, 141, 1978. 12. SEELIG, R.,and SEELIG, H.P. The possible role of serum complement system in the formal pathogenesis of acute pancreatitis. Acta Hepato-Gastroenterol. 22, 263, 1975. 13. MINTA, J.O., NAN, D., and MOVAT, H.Z. Kinetic studies on the fragmentation of the third component of complement (C3) by trypsin. J. Immunol. 118, 398, 1977. 14. BARRETT, A.J., and STARKEY, P.M. The interaction of &-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem. 5.133, 709, 1973.