Agents and Actions, vol. 29, 3/4 (1990)

0065-4299/90/040388-06 $1.50+ 0.20/0 9 1990 Birkh/iuser Verlag, Basel

Inactivation of l-proteinase inhibitor by Cu(II) and hydrogen peroxide N.S. Kwon, P.C. Chan and L. Kesner 1 Department of Biochemistry, State Universityof New YorkHealth ScienceCenter at Brooklyn, Brooklyn,N.Y 11203,USA

Abstract

When cq-proteinase inhibitor was treated with 1 5 g M CuSO4 in the presence ofH202 (250-1000 gM), its elastase inhibitory capacity was markedly decreased. Several other metal ions tested had either very little or no effect. The Cu(II)-catalyzed decrease in the inhibition of elastase activity can also be demonstrated in dialyzed plasma. These results are consistent with the hypothesis that in several pathological conditions in which extracellular copper levels are elevated, Cu(lI)-catalyzed peroxidation of cq-proteinase inhibitor may occur at sites of inflammation where H20 / is secreted as a major product by activated phagocytes.

Introduction

~l-Proteinase inhibitor (cq-PI) is a major serine protease inhibitor in the plasma. One of its primary physiological functions is to protect connective tissue from degradation by elastase, an enzyme which is secreted by activated phagocytes [1, 2]. ~I-PI is responsible for over 90% of the serum anti-elastase activity [3]. An imbalance between the inhibitor and elastase has been postulated for the tissue destruction in pulmonary emphysema and rheumatoid arthritis [2, 4-7]. cq-PI can easily be inactivated by various chemical oxidants such as N-chlorosuccinimide [8, 9], chloramine T [8], ozone [10], cigarette smoke [8, ll, 12], and hypochlorous acid [13]. Phagocytes, upon stimulation, have also been shown to oxidize the inhibitor by releasing myeloperoxidase and various reactive oxygen species [13-15]. 1 To whomall correspondenceshould be addressed.

Copper is an essential trace metal ion. It is a component of several enzymes including lysyl oxidase and superoxidase dismutase, which are important for repair and defence mechanisms. On the other hand, under certain conditions it may also serve to magnify the deleterious effect of reactive oxygen species, especially H20 2 [16-19]. Moreover, increased levels of copper have been observed in the serum and synovial fluid of patients with rheumatoid arthritis [20, 21] and in the serum of cigarette smokers [22]. The present study is focused on the catalytic role of trace amount of Cu(II) on the inactivation of cq-PI by H202. Materials and methods

Materials Elastase (porcine pancreatic, EC 3.4.21.36, type II-A), N-succinyl-L-alanyl-L-alanyl-L-alanine p-nitroanilide and ATP were purchased from Sigma Chemical Co.; hydrogen peroxide from Aldrich

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Chemical Co.; and catalase (beef liver) from Boehringer Mannheim Biochemicals. el-PI was purified from freshly prepared platelet-depleted human plasma according to the method of Travis and Johnson [23]. The human plasma was kindly provided by Dr. T.C. Detwiler's laboratory in our department.

ooll

~o

ii

-o

9

Treatment of ~:-PI with Cu(II) and 1/202 e~-PI (50 pg/ml, 0.94 gM) was preincubated with varying concentrations of CuSO4 for 5 min in a water bath at 37 ~ The CuSO4 was dissolved in water and added to a buffered %-PI solution. H 2 0 2 was then added to the mixture and incubated for 30 rain at 37~ The reaction was stopped by addition of EDTA (0.5 m M ) and catalase (10 gg/ml). The treatment was performed in 5 m M sodium phosphate buffer, pH 7.4. The concentration of %-PI was measured by using an extinction coefficient of A (1%, 1 c m ) = 5.3 at 280 nm [24]. H 2 0 2 was diluted from a 30% stock solution whose concentration was determined by the method of Allen et al. [25]. Various dilutions were made in 5 m M sodium phosphate, pH 7.4.

Treatment of dialyzed plasma with Cu (II) and H20 2 H u m a n plasma was mixed with 5 m M EDTA, and dialyzed against 100 volumes of a phosphate buffered saline solution (5 m M sodium phosphate, 150 m M NaC1, p H 7.4) for 5 days at 4~ The buffer was changed every 9 - 1 5 hr. The dialyzed plasma (50 gl/ml) was treated with CuSO4 and H z O 2 in the phosphate buffered saline by a similar procedure as described above.

0

~

0

500 H202 (yM)

L

I000

Figure l

Cu(II)-catalyzed peroxidation %-PI. %-PI (50#g/ml) was treated with various concentrationsof CuSO4 (o, none; e, 1 gM; A, 2 gM; A, 5 gM) and H202 at 37~ The reaction was stopped by adding EDTA (0.5 raM) and catalase (10/~g/ml) after 30 rain. Elastase was incubated with the treated %-PI, and its activitywas assayed. Elastase inhibitory activity of the treated %-PI was expressed as % of control. The control was preincubated under similar condition in the absence of CuSO4 and H202.

change of absorbance at 410 nm for 1 min using a Cary 210 Spectrophotometer (Varian). Elastase was dissolved in Tris buffer at the concentration to give an increase of absorbance of 0.16___0.02 per rain in the absence of inhibitor. Control inhibitor incubated without CuSO4 and H 2 0 2 reduced the elastase activity by 6 5 - 7 6 % . Elastase inhibitory activity of treated inhibitor was expressed as percent of the control. New plastic test tubes (16 x 8 2 m m , Sarstedt 55.524) were used for the treatment of inhibitor and incubation with elastase. Less elastase and %-PI activities were observed in glass or used plastic tubes, probably due to nonspecific protein adsorption.

Assay for elastase inhibitory capacities of ~I-PI and plasma The elastase inhibitory activity of %-PI or plasma was measured by the method of Travis and Johnson [23] with slight modification. Typically, 200 gl of porcine pancreatic elastase was incubated with 100 ~tl of the treated or untreated inhibitor for 10 rain at 25 ~ 680 gl of Tris-HCl buffer (0.2 M, p H 8 . 0 ) and 150gg of N-succinyl-L-alanyl-Lalanyl-L-alanine p-nitroanilide (in 20 pl dimethylsulfoxide) were added to start the reaction. Elastase activity was determined by measuring the

Results

Inactivation of ~:-PI by Cu (II)/H20 2 When ~:-PI (50 gg/ml) was treated with CuSO4 (1, 2 or 5 pM) and H 2 0 2 (250, 500 or 1000 pM) for 30 rain at 37~ the inhibitor activity was significantly decreased (Fig. 1). In the presence of 500 p M H202, 1, 2 or 5 g M CuSO 4 lowered the ~:-PI activity to 95, 81 or 40% of the control, respectively. Greater destruction of ~I-PI was de-

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Agents and Actions, vol. 29, 3/4 (1990) Table 1

w ;-

Effects of metal ions on the peroxidation of e~-Pl 1.

~176 9

z5

0 0

9 , 60 TIME (MIN)

9 -------- 0 120

Figure 2

Time course of ~j-PI inactivation by Cu(II)/H202. ~I-PI (50/~g/ml) was incubated with 5 gM CuSO4 and 500 gM H202 at 37~ At 15 rain the reaction mixture was divided into 3 equal fractions. EDTA (0.5 raM) was added to Fraction (a) (A), catalase (10 ffg/ml) to Fraction (b) (o), and nothing was added to Fraction (c) (o). The incubation was continued at 37 ~ and at various intervals aliquots were taken from each fraction. Catalase was added to (a), EDTA to (b) and both catalase and EDTA to (c). They were then assayed for ~a-PI activity.

tected at higher c o n c e n t r a t i o n s o f H 2 0 2. N e i t h e r C u S O 4 n o r H 2 0 2 alone in the c o n c e n t r a t i o n r a n g e tested s h o w e d a n y significant effect.

Time course of ~ - P I inactivation In a time c o u r s e study, cq-PI was i n c u b a t e d with 5 g M C u S O ~ a n d 500 ~tM H202, the r e a c t i o n was s t o p p e d at v a r i o u s times by a d d i n g E D T A and catalase, and activity o f the i n h i b i t o r was then m e a s u r e d . T h e r e m a i n i n g cq-PI activity decreased to 4 8 % o f the c o n t r o l at 15 min, 3 0 % at 30 min, and 9 % at 60 m i n o f i n c u b a t i o n (Fig. 2). T h e r e was no detectable i n h i b i t o r y activity after 120 rain. W h e n E D T A a l o n e was a d d e d after 15 m i n o f inc u b a t i o n , the i n a c t i v a t i o n was arrested i m m e diately. H o w e v e r the a d d i t i o n o f catalase alone at that time did n o t stop the i n a c t i v a t i o n completely.

Effects of other metal ions Several o t h e r t r a n s i t i o n metal ions wer c o m p a r e d with Cu(II) for their ability to catalyze the cq-PI inactivation. % - P I was treated w i t h v a r i o u s m e t a l ions in the presence o f H 2 0 2 (Table 1). U n d e r the

Metal solution (5 gM)

~I-PI activity (% of control)

CuSO4 ZnSO 4 FeSO4 FeSO~ + ATP FeC13 FeC13+ ATP HgCIz A1CI3 MnSO 4 NiSO~ MgC12 CdCI2 Cr(NO3) a

39 93 96 89 98 101 94 95 96 96 96 98 99

1 cq-PI (50 pg/ml) was treated with 5 gM of the metal salt solution and 500 gM H~O2, and assayed for the cq-PI activity. Where indicated ATP (50 laM) was premixed with either FeSO4 or FeCI3 before adding to el-PI. The activity was not affected by ATP alone in the presence or absence of H202 at tested concentrations. In the absence of added metal ions H202 alone did not show a significant effect.

c o n d i t i o n s described, C u S O 4 caused a r e d u c t i o n o f ~I-PI activity to 3 9 % o f control. O t h e r m e t a l salts tested h a d either very slight or n o significant effect. A T P has been k n o w n to a u g m e n t the h y d r o x y l radical ( H O . ) f o r m a t i o n in a m i x t u r e of i r o n and H 2 O 2 by f o r m i n g c o m p l e x e s w i t h the i r o n [26]. W h e n 50 g M o f A T P was a d d e d to F e S O 4 , o n l y a slight increase in ~I-PI i n a c t i v a t i o n was observed, while there was no effect in the presence o f FeC13.

Protective effect of copper chelators T h e effects o f v a r i o u s c o p p e r c h e l a t o r s were tested o n the i n a c t i v a t i o n o f cq-PI by Cu(II)/H20 z. T h e c h e l a t o r s (50/aM) were m i x e d w i t h ~I-PI (50 ~g/ ml) b e f o r e a d d i n g 10 g M C u S O 4 and 200 g M H 2 0 2. A f t e r 30 rain i n c u b a t i o n the cq-PI activity was m e a s u r e d . C h e l a t o r s were dissolved in w a t e r a n d a d j u s t e d p H to 7.4 with either N a O H or HC1. T h e activity o f C u ( I I ) / H z O z treated cq-PI was 14% o f n o n - t r e a t e d c o n t r o l w i t h o u t chelator. W h i l e all tested c h e l a t o r s were effective to s o m e degree o n the p r o t e c t i o n to C u ( I I ) - c a t a l y z e d pero x i d a t i o n , E D T A a n d histidine were the m o s t effective (Table 2).

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tions were needed to obtain a similar extent of inactivation (compare Fig. 1 and Fig. 3). Elastase inhibitory capacity of the plasma was lowered to 94% of the control by 4 m M H 2 0 z in the absence of added copper. Addition of 10, 20 or 50 gM CuSO4 enhanced the inactivation to 89, 77 or 27% of the control, respectively. There was no detectable elastase activity in the dialyzed plasma.

so

N

25

..3 U.I

0

Discussion '

0

2 HRO2 (mM)

'

4

Figure 3 Inactivation of plasma elastase inhibitory capacity by Cu(II)/ H202. Fresh human plasma was mixed with 5 mM EDTA, and dialyzed against 5 mM sodium phosphate containing 150 mM NaC1, pH 7.4. The dialyzed plasma (50 pl/ml) was treated with various concentrations Of CuSO 4 (o, none; e, 10 oM; A, 20 gM; A, 50 gM) and H 2 0 z (1-4 m3~. The reaction was stopped by adding 5 mM EDTA and catalase (50 pg/ml), and elastase inhibitory capacity of the plasma was assayed.

Table 2

Effects of copper chelators on Cu(II)-catalyzed peroxidation of cq-PI ~. Chelator (50 gM)

%-PI activity (% of control)

(none) EDTA L-Histidine Sodium citrate L-Penicillamine L-Alanine

14 99 98 84 61 35

1 % - P I (50 I-tg/ml)was treated with 10 ~tM CuSO 4 and 200 gM H20 2 in the presence of a chelator as indicated.

Inactivation of plasma eIastase inhibitory capacity by CU ( II)-catalyzed peroxidation Various plasma proteins, particularly albumin and ceruloplasmin, are capable of binding Cu(II) and may thus influence the oxidative inactivation of cq-PI. Dialyzed plasma was prepared and then treated with various concentrations of CuSO4 (10, 20 or 50 ~tM) and H202 (i, 2 or 4 m M ) as described in Materials and methods. The elastase inhibitory capacity of the plasma was assayed with added elastase and found to be still affected by Cu(II) and H 2 0 2 (Fig. 3). However, much higher concentra-

Cu(II)-catalyzed peroxidation of ~I-PI was investigated in the present study. It was found that Cu(II) in the concentrations of 1 to 5 BM markedly enhanced the inactivation of el-PI in the presence of less than l m M H202 (Fig. 1). Longer treatment times were more damaging to the activity of the inhibitor, and the inactivation was stopped immediately by the addition of EDTA (Fig. 2). Among various transition metal ions tested, Cu(II) was the only one which showed prominent catalytic activity on %-PI peroxidation (Table 1). The Cu(II)-catalyzed peroxidation was also demonstrated in dialyzed plasma which contains %-PI along with other copper binding proteins (Fig. 3). The serum concentration of non-ceruloplasmin copper in rheumatoid arthritis patients was reported to be as high as 103 gg/100 ml (16 ~tM), while the normal level was 7 gg/100 ml [20]. The concentration of copper in the synovial fluid of patients with rheumatoid arthritis was measured as 13.8 + 4.8 g M [21]. A substantial fraction of this copper may be available for the catalysis of %-PI peroxidation. In physiological systems in which HzO 2 is released, the localized concentrations of the immediate environment have been estimated to be in m M range [19, 27]. The levels of Cu(II) and H 2 0 2 used in this study are close to those physiological concentrations in a disease state. Thus, inactivation of cq-PI by copper and H 2 0 2 could disturb the %-PI-elastase balance at sites of inflammation. Accumulation of small localized effects over a long period of time may lead to cumulative connective tissue destruction in the lung or joints. The mechanisms of copper-catalyzed peroxidation are still not well understood. Several investigators have proposed various reactions or intermediates, for example copper-oxygen complexes [28], a Fenton-type reaction [29], and Cu(III)-dependent reactions [30]. In the presence of chloride ions, a

392

myeloperoxidase-mimetic reaction that generates hypochlorous acid also has been suggested [19]. One of these mechanisms or the combined effects of some of them may be responsible for the e~-PI peroxidation. Alternatively, it is also possible that Cu(II) is bound at certain sites on the inbitor, and H 2 0 2 may react with the bound copper to generate HO., copper-oxygen complexes or other reactive species [31-33], resulting in the destruction of its inhibitory activity. In the study with copper chelators, Cu(II)-catalyzed peroxidation of %-PI (0.94 laM) was abolished by EDTA and histidine, but the peroxidation was not completely prevented by sodium citrate, penicillamine and alanine (Table 2). The concentrations of copper chelators were more than 50 times that of cq-PI. These data suggest that ~ - P I has a high affinity for copper, and thus may still bind copper ions even in the presence of other chelators or plama proteins. Thus the copper ions bound to al-PI may make the inhibitor more susceptible to destruction by H202 derived from phagocytes. Received 10 May 1988; accepted by I Otterness, 3 October 1988

References [1] J. Travis and G.S. Salvesen, Human plasma proteinase inhibitors. Annu. Rev. Biochem. 52, 655 709 (1983). [2] H. Heidtmann and J. Travis, Human cq-proteinase inhibitor. In Proteinase Inhibitors (Eds. A.J. Barrett and G. Salvesen) pp. 441 456, Elsevier, Amsterdam 1986. [3] K. Ohlsson, Interaction of granulocyte neutral proteases with alpha-l-antitrypsin, alpha-2-macroglobulin and alpha-l-antichymotrypsin. In Neutral proteases of Human Polymorphonuclear Leukocytes (Eds. K. Havemann and A. Janoff) pp. 167-177, Urban and Schwarzenberg, Baltimore 1978. [4] C.-B. Laurell and S. Eriksson, The electrophoretic cq-globulin pattern of serum Cq-antitrypsin deficiency. Scand. J. Clin. Lab. Invest. 15, 132-140 (1963). [5] A. Janoff, B. Sloan, G. Weinbaum, V. Damiano, R. A. Sandhaus, J. Elias and P. Kimbel, Experimental emphysema induced with purified human neutrophil elastase: tissue localization o.1"the instilled protease. Am. Rev. Respir. Dis. 115, 461-478 (1977). [6] R.M. Senior, H. Tegner, C. Kuhn, K. Ohlsson, B.C. Starcher and J. A. Pierce, The induction of pulmonary emphysema with human leukocyte elastase. Am. Rev. Respir. Dis. 116, 469-475 (1977). [7] P.S. Wong and J. Travis, Isolation and properties of oxidized alpha-l-proteinase inhibitor from human rheumatoid synovial fluid. Biochem. Biophys. Res. Commun. 96, 1449 1454 (1980).

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Agents and Actions, vol. 29, 3/4 (1990) hosts for facultative intracellular pathogens. In Basis and Clinical Aspects of Granulomatous Diseases. (Eds. D.L. Boros and T. Yoshida) pp. 67-77, Elsevier North Holland, New York 1980. [28] L.L. Ingraham, Model copper oxidases: the cupric ion-catalyzed hydrogen peroxide oxidation of catechol. Arch. Biochem. Biophys. 81, 309 318 (1959) [29] J.M.C. Gutteridge and S. Wilkins, Copper salt-dependent hydroxyl radical formation: damage to proteins acting as antioxidants. Biochim. Biophys. Acta 759, 38-41 (1983). [30] A. Levitzki, M. Anbar and A. Berger, Specifie oxidation of peptide via their copper complexes. Biochemistry 6, 3757 3765 (1967).

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