Reversible Inhibition of Neutrophil Elastase by Thiol-modified a- 1 Protease Inhibitor*

We have modified the single cysteine residue of al- protease inhibitor (01-PI) with HgCL, methylmethane thiosulfonate, oxidized glutathione (GSSG), and N-(l- anilinonaphthyb4)maleimide (ANM). Whereas native al-PI combines rapidly and quasi-irreversibly with neutrophil elastase, the thiol-modified a1-PI derivatives are dissociable reversible competitive inhibitors of the enzyme, with values of Ki in the range of 6-7 nM. Removal of the thiol modifications restores the rapid irreversible mode of inhibition. Once native al-PI has combined with neutrophil elastase, the enzyme- inhibitor complex retains a reactive thiol group, but the two proteins can no longer be dissociated by sub- sequent reaction with ANM, even after exposure to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From kinetic measurements of fluorescence, ANM-modified a1-PI combines with neutrophil elastase via an apparent bimolecular process with a second order rate constant on the order of lo5 M” s-’. We estimate a dissociation rate constant on the order of s-’. The emission of ANM-modified al-PI is in- creased in intensity and blue shifted from the maxi-mum in ANM-modified cysteine, consistent with a pre- dominantly nonpolar environment. u,. ANS-, N-(l-anilinonaphthyl-4)succinimido-; GS-, y-glutamyl- cysteinylglycine (1/2 oxidized glutathione); MS-, thiomethyl-. All derivatives were separated from excess modifying reagent except for the GS-derivative, which was measured in the presence of 1 WM GSSG. The GS- and MS- groups were removed with 1 mM DTT, and low molecular weight thiols were then separated from the al-PI by centrifugation through a Sephadex G-25 column. Control rates were determined in the presence of 30 nM modifying reagents. No subsequent changes in amidolytic activity of elastase in the presence of modified al-PI were observed over the first hour of incubation.


Reversible Inhibition of Neutrophil Elastase by Thiol-modified a-1 Protease Inhibitor*
Suresh C. Tyagi From the Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11 794 We have modified the single cysteine residue of a lprotease inhibitor (01-PI) with HgCL, methylmethane thiosulfonate, oxidized glutathione (GSSG), and N-(l-anilinonaphthyb4)maleimide (ANM). Whereas native al-PI combines rapidly and quasi-irreversibly with neutrophil elastase, the thiol-modified a1-PI derivatives are dissociable reversible competitive inhibitors of the enzyme, with values of K i in the range of 6-7 nM. Removal of the thiol modifications restores the rapid irreversible mode of inhibition. Once native a l -PI has combined with neutrophil elastase, the enzymeinhibitor complex retains a reactive thiol group, but the two proteins can no longer be dissociated by subsequent reaction with ANM, even after exposure to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From kinetic measurements of fluorescence, ANM-modified a1-PI combines with neutrophil elastase via an apparent bimolecular process with a second order rate constant on the order of lo5 M" s-'. We estimate a dissociation rate constant on the order of s-'. The emission of ANM-modified al-PI is increased in intensity and blue shifted from the maximum in ANM-modified cysteine, consistent with a predominantly nonpolar environment.
Association with neutrophil elastase results in an additional blue shift with further increase in intensity, consistent with a further decrease in polarity of the environment of the cysteine. Modification with methylmethane thiosulfonate or GSSG results in a small decrease in quantum yield and a red shift in the tryptophan emission spectrum of the modified inhibitor, suggestive of increased polarity of the environment of at least 1 of the 2 tryptophan residues in al-PI. These changes are reversed by dithiothreitol and are consistent with a conformational change which transforms the inhibitory activity from a rapid, irreversible mode in native a l -PI to a dissociable competitive mode in the mixed disulfide derivatives.
A number of plasma proteins with potent and specific serine protease inhibitory activities have been found to possess significant sequence homologies and have been grouped together as a superfamily of "serpins" (1). Examples of these serpins include al-antichymotrypsin (2), a2-antiplasmin (3), antithrombin I11 (4), and C1-inhibitor ( 5 ) . The most abundant * This work was supported by Grant HL-14262 from the United States Public Health Service and by grants from the New York State Office of Science and Technology (Biotechnology Center, State University of New York at Stony Brook) and Cortech, Inc. (to S. R. Simon). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. circulating serpin is al-protease inhibitor (al-PI)', a glycoprotein of M, = 52,000 (6). While a l -P I can inhibit a number of serine proteases, its highest specificity is for human neutrophil elastase, a protease which can degrade a number of proteins of the interstitial extracellular matrix and which may be released in sufficient levels during inflammatory conditions to produce tissue injury (7-11). The circulating level of a1-PI is around 1.3 mg/ml in normal individuals (12) but may rise significantly during acute phase reactions to inflammatory stimuli. It has been assumed that the major role of circulating a l -P I is to irreversibly inactivate any elastase which might be released from activated or senescent neutrophils into the blood. Such inactivation occurs through formation of a stable 1:l enzyme-inhibitor complex involving the active site of neutrophil elastase (13). The stability of this complex has been ascribed to a putative conformational change which is triggered by proteolytic cleavage of the bond between residues Met"58-Ser359 (14).
Chemical modification of specific amino acid side chains in al-PI has been previously reported. Modification of lysine and arginine side chains has minimal effect on inhibitory activity (15)(16)(17)(18)(19)(20), whereas modification of a single tyrosine residue by tetranitromethane or N-acetyl imidazole prevents formation of a stable enzyme-inhibitor complex (21). The most physiologically important amino acid modification previously investigated is oxidation of 2 of the 8 methionine residues in the protein (22), which can be accomplished not only by chemical oxidants but also by the action of reactive oxygen species liberated by activated neutrophils (23) or purified neutrophil myeloperoxidase (24). This modification results in a marked loss of affinity of a l -P I for neutrophil elastase and a thousand-fold decrease in the rate of association of enzyme and inhibitor, although the complex which is formed is still quite stable (25).
There is a single cysteine residue in al-PI, which has been localized at position 232 in the amino acid sequence by peptide and cDNA sequencing (6) as well as by x-ray crystallographic analysis (26). This single thiol is capable of forming mixed disulfides with such plasma constituents as cysteine, glutathione, and other plasma proteins with free thiols, such as IgA (27). A variable percentage of the circulating a l -P I has its cysteine tied up in such mixed disulfides; this fraction is in dynamic equilibrium with the remaining fraction of the protein in which the cysteine sulfhydryl is free (28). While the availability of a free thiol group has been previously used to advantage in the purification of a l -P I (29), no functional The abbreviations used are: al-PI, al-protease inhibitor; MMTS, methylmethane thiosulfonate; GSSG, oxidized glutathione; GSH, reduced glutathione; ANM, N-( 1-anilinonaphthyl-4)maleimide; DTT, dithiothreitol; MeOSucAAPVpNa, methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide; BAL, 2,3-dimercaptopropanol (British anti-Lewisite); PBS, Dulbecco's phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoreses. 5279 studies on thiol-modified inhibitor have been reported. Since circulating al-PI is heterogeneous with respect to the availability of the free thiol a t Cys232, we chose to investigate the effect of modification of this residue i n vitro on the conformation and inhibitory properties of the protein.
Chemical Modification of a1 -PI Modification of a1-PI with various reagents was carried out by mixing a 1 mg/ml solution of inhibitor in 0.3 M phosphate-buffered saline (PBS), pH 7.4, with 0.1 mM reagent at 4 "C for 12 h. The concentration of solutions of MMTS, which could not be conveniently prepared gravimetrically, was determined by measuring the decrease in absorbance at 412 nm after addition of an aliquot of MMTS solution to a stock solution of 5-mercapto-2-nitrobenzoic acid. Oxidized al-PI was prepared by reacting 10 p~ al-PI with a 20-fold excess of chloramine T in the same buffer at 25 "C for 20 min. Excess reagents were removed by microcentrifugation through 0.8 X 10-cm columns of Sephadex G-25 (30) previously equilibrated with 0.3 M PBS.
Modification of the complex between native al-PI and neutrophil elastase with ANM was carried out by first allowing 2.1 nmol of al-PI and 1.7 nmol of elastase in 50 pl to combine for 30 min at 25 "C and then, after addition of 0.1 mM phenylmethylsulfonyl fluoride to stop proteolysis by any free enzyme, reacting the mixture with 2.5 nmol of ANM at 4 "C for 12 h as above. Excess reagents were removed by microcentrifugation through columns of Sephadex G-25 as described.

Neutrophil Elastase Activity Assays
Amidolytic activity of neutrophil elastase was assayed with Me-OSucAAPVpNa (31). Release of p-nitroaniline was monitored by recording absorbance at 405 nm in a ThermoMax multiwell microplate reader (Molecular Devices, Palo Alto, CA) operating in the kinetic mode. The substrate concentration was varied from 0.15 to 0.6 mM. Neutrophil elastase concentrations ranged from 10 to 500 nM. All measurements were carried out in 0.3 M PBS buffer, pH 7.4, containing 0.01% Triton X-100. Kinetic data were collected for 5 min, during which period reaction rates remained linear. Activities in the presence of thiol-modified a l -P I were all normalized to controls determined in the absence of any a1-PI but in the presence of an amount of thiol-modifying reagent equal to that used in preparing the modified inhibitor.

Inhibitory Properties of Modified al-PI
Stoichiometry of Inhibition-Various concentrations of thiol-modified al-PI were mixed with 1.5 p M neutrophil elastase in 0.3 M PBS, pH 7.4, containing 0.01% Triton X-100 at 25 "C. Aliquots of 50 pl were removed from the incubation mixtures and added to 100 p1 of a 0.45 mM solution of MeOSucAAPVpNa in the same buffer. The amidolytic rate was observed in the ThermoMax microplate reader as described above. The final elastase concentration during the assay was 500 nM and the final substrate concentration was 0.3 mM.
Determination of Inhibition Constant-Various concentrations of thiol-modified al-PI ranging from 7.5 to 500 nM final concentration were mixed with 30 nM neutrophil elastase at 25 "C prior to addition of MeOSucAAPVpNa to give a final substrate concentration ranging from 0.15 to 0.6 mM. All reactions were carried out in 0.3 M PBS, pH 7.4, containing 0.01% Triton X-100. Data were analyzed according to the methods of Green and Work (32), Henderson (33), Bieth (34), and Empie and Laskowski (35) for quantitating inhibition under conditions in which a substantial fraction of the inhibitor is bound to the enzyme.

SDS-PAGE
SDS-PAGE was performed on vertical slabs according to a modification of the procedure of Laemmli (36) using 10% acrylamide, 0.3% methylene-bisacrylamide at pH 8.8 in the absence of any thiolreducing agents. All solutions contained 0.1% SDS.

Spectroscopic Methods
Absorption spectra were recorded with a Hewlett-Packard 8452A diode array spectrophotometer. The concentrations of a set of native a l -P I standards were determined by absorbance, using a value of & nm = 5.0 (29) and these samples were then used to generate a standard curve employing the protein assay procedure of Bradford (37). This standard curve was also used to determine the concentrations of various thiol-modified al-PI derivatives. Neutrophil elastase concentrations were determined by absorbance, using a value of & nm = 9.85 (38).
Fluorescence spectra were recorded or. a computer-controlled Spex Datamate spectrofluorimeter. The excitation and emission slits were adjusted for 5-nm bandpass width. Spectra were recorded at 1-nm intervals and were corrected for base line and instrument response. Samples were prepared and incubated for appropriate times prior to measurement at 25 "C in 0.3 x 0.3-cm micro cells.
Tryptophan fluorescence was determined with X,, at 295 nm, and the quantum yield was calculated from the area of the emission spectrum by the method of Parker and Rees (39) using the equation where D is the absorbance, A is the area of the emission spectrum, and Q is the quantum yield of the protein, P, and reference, R, respectively. A reference solution of tryptophan, for which a value of QR = 0.2 was assumed, was employed (40). Protein solutions used for these measurements had an absorbance of less than 0.05 to avoid inner filter effects. For the determination of the tryptophan emission spectrum of GSSG-modified al-PI, a solution of 25 p M a l -P I was reacted with 100 PM GSSG for 30 min prior to recording the fluorescence without removal of the excess reagent, and the spectrum was corrected by subtracting a blank containing GSSG alone in buffer. The -SG group was then removed by reduction with 1 mM DTT, and the protein was separated from the thiols by centrifugation through a Sephadex G-25 column as described above prior to redetermination of the emission spectrum.

Association of ANM-modified al-PI with Neutrophil Elastase
The association of ANM-modified al-PI with neutrophil elastase was followed by recording emission intensity at 430 nm with excitation at 355 nm. Concentrations of a l -P I were varied from 0.5 to 1 p~, and concentrations of elastase were varied from 0.2 to 0.4 PM, with al-PI concentration always maintained at least twice that of elastase. All reactions were carried out in 0.3 M PBS buffer, pH 7.4, at 25 "C. Data were analyzed assuming a simple reversible bimolecular reaction. Under conditions in which excess inhibitor is present, the integrated form of the association reaction given by Jencks (41) can be used to obtain an estimate of the association rate constant, k, In this expression, I, and E, are the initial concentrations of al-PI and elastase, respectively, and x is the concentration of the al-PIelastase complex at any time, t. Our measure of x is based on the relative fluorescence of the mixture of a1-PI and neutrophil elastase as a function of time: where F is the fluorescence intensity at time t, F, is the initial intensity, and F, , , is the extrapolated final intensity. Data were fit to the model by a nonlinear least squares regression program (Enzfitter, Elsevier).

RESULTS
Chemical Modification of a1 -PI-The stoichiometry and specificity of modification of al-PI by HgC12, MMTS, and ANM was studied by spectroscopic measurements. Reaction of Hgi2 with thiols is associated with an absorption band with X , , , of 255 nm and a molar extinction coefficient, e255 nm of 8 X lo3 M" cm" (42). From difference spectroscopy measurements of HgC12-modified a1-PI uersza native a1-PI, we confirmed that a single S-Hg bond was formed at completion of the reaction. We confirmed the stoichiometry of thiomethylation of al-PI by MMTS by reacting the modified protein (which had been separated from excess reagent by gel filtration) with 5-mercapto-2-nitrobenzoate and measuring the decrease in absorbance at 412 nm and the increase in absorbance at 330 nm as described previously (43). Again, by difference spectroscopy we could detect the presence of a single disulfide bond/molecule of modified al-PI after reaction of the protein with MMTS. Incorporation of ANM into al-PI could be measured after removal of excess free reagent by determining the increase in absorbance at 351 nm due to the N-(anilinon-aphthy1)succinimidyl group (44). Using the reported molar extinction coefficient, c351 " , , , of 13.18 X IO3 M" cm", we calculated that a single ANM molecule had reacted with al-PI. The emission spectrum of the ANM-a1-PI adduct was consistent with formation of only a S-succinimidyl product, without any evidence for additional reaction of the imide ring with a-or e-amino groups (45).
Effect of Thiol Modification on Activity-If native al-PI is added to neutrophil elastase simultaneously with the chromogenic substrate MeOSucAAPVpNa, a rapid decrease in the rate of amidolysis can be observed as the inhibitor inactivates the enzyme. In Fig. 1, the rapidly progressive inhibition of 45 nM elastase by a 6-fold excess of native al-PI is compared to the inhibition by the same concentration of HgC12-modified a1-PI, also added simultaneously with the substrate. The modified inhibitor does not progressively inactivate the enzyme, but the rate of amidolysis is clearly reduced. Free HgClz has virtually no effect on elastase-catalyzed amidolysis. To demonstrate that modification with HgC12 had not simply denatured a fraction of the a1-PI, we incubated 270 nM Hgmodified a1-PI with 1 mM BAL for 30 min, and recovered about 40% of the rapidly progressive quasi-irreversible inhibitory activity characteristic of native al-PI. This indicates that the effect of thiol modification of the inhibitor is reversible.
In Table I, we compare the effect of modification of al-PI by HgC12, MMTS, ANM, and GSSG on inhibitory activity.  at 25 "C, in the presence and absence of native or modified al-PI. Five min prior to addition of substrate, 30 nM native or modified a l -PI was added to the enzyme and the subsequent amidolytic rate, u, was compared to the rate observed in the absence of any added a l -PI, u,. ANS-, N-(l-anilinonaphthyl-4)succinimido-; GS-, y-glutamylcysteinylglycine (1/2 oxidized glutathione); MS-, thiomethyl-. All derivatives were separated from excess modifying reagent except for the GS-derivative, which was measured in the presence of 1 WM GSSG. The GS-and MS-groups were removed with 1 mM DTT, and low molecular weight thiols were then separated from the al-PI by centrifugation through a Sephadex G-25 column. Control rates were determined in the presence of 30 nM modifying reagents. No subsequent changes in amidolytic activity of elastase in the presence of modified al-PI were observed over the first hour of incubation. In these studies, we employed 30 nM neutrophil elastase and 30 nM native or modified a1-PI, which were mixed 5 min before addition of 0.6 mM MeOSucAAPVpNa. Whereas native al-PI rapidly inhibited over 98% of the elastase, we could detect 58-71% residual amidolytic activity after incubation with the modified inhibitors at these concentrations. This residual activity remained unchanged after preincubation with the inhibitors for up to 1 h. Addition of up to 100 ~L M of any of the modifying reagents directly to neutrophil elastase reduced its amidolytic activity by only about 2%. Simultaneous addition of stoichiometric amounts of both native al-PI and MMTS-modified a1-PI to neutrophil elastase resulted in no detectable competition by the modified inhibitor for the rapid and complete inhibition by the native al-PI. We tested the reversibility of the effects of modification with MMTS and GSSG on al-PI activity by incubation with 1 mM DTT and subsequent removal of free low molecular weight thiols. About 86% of the quasi-irreversible inhibitory activity characteristic of native al-PI is recovered after removal of the thiomethyl group from MMTS-modified a1-PI. Reduction of the mixed disulfide between al-PI and glutathione with DTT resulted in restoration of 93% of the inhibitory activity characteristic of native al-PI.
In order to show that the partial reduction in activity observed for the modified inhibitors in Table I reflects a reversible mode of binding, rather than a mixture of inactive molecules and molecules with full inhibitory activity, we subjected mixtures of neutrophil elastase and both native and modified a1-PI to SDS-PAGE, as shown in Fig. 2A. The complex between native a1-PI and elastase is so tight that it survives the separating force of electrophoresis under denaturing conditions (38). Some degradation of the native al-PI to lower molecular mass fragments by neutrophil elastase can be seen, but no 52-kDa protein at the position of undegraded al-PI can be detected. No complex can be seen after SDS-PAGE, either by protein staining with Coomassie Blue or by the fluorescence of the anilinonaphthyl group, however, when neutrophil elastase and ANM-modified al-PI are incubated together after reaction of the thiol group on the inhibitor. The possibility that the ANM-modified al-PI was rapidly degraded to an inactive cleaved product can be ruled out since the modified inhibitor in mixtures with neutrophil elastase still migrates at the same position as in the absence of the enzyme, as long as SDS-PAGE is performed within 10 min a t 37 "C after mixing enzyme and modified inhibitor. Prolonged incubation of elastase and modified a1-PI does result in slow degradation of the inhibitor (data not shown). If native and ANM-modified a1-PI are mixed simultaneously with neutrophil elastase and the mixture is then subjected to SDS-PAGE, all the elastase is complexed with the native al-PI and all the ANM-modified inhibitor is found separated from the complex at its usual migratory distance. . To demonstrate that the quasi-irreversible complex between native al-PI and neutrophil elastase retains reactivity of the free thiol but cannot be dissociated by subsequent thiol modification, we reacted approximately stoichiometric quantitities of unmodified al-PI and elastase for 30 min and then, after addition of phenylmethylsulfonyl fluoride to minimize subsequent degradation of proteins by any free elastase, we incubated the reaction mixture with an excess of ANM for 12 h. After removal of low molecular weight materials by gel filtration, the protein mixture was subjected to SDS-PAGE. As shown in Fig. 2B, two bands could be visualized both by fluorescence and by Coomassie staining for protein, at the mobilities for the al-PI-elastase complex and for the major degradation fragment of a1-PI. A third band at the mobility for free neutrophil elastase could also be seen on Coomassie staining. The presence of a fluorescent band at the position of the al-PI-elastase complex confirms that this preformed complex still has a reactive thiol group, but the extremely tight linkage between the proteins is not broken by subsequent modification of the cysteine.
Kinetics of Inhibition of Neutrophil Elastase by Modified al-PI-Although thiol-modified derivatives of a1-PI do not rapidly form virtually irreversible complexes with neutrophil elastase, they are moderately effective as reversible inhibitors. As shown in Fig. 3, the amidolytic activity of 30 nM neutrophil elastase is effectively inhibited by an %fold excess of MMTSmodified al-PI. In this concentration range, a significant fraction of the total inhibitor added is bound to the enzyme, so binding can be analyzed by the partition ratio method as described by Silverman (46). We have measured the extent of inhibition as a function of modified a1-PI concentration a t several different fixed concentrations of MeOSucAAPVpNa and established that the data is consistent with a model of simple competitive inhibition (32-35). Henderson plots a t two different substrate concentrations are shown in the inset to Fig. 3 and conform to the following equation for competitive inhibition by a species which is substantially bound by the enzyme: From determinations of amidolytic activity in the absence of inhibitor, we have obtained a value of K, for MeOSuc-AAPVpNa of 0.21 f 0.03 mM. From Henderson plots obtained for other thiol-modified derivatives of a1-PI, we have calcu- lated values of Ki of 6-7 nM for all the a1-PI derivatives we have studied. Extent of inhibition is unaffected by order of addition of substrate and inhibitor and has already reached a steady state within the time limit of manual mixing of components, indicating that a scheme of rapid equilibria can be used to describe the inhibition, as shown below: where K: = k-l/kl.
Stoichiometry and Reversibility of Inhibition-At 500 nM neutrophil elastase (about 80 times the value of Ki for modified a1-PI), addition of increasing amounts of modified inhibitor gives rise to a roughly linear titration curve. One such curve, obtained with HgClz-modified a1-PI, is shown in Fig.  4. The linear portion of the curve has a slope of 1.01 f 0.01; the X intercept value is consistent with a stoichiometry of inhibition of 1:l. If a mixture of 500 nM elastase and 500 nM HgClp-modified a1-PI is assayed directly for amidolytic activity, only about 1% of the activity of the same concentration of enzyme alone can be detected. If, however, the mixture is diluted 50-fold, so that both enzyme and inhibitor are now present at 10 nM, in the presence of 0.3 mM MeOSuc-AAPVpNa about 75% residual activity can now be detected, indicating that the complex between neutrophil elastase and the modified al-PI is dissociable with restoration of amidolytic activity.
Fluorescence of ANM-modified a1 -PI-The fluorescence of the anilinonaphthyl group in ANM is sensitive both to addition of thiols across the double bond of the maleimide ring and to the environment of the conjugated ring system (44,47). gesting that the environment of the anilinonaphthyl ring system is more nonpolar in the protein derivative than in the aqueous solution of the amino acid adduct. Upon addition of an approximately stoichiometic concentration of neutrophil elastase, the emission of the anilinonaphthyl ring system is still further blue shifted with an enhancement in fluorescence intensity and a new X , , , at 430 nm. At a concentration of 500 nM, the inhibitor is largely bound to the enzyme as we have shown above. We conclude, therefore, that binding of modified d -P I to elastase further increases the hydrophobicity of the environment around the anilinonaphthyl ring system which is coupled to the thiol group in the inhibitor.
If a1-PI is added to buffer containing 7 M urea or 3 M guanidinium chloride, reaction with ANM is complete within 10-15 min, and the fluorescence of the modified protein resembles that of the adduct with free cysteine (data not shown). We conclude that unfolding of al-PI in these denaturing solvents increases accessibility of the thiol group in the protein to the fluorogenic maleimide, and the resulting Ssuccinimidyl adduct remains fully accessible to these aqueous media.
Kinetics of Combination of ANM-modified a l -P I with Neutrophil Elastase-The blue shift in X , , , in the emission of the anilinonaphthyl ring system from 448 to 430 nm seen upon addition of ANM-modified-al-PI to neutrophil elastase can be followed over time, as shown in Fig. 5. There is a significant change in emission over the first 2 min after mixing enzyme and inhibitor which can be fit to a simple model of second order kinetics. The inset to Fig. 5 shows the fit to a transform of the data which gives linear plots for second order reactions. The apparent second order rate constant for formation of the E-I complex was calculated to be 1.41 5 0.13 X lo5 M-' s-' .
Varying the concentrations of elastase and modified a1-PI from 200 to 600 nM, at either constant or varying ratios of enzyme to inhibitor gave identical values for this apparent second order rate constant.

Effects of Oxidation and Thiol Modification on Endogenous
Fluorescence of al-PI-We have examined the endogenous fluorescence arising from the 2 tryptophan residues in al-PI before and after oxidation with chloramine T or modification of the protein with MMTS or GSSG. With excitation at 295 nm, native al-PI has a characteristic emission spectrum with Excitation was at 355 nm and emission intensity at 430 nm was recorded at 2-s intervals. Excitation and emission slits were set for 5 nm bandpass. The curve represents a least squares fit of the data to a second order process computed by nonlinear regression. Inset, linear representation of the data according to the equation as N-chlorosuccinimide or chloramine T, leads to marked reduction in the intensity of emission from tryptophan. However, the X, , , of the residual tryptophan emission is only slightly red-shifted to 333 nm. In contrast to the effect of oxidation, there is only a small reduction in the quantum yield, Qp, from 0.08 in the native inhibitor to 0.07 in the MMTS-modified inhibitor. More striking is the marked red shift in X, , , in the emission spectrum, from 330 nm in the native protein, to 343 nm in the MMTS-modified protein, and to 341 nm in the glutathione-modified protein. This red shift suggests that at least 1 of the tryptophan residues in al-PI has become more accessible to the aqueous solvent after reaction of the thiol group to form a mixed disulfide. Upon addition of excess DTT to the glutathione-modified protein, the X, , , in the emission spectrum was shifted back to 335 nm, indicating that this change in tryptophan fluorescence, like the effect on elastase-inhibitory activity, was reversible.

DISCUSSION
The biological role of the single cysteine at position 232 in a1-PI is still open to conjecture, although it has been proposed that the mixture of free and thiol-blocked a1-PI in plasma contributes to an overall redox buffering system of free thiols and disulfides (28). In this paper we show that a marked change in elastase-inhibitory properties of a1-PI occurs after modification of the protein with a number of thiol-specific reagents and that this change in activity is correlated with changes in spectral properties suggestive of a protein conformational change. Removal of the thiol modifications with BAL or DTT restores the inhibitory activity characteristic of the native protein, strongly suggesting that the altered properties of the derivatives were due to thiol modification alone. Moreover, we have been able to use an environment-sensitive thiol-specific probe to follow the kinetics of binding of thiolmodified al-PI to neutrophil elastase.
Inhibition of neutrophil elastase by native a1-PI is generally explained by attack of the enzyme at the Met358-Ser359 peptide bond in a conformationally constrained loop within the inhibitor, leading to formation of a stable enzyme-inhibitor complex (14). This complex is formed rapidly by a mechanism involving a rate-limiting second order step and decomposes over days in a first order process to release free elastase and a product of al-PI which has been cleaved at the Met358-Ser359 bond. The second order formation rate constant for the enzyme-inhibitor complex has been estimated to be at least IO7 M" s-', while the dissociation constant has been estimated to be no more than 2 X s-', consistent with an overall apparent Ki value of M (7). Oxidized al-PI combines with elastase at a slower rate than the native inhibitor, with a second order formation rate Constant estimated around lo4 mass as the cleaved product of the native inhibitor with a first order rate constant about s-', indicating that its mechanism of inhibition also most likely involves attack at the Met358-Ser3sg bond (25).
The detailed mechanism for association and dissociation of thiol-modified a1-PI derivatives and neutrophil elastase is not known. However, we can conclude from the kinetics of the change in fluorescence of ANM-modified al-PI upon mixing with elastase that association of enzyme with the modified inhibitor must proceed via a rate-limiting second order kinetic step with a rate constant for combination of lo5 oxidized protein but two orders of magnitude slower than the native inhibitor. From this association rate and the steadystate measurements of elastase-catalyzed amidolysis in the "1 s-l , and dissociates as a fragment of the same molecular "I s-' , about an order of magnitude faster than that of the presence of modified al-PI, we conclude that the modified inhibitors interact reversibly with the enzyme as simple competitive inhibitors with values of Ki in the range of 6-7 X IO-' M, and dissociation constants at least three orders of magnitude faster than the native inhibitor and two orders of magnitude faster than the oxidized inhibitor.
Not only are the kinetic constants for association and dissociation of thiol-modified al-PI different from those for native or oxidized a1-PI, but the fully reversible nature of the inhibition of the thiol-modifed protein is also distinctive. While we have no direct evidence that the methionine in these thiol-modified derivatives has not been oxidized, the recovery of rapid, quasi-irreversible inhibitory activity after incubation of HgClp-modified a1-PI with BAL or of MMTSor GSSGmodified al-PI with DTT suggests that native al-PI has been regenerated. Incubation with thiols alone will not reduce oxidized forms of methionine. After prolonged incubation of neutrophil elastase with thiol-modified derivatives of a1-PI over several hours, we do observe degradation of the modified inhibitor as evidenced by a shift to lower apparent M, on SDS-PAGE. We are currently studying this proteolytic degradation in greater detail, but the kinetics of degradation are obviously much slower than the first order dissociation of the modified al-PI-elastase complex to regenerate native inhibitor. We can detect the incorporation of thiol-modifying reagent into the preformed native al-PI-elastase complex by SDS-PAGE, but the subsequent modification of the cysteine after complex formation between unmodified inhibitor and enzyme does not convert the complex to an easily dissociable form. The only species other than the undissociated complex which can be seen on SDS-PAGE after prolonged reaction of native al-PI-elastase complex with ANM are degradation fragments of the inhibitor. This result offers strong support for our conclusion that the nature of the reaction between elastase and native al-PI, with its apparent attendant cleavage of the Met35s-Ser359 bond and presumed formation of a covalent acyl-enzyme complex, is fundamentally different from the reversible noncovalent association of elastase and previously thiol-modified al-PI.
The environment of Cys232 in a1-PI appears to be hydrophobic, as indicated by the marked enhancement and blue shift of the fluorescence of ANM-modified al-PI compared with ANM-modified cysteine. This hydrophobicity is consistent with the sterically hindered access to the thiol of Cys232 which has been postulated to account for the failure of a1-PI to form disulfide-linked dimers or to react with the free sulfhydryl group of serum albumin (28). The hydrophobicity of this environment is even more enhanced when the modified inhibitor binds to neutrophil elastase. Since we know from the x-ray crystallographic structure of al-PI (26) that Cys232 is some distance from the active site loop, we can conclude that a conformational change in the inhibitor associated with binding to elastase extends to the region around CYS'~~. We detect this conformational change in the modified inhibitor by using the modifying reagent as a reporter group and must therefore add the cautionary note that the modified inhibitor may be bound to elastase in a different manner than the native protein. Therefore, the apparent conformational change associated with combination of modified al-PI with neutrophil elastase may not be equivalent to the putative structural changes associated with formation of the complex between native d -P I and elastase. However, the fact that we can subsequently react the preformed al-PI-elastase complex with ANM is consistent with the hypothesis that Cys232 is not directly involved in the interaction of native al-PI with elastase.
The transformation of the mechanism of inhibition by al-PI associated with modification of CYS*~' appears also to be associated with a conformational change in this protein, as reflected by the marked red shift in endogenous fluorescence from the tryptophan side chains upon reaction with MMTS or GSSG. Since the shift in tryptophan emission is reversible upon reduction of the dithio-adducts with DTT, like the change in the mode of inhibition, it cannot be ascribed to denaturation of the protein. This red shift resembles the change in emission associated with transfer of a tryptophan side chain from a predominantly non-polar environment to an environment which is partially accessible to aqueous solvent (48). Since Trp238 is relatively close to CysZ3', it is possible that this residue contributes more to the observed red shift than Trplg4, but we have no direct verification of the relative contributions of the 2 residues to the spectra of the native and modified protein. We observed no similar red shift in tryptophan emission when we oxidized al-PI, although the marked reduction in emission intensity suggests that oxidants may attack the tryptophan ring system. Therefore, the apparent conformational changes induced by modification of CysZ3' in al-PI do not occur upon oxidation of Met358. We are now actively investigating the possibility that the conformational change induced by modification of C Y S~~' directly affects the conformationally constrained loop including residues Met358-Ser359 to convert al-PI from a quasi-irreversible inhibitor to a reversible competitive inhibitor of neutrophil elastase.
Since a variable fraction of the circulating al-PI has its thiol group modified by cysteine or glutathione, the physiological significance of the properties of these modified forms of the inhibitor should be considered. The concentrations of CysSH and CysSSCys in plasma have been reported to be 20 and 40 PM, respectively. This substantial concentration of free thiol would be expected to facilitate ongoing disulfide exchanges among all potentially reactive species, ensuring that the thiol-modified al-PI in plasma would always be in dynamic equilibrium with the unmodified inhibitor. Indeed, this dynamic equilibrium has been demonstrated in vivo by showing that [3sS]cysteine which is linked to a1-PI through a disulfide bond exchanges in the plasma with a dialyzable pool of the free amino acid and appears in the urine, while the protein is not excreted (28). Since the unmodified and disulfide-bridged forms of a1-PI are in continual exchange in the plasma, it is possible for all the inhibitor to combine through the unmodified form with neutrophil elastase to form quasiirreversible complexes. Such exchange would not be expected to occur to a significant extent in the disulfide-bridged species of al-PI which we prepared and studied in the absence of free thiols.