Eur. J . Biochem. 140, 105-111 (1984) t FEBS 1984

Interaction of human a,-antichymotrypsin with chymotrypsin

Anne LAINE, Monique DAVRIL, and Annette HAYEM

Unit6 16 de l'lnstitut National de la Sante et de la Recherche MCdicale, Lille

(Received November 14, 1983) - EJB 83 1219

Human a,-antichymotrypsin reacts with bovine chymotrypsin to form an equimolar complex and this reaction is accompanied by the formation of a free, modified form of the inhibitor. Time-course studies, performed on mixtures containing an excess of native inhibitor and kept at 0°C or at 25 "C, show that the equimolar complex dissociates spontaneously ; this dissociation results in the release of inactive modified a,-antichymotrypsin and of some active enzyme, which is able to recycle with active inhibitor in excess. When all the native inhibitor is used up, the released active enzyme degrades the remaining intact complex into intermediate forms. At the endpoint of the reaction only inactive modified inhibitor and some active chymotrypsin remain. Immunochemical data indicate that, in the complex, a steric hindrance of the antigenic determinants of the inhibitor prevents the formation of the precipitate with specific antiserum. Inactive modified inhibitor, which has dissociated from the complex, has retained antigenic determinants of the native a,-antichymotrypsin.

a,-Antichymotrypsin is probably one of the most specific human serum antiproteases since it is known specifically to control the activity of chymotrypsin-like proteases from phagocytic cells (neutrophils, basophils, tissue mast cells) [l]. a,-Antichymotrypsin forms an equimolar complex with hu- man chymotrypsin-like enzymes such as leukocyte cathepsin G or pancreatic chymotrypsin [2,3].

More recently [4] interactions of El-antichymotrypsin with leukocyte cathepsin G and bovine chymotrypsin were compared using circular dichroism spectroscopy and analyti- cal electrophoresis. It was shown that, in both cases, forma- tion of an equimolar complex occurred and that, moreover, these complexes dissociated within few days even if they were kept at 0°C.

The reaction of human serum a,-antichymotrypsin with bovine chymotrypsin was studied as a model for the reaction between this inhibitor and chymotrypsin-like enzymes. After preliminary studies on mixtures containing different inhibitor- to-enzyme molar ratios, this report is focused on the time- course of formation and dissociation of the complex when the inhibitor is in excess over the enzyme. First we started from previous experiments in which the mixture was kept at 0 "C [4], then we performed the reaction at 25 "C to avoid a temperature difference between the incubation step and the analyses. Taking into account the fact that formation of such a complex was primarily an interaction between two proteins, we fol- lowed each of the two components during the reaction using some of their own characteristics : esterase activity visualiza- tion and spectrophotometric measurements for the enzyme and immunochemical analyses with specific antiserum for the inhibitor. Moreover, methods such as sodium dodecyl sulfate (SDS) and alkaline polyacrylamide gel electrophoreses were carried out to obtain information about all the components present in the reaction mixture.

Abbreviatians. PMSF, phenylmethylsulfonyl fluoride; Suc-Ala- Ah-Pro-Phe-NA, succinyl-r -alanyl-r -alanyl-propyl-r -phenylalanyl- p-nitroanilide; SDS, sodium dodecyl sulfate.

Enzyme. a-Chymotrypsin (EC 3.4.21 .I).

MATERIALS AND METHODS

Muterials

a, -Antichymotrypsin was purified as previously described [5]. Its purity was determined by SDS/polyacrylamide gel electrophoresis, which showed only one band (M,= 58000) with or without prior reduction, and by amino-terminal residue analysis. After manual Edman degradation [6] the amino-terminal residue was determined to be Asn (instead of Arg previously reported [5]). A very low yield (25%) was obtained because of the poor solubility in the coupling buffer and of the large proportion of carbohydrates (24%) in the molecule, which could explain the error in our pre- vious determination. a,-Antichymotrypsin was kept frozen in 0.01 M sodium phosphate buffer (pH 7.4), 0.3 M NaCl, 0.02% NaN, . Bovine a-chymotrypsin from Sigma (N"-tosyl- L-lysine-chloromethyl-ketone-treated, type VII) was dissol- ved just before use in the same buffer. Spectrophotometric measurements using absorption coefficients ,4;;4 of 20.5 [7] and 6.2 [2] were used to determine respectively chymotrypsin and a,-antichymotrypsin concentrations. The latter value was in good agreement with the concentration determined by rocket immunoelectrophoresis according to Weeke [8] using dilutions of a standard serum obtained from Behring.

Chymotrypsin was estimated to be 70% active by titration withp-nitrophenyl acetate according to Kezdy and Kaiser [9]. The percentage of active inhibitor in our preparation was estimated to be 85 -90%, the criterion for the activity being based on electrophoretic separation of active and inactive a,- antichymotrypsin [5]. The inhibitor-to-enzyme molar ratios were expressed taking into account the protein content of the two components. In experiments critically dependent on the amount of active material (i. e. in analysis of the stoichiometry of the inhibition) only the active material content was taken into account.

Chemicals were of analytical grade. Phenylmethylsulfonyl fluoride (PMSF) was from Sigma; a 100mM solution was made in isopropanol.

106

Methods

Spectrophotometric assays of chymotrypsin activity were performed on an Uvikon spectrophotometer (Kontron), ac- cording to Nakajima et al. [lo] using Suc-Ala-Ala-Pro-Phe- NA (Bachem) as substrate.

Polyacrylamide gel electrophoresis were carried out on slab gels as previously described [5]. The stained gels were scanned with a Vernon PH 1 4 densitometer.

Analytical electrophoresis was performed in 1 % agarose gel (Indubiose A 45, Industrie Biologique Francaise) in barbital buffer (0.02 M sodium barbital, 0.0042 M barbital, pH 8.6, ionic strength 0.02). After precipitation of the pro- teins in ethanollwaterjacetic acid (4.5/4.5/1 by vol.) for at least 4 h and drying, the slab was stained for protein with Coomassie brilliant blue R 250. Enzymatic activity visualiza- tion was performed after electrophoretic separation as fol- lows: the slab was washed for 10 min in 0.1 M sodium phosphate buffer pH 7.4, then it was immersed in substrate solution at room temperature (25 "C) according to Uriel and Berges [I I]: components with an esterase activity appeared as pink-coloured spots on an unstained background.

Crossed immunoelectrophoresis [ 12 ] was performed using buffer and agarose gel as for analytical electrophoresis at pH 8.6. It was also used for quantitative assays according to Weeke [I 31. Specific antiserum against a,-antichymotrypsin was from Dako.

For a series of samples analyzed by the electrophoretic or immunoelectrophoretic method, the same initial amount of native a,-antichymotrypsin was applied.

RESULTS

Stoichiometry of the inhihition

In order to determine the stoichiometry of the reaction between a,-antichymotrypsin and bovine chymotrypsin we performed spectrophotometric enzymatic assays. Varying amounts of a, -antichymotrypsin were preincubated with a constant amount of chymotrypsin for 5 min at 25 "C. The residual enzyme activity was measured on aliquots by the spectrophotometric method (Fig. 1). A linear inhibition curve is observed up to 25% residual activity. Extrapolation of the linear part of the curve leads, for a null enzymatic activity, to an inhibitor-to-enzyme molar ratio somewhat higher than equimolar (1,4611). Moreover no complete elimination of chymotrypsin activity is obtained even when the molar ratio is higher than 2 i l .

Electrophoretic anul~~.si.s of various irzhihitor-to-et~z!,mr molar ratios

We undertook a study similar to the one previously carried out when a,-antichymotrypsin was allowed to react with cathepsin G [3]. Four mixtures of a,-antichymotrypsin and bovine chymotrypsin with different inhibitor-to-enzyme molar ratios (0.35/1,0.7/1, 1.4/1, 3.5/1) were incubated for I0 min at 0 "C. After PMSF addition they were studied by both SDS and alkaline polyacrylamide gel electrophoresis. Most results (data not shown) were similar or identical to those obtained when a,- antichymotrypsin reacted with cathepsin G. Formation of an equimolar complex was obtained. It was accompanied by the appearance of a free, modified form of the inhibitor but this occurred to a lesser extent than in [3]. Moreover in the presence

Molar rat io

Fig. 1. Inhibition of' chymotrypsin by cc,-antichymotrypsin. Increasing amounts of inhibitor were added to constant amounts of titrated enzyme (0.74 pM active in the mixtures) and preincubated in phosphate/saline buffer, pH 7.4 for 5 min at 25 "C. Residual chymo- trypsin activity was measured on aliquots after dilution 1 : 100 in 0.1 M Hepes buffer, 0.5 M NaCI, pH 7.5 in the presence of 9.8% dimethyl- sulfoxide after addition of 0.1 mM Suc-Ah,-Pro-Phe-NA (final concentration) as substrate [lo]. The molar ratios were calculated taking into account the active material contents (85% for the inhibitor and 70% for the enzyme)

of SDS, liberation of a fragment having the same migration as a peptide of M , near 5000-6000 was also obtained. As previously shown [4] the unreduced a,-antichymotrypsin

chymotrypsin complex had a M, equal to 77000. If samples had been previously reduced, only the C chain of chymotrypsin (residues 149 - 245) would have remained linked to the inhibitor, so that the observed complex then had a smaller M , (67000).

Time-course studies

Our previous experiments [4] showed that consumption of the active inhibitor, initially present, occurred when the incubation time increased ; moreover the system was evolving with time. So we undertook a time-course study following especially the residual enzymatic activity. The use of an excess of inhibitor in the reaction mixture was to eliminate some unwanted proteolysis of the products.

a,-Antichymotrypsinlchymotrypsin mixture kept at 0 C

To make the connection with our previous experiments [4] we first performed the incubation at 0°C. a,-Antichymo- trypsin was incubated with bovine chymotrypsin to obtain an inhibitor-to-enzyme molar ratio equal to 1.6/1.

Spectrophotometric measurements of remaining chymo- trypsin activity were performed on aliquots after 10 min, 1 h, 27 h, 48 h, 6 days and 11 days; the results, expressed as percentages of the initial activity of the chymotrypsin solution without inhibitor, are respectively 7 % , 20/:, S%, 10.5%, 22.5% and 24%. At various time intervals (10 min, 1 h, 3 h, 5 h, 27 h, 48 h, 72 h, 6 days and 11 days) an aliquot from this mixture was applied in duplicate on to an agarose slab gel at p H 8.6. This electrophoretic system was chosen instead of polyacrylamide gel at pH 8.3 since free chymotrypsin, having a slightly cathodal migration, could be easily visualized using its esterase activity. After electrophoresis, one lane was used for protein staining (Fig. 2A) and the other one for esterase activity visualization (Fig. 2C).

107

I ' O 0 I

chv

10 rnin

t h

3 h

5 h

17 h

46 h

72 h

6 dwr

I1 d o n - -+

+

B

Time (min)

Time ( h l

Fig. 3. Enzyme activities measured on uliquots withdrawn at diflerent time intervals from a mixture ojcc,-antichymotrypsin with chymotrypsin (molar ratio 211, expressed as total protein content) kept at 25 "C. Enzyme concentration in the mixture was 3.45 pM. Conditions for spectrophotometric assay were as in Fig. 1: aliquots were diluted 1 : 100 and measured simultaneously with a suitable dilution of a reference chymotrypsin solution. Residual activities are expressed as percentages of this reference chymotrypsin activity (initial activity) as a function of incubation times of the inhibitor/enzyme mixture. Inset: time (min)

been perturbed. We tried to investigate this point in the following experiments.

Fig. 2. Time-course study by ugurose gel electrophoresis of a mixture of cc,-antichymotrypsin with chymotrypsin (molar ratio 1.6/1), which was kept at 0 <C. (A) Protein staining with Coomassie brilliant blue. (B) Staining for protein and precipitate patterns, obtained on crossed immunoelectrophoresis with antiserum against a,-antichymotrypsin, of native inhibitor (I) and inactive inhibitor formed after an incubation of 11 days with chymotrypsin (m). (C) Esterase activify visualization. a,-Achy, cc,-antichymotrypsin; chy, chymotrypsin (0.1 5 pg); the other samples contained initially 0.75 pg chymotrypsin at the starting point of the reaction

After 10 rnin of incubation, the major component is the complex between a,-antichymotrypsin and chymotrypsin; it is less anodic than the native inhibitor (Fig. 2A). When the incubation time increases, the disappearance of the complex is paralleled by the increase of modified inactive inhibitor, which migrates faster than native inhibitor. After 11 days of incuba- tion at 0 "C, this modified inhibitor is the major component, it has no inhibitory activity left but reacts with antiserum against cc,-antichymotrypsin (Fig. 2B). It is noticeable that in all the samples some esterase activity is seen at the same level as the complex. The decrease in the intensity of this spot after 27 h, and later, is paralleled by the reappearance of some esterase activity corresponding to free chymotrypsin. These experi- ments thus bring evidence that chymotrypsin dissociates in a partly active form from the complex; this dissociation is relatively slow at 0 "C since at least 11 days are needed to reach the complete dissociation for the molar ratio we studied.

In fact visualization of esterase activity on the mixture previously kept at 0 "C required at least 40 rnin of incubation at room temperature (25 "C) after electrophoresis, which had also been performed at room temperature, thus the equilibrium of the mixture going through a temperature difference might have

a,-Antichymotrypsin/chymotrypsin mixture kept at 25 "C

The rate of the reaction was investigated by studying an eel- antichymotrypsin/chymotrypsin mixture which was kept and analyzed at 25 "C. The inhibitor-to-enzyme molar ratio was 211, i.e. higher than the previous one, in order to know more precisely what was going to happen to the excess of active inhibitor using alkaline polyacrylamide gel electrophoresis.

Preliminary studies were performed by spectrophotometric measurements on samples withdrawn at various time intervals. Results are shown in Fig. 3. The curve shows a rapid loss in enzyme activity followed by a reappearance after 1 h of incubation and later. We had checked before that the activity of the chymotrypsin solution was stable at 25 "C, during at least 24 h. Since the reappearance of enzymatic activity seemed to occur very rapidly, even if inhibitor was in excess at the beginning of the reaction, the samples to be studied by polyacrylamide gel electrophoresis or by crossed immuno- electrophoresis were prepared as follows : ten identical mix- tures (molar ratio 2/1) were started off at different times (52 h, 24 h, 6 h, 210 min, 90 min, 60 min, 30 min, 10 min, 5 min, 1 min) before the addition, at the same moment, of a hundredfold molar excess of PMSF over chymotrypsin initi- ally present. An identical mixture was further studied after 10 days and one month of incubation at 25 "C. After 10 rnin of PMSF action, the mixtures were immediately studied by alkaline and SDS/polyacrylamide gel electrophoresis and by crossed immunoelectrophoresis with specific antiserum against a, -antichymotrypsin. We checked whether the reproducibility in the preparation of all the mixtures was good by performing spectrophotometric enzymatic measurements. A control mix- ture was made with the same molar ratio of a,-antichymo- trypsin to chymotrypsin which had been, before mixing, inactivated with a hundredfold molar excess of PMSF for 10 rnin at 25 "C.

108

A

B

slot c

slot 1

slot 6

slot 8

+ slot 10

c

M ' E C I 1 2 3 4 5 6 7 8 9 l O I M

hr 90 000

77 000

58 000

26 000

20 000

10 000

c 1 2 3 4 5 cm from the

top of the gel

Fig. 4. SI~Slpol~~uc,r)'IuIMic/p gel elt~ctrophowsis on a 5 -30% gradient gel. M and M'= M , markers: phosphorylase h (94000) bovine albumin (67000). ovalbumin (43000), carbonic anhydrase (30000), trypsin inhibitor (20 100) and a-lactalbumin (14400). Only M' is reduced. E, bovine chymotrypsin; C , control mixture = al-antichymotrypsin +inactivated chymotrypsin ; I, a,-antichymotrypsin ; 1 - 10 = samples withdrawn from mixtures of a,-antichymotrypsin with chymotrypsin (molar ratio 2/1) kept for various incubation times at 25 'C and then with PMSF added: 1, 1 min; 2, 5 mint 3. 10 min; 4, 30 min; 5,60 min; 6,OO min; 7, 210 min; 8 ,6 h; 9,24 h; 10, 52 h. (A) Electrophoresis pattern. The arrow indicates the mobility of the diffuse band migrating as a peptide of Mr near 5000-6000; 2-mercaptoethanol present in slot M' has an effect on chymotrypsin in slot E. (B) Densitometric traces are shown for slots C; 1, 6, 8 and 10

+

S I C 1 2 3 4 5 6 7 0 9 1 0 1 s

Fig. 5 . Alkalinegel r lc~c~roplzoresi~ on 1O"/,polyucrylamide g e l a t p H 8.3. S, normal human serum; 1 . C and slots 1 -10 are as in Fig. 4. a, a,- antichymotrypsin . chymotrypsin complex; a ' , intermediate component; b, active ti,-antichymotrypsin; c, inactive %,-antichymotrypsin

Fig. 4A shows the pattern obtained by SDS/polyacryl- amide gel electrophoresis for ten different incubation times. In part B of Fig. 4 the mobilities of the reference proteins were plotted versus the logarithm of their M , . Densitometric traces are shown for slots C, 1, 6, 8 and 10. The M , values of the different components were then estimated : native inhibitor, 58000; chymotrypsin, 26000; complex, 77000. A weak band of chymotrypsin remains after 1 min (slot 1) and is hardly visible u p t o 90 min (slot 6), thereafter the intensity of this band increases. The amount of complex decreases markedly after more than 210 min (slot 7). There is a small amount of complex left after 6 h, and it has completely disappeared after 24 h (slot 9). In all the mixtures a band having about the same

migration as a1 -antichymotrypsin is observed : this will be later interpreted with regard t o the results obtained by electro- phoresis in alkaline polyacrylamide gel.

Fig. 5 shows the patterns obtained by alkaline poly- acrylamide gel electrophoresis of the same mixtures. Some active inhibitor (band b) is seen for a t least 3 0 m i n of incubation but its amount decreases from 1 min t o 30 min (slots 1-4). Concerning the complex (band a ) results are in agreement with those shown in Fig. 4: complex is observed up to 210 min (slot 7) and a very small amount of complex remains after 6 h. A component migrating as a n intermediate (band a') between the complex and the inactive inhibitor appears then (slot 8): it might be due t o degraded complex. Its

109

amount decreases after 24 h and 52 h (slots 9 and 10) while the amount of inactive a,-antichymotrypsin (band c) increases markedly.

After 10days of incubation and later, the pattern ob- tained by SDS/polyacrylamide gel electrophoresis (Fig. 6A) exhibits only one band of Mr=55000 and a weak band of chymotrypsin (slot m). In alkaline polyacrylamide gel electro- phoresis (Fig. 6B) only modified inactive inhibitor, migrating faster than native inhibitor, is observed. We have never observed, in these conditions, a transformation or a degrada- tion going further than the inactive form of the inhibitor of MI = 55000.

The results obtained in Fig. 5 explain why the peak in the range of M , = 55000 - 58000 in the densitometric traces of the SDS electrophoretic patterns (Fig. 4B) shows some shifts from one slot to another: different molecular species having a M, in the same range coexist. For example, in slot 8, the material in this peak has a slightly higher M, than that in slot 6 ; this is probably due to the presence of some degraded complex seen in Fig. 5 (slot 8). In slot 10 (Fig. 4B) the peak has shifted to smaller M,: there is less degraded complex and the modified inhibitor becomes the predominant component as seen in Fig. 5 (slot 10). After 10 days of incubation and later (Fig. 6) only modified inhibitor exists, the corresponding band is well defined after SDS electrophoresis and its MI (55000) is easier to estimate.

Some of the patterns, obtained by crossed immunoelectro- phoresis, using specific antiserum against a,-antichymotryp- sin, are shown in Fig. 7 and compared with that of native a,- antichymotrypsin. Sample (b) was applied in duplicate and, after the first-dimension electrophoresis, one lane was used for esterase activity visualization as in Fig. 2C. Quantification of the precipitation areas was performed and the results were expressed as percentages of the precipitation area given by an equivalent amount of native a, -antichymotrypsin. The values obtained show a marked decrease of the precipitation area when a,-antichymotrypsin is mixed with chymotrypsin : up to at least 60 min of incubation the precipitation area represents about 40 -50% of the reference one. In sample (b) (1 min) the precipitation curve shows a peak with a small cationic shoulder. A spot with an esterase activity is easily seen at the same level as this cationic shoulder: it corresponds to the complex shown in Fig. 2. After 210 min of incubation (samplee) and later, the peak gets higher and broader. In samples (g) and (h) the precipitation areas obtained after 24 h and 52 h represent respectively 11 5 and 125% of the reference one. Moreover the migration rate has increased when com- pared with the reference one and the precipitate does not have the same morphological appearance as that of native inhibitor. After 10 days of incubation at 25 "C, the mixture gives a precipitation area equal to about 130% of the reference area.

DISCUSSION The results obtained using the different methods dem-

onstrate that the equimolar complex formed between a,-an- tichymotrypsin and chymotrypsin dissociates spontaneous- ly even if the mixture has been made in the presence of an excess of inhibitor. A modified inactive inhibitor and some active enzyme are released from the complex. The enzyme can recycle and thus is able to attack more than a single a,-antichymotrypsin molecule, using up the excess of in- hibitor. The dissociation is slow at 0 "C but occurs rapidly at

A

+

I m

C \

I m

t *r

10 000

I 2 3 4 5 6 cm from the top of the gel

Fig. 6. Polyacrylumide gel electrophoresis patterns of native inhibitor ( I ) and of a mixture of u,-antichymotrypsin and chymotrypsin (molar ratio 211) kept for 10 days at 25 "C (m) . Identical results were obtained on a mixture kept for one month at 25 "C. (A) SDS/polyacrylamide gel electrophoresis on a 5 -30% gradient gel. (B) Alkaline polyacry- lamide gel electrophoresis. (C) Densitometric traces of the slots shown in (A). The mobilities of the M, markers (not shown) were plotted versus log M ,

25 "C; thus the esterase activity shown at the same level as the complex (Fig. 2) is only due to dissociation on the spot during the revelation procedure. The same phenomenon of dis- sociation, with recycling enzyme, has already been described for the antithrombin-111. trypsin complex [14].

In the present time-course studies the reappearance of an increasing enzymatic activity is shown when all the excess of active inhibitor is used up and transient partially degraded complexes appear. Using thin-layer polyacrylamide gel iso- electric focusing, Gianazza and Arnaud [15] showed that, in the presence of excess enzyme, incubation of human plasma with bovine chymotrypsin resulted in the formation of a

110

+

+

E AV

+

ti- ) +

Fig. I. Crossed i m r n i m o e k ~ c . t r ~ ~ p h ~ r e ~ ~ . s patterns obtained with anti- .serum aguinst a,-antic~h~motr)psin. a, native cc,-antichymotrypsin; b -h, samples withdrawn from mixtures of a,-antichymotrypsin with chymotrypsin (molar ratio 2,l) kept for different time intervals at 25 ‘C: b, 1 min;c, 10 min: d, 60 min; e, 210 min; f, 6 h ; g, 24 h ; h, 52 h. EAV= esterase activity visualization after the first-dimension elec- trophoresis. The bar :it the bottom of each photograph indicates the localization of the maximum of the precipitin arc obtained with native x1 -antichymotrypsin ( a )

secondary complex between a, -antichymotrypsin and chymo- trypsin while in tho presence of excess inhibitor a primary complex with a higher pI was obtained.

A system using equimolar quantities of enzyme and inhibitor would not have been simpler since, even ifthe mixture had been prepared in calculated equimolar quantities at the starting point of the reaction, the system would very quickly have developed an excess of enzyme because of the complex dissociation and the release of active enzyme.

With all the data presented above we can suggest that a,- antichymotrypsin has a temporary inhibitory behaviour for bovine chymotrypsin as defined by Bieth [16].

Similar behaviour was previously shown for other serine protease inhibitors from human plasma. Some plasmin ac- tivity has been generated from a,-antiplasmin. plasmin com- plex [I 71. It was shown that the slow spontaneous dissociation of the antithrombin-111. thrombin complex resulted in active thrombin and inactive antithrombin 111 [18]. The authors concluded that the inactivation of thrombin by antithrombin 111 is a form of so-called temporary inhibition. Instability of the protease. inhibitor complex and temporary inhibitor be- haviour were also demonstrated for a,-protease inhibitor reacting with porcine elastase [19,20] or porcine trypsin [2 1 ,221.

Whereas a totally consistent model for a,-antichymo- trypsin reactions is not possible at this time, the most comprehensive scheme that can be proposed is that shown in the following equation:

k , kz k3 E f I +- E I + EI* --+ E+1*

where E is the enzyme, I the inhibitor, EI an enzyme-substrate type of complex, EI* the covalent complex which can be vizualized by electrophoreses and I* the modified inhibitor. Such an equation was suggested concerning a, -protease inhibitor by other authors [22] who established that the k - , path occurred. In our system, this k - l path has not yet been established. Moreover the very limited specificity of a,- antichymotrypsin does not allow us to use a similar stratagem to determine whether active inhibitor is released from the complex. Recently Lobermann et al. [23] showed that complex dissociation and therefore the temporary inhibitor behaviour they observed for the a,-protease-inhibitor . chymotrypsin complex did not occur if the complex had been treated with diisopropyl fluorophosphate or if a,-protease inhibitor was pretreated with this synthetic inhibitor before complex forma- tion. A protease contaminant of a,-protease inhibitor would be the cause of the spontaneous dissociation. In the system we studied this hypothesis does not seem to be valid. If a serine protease contaminant initially present is the cause of the spontaneous complex dissociation, the process must be de- finitively stopped when PMSF is added to the mixture. In fact our preliminary results show that, after PMSF addition, the consumption of active inhibitor, which occurs because of the complex dissociation, is only momentarily stopped, i.e. for as long as PMSF is active in the mixture; it is known that PMSF does not keep its activity for a long time in aqueous solution at pH 7.5 [24]. Active enzyme released thereafter makes the reaction continue. Nevertheless the elucidation of whether there is an inherent instability of the complex or whether the breakdown of the complex is only due to proteolytic activity of released enzyme awaits further experiments. It will be of great interest, from a physiological point of view, to investigate the fate of cathepsin G when a,-antichymotrypsin . cathepsin G dissociates spontaneously as already described [3,4].

Immunochemical data. presented in this paper, show that there is in the complex some steric hindrance of the antigenic determinants of the inhibitor, which prevents the formation of the precipitate. After complete dissociation the inhibitor species obtained is the inactive inhibitor; it is able to react with antibodies against native inhibitor, thus it must have retained antigenic determinants of native inhibitor. However, the precipitation area obtained is higher than that obtained with an equivalent amount of native inhibitor; this may be due either to the greater electrophoretic mobility of this com- ponent at pH8 .6 or to some change in the antigenic de- terminants of the molecule. The difference in the morpho- logical appearance of the precipitate also suggests this kind of phenomenon. It is known that immunochemical results are closely related to morphological changes (251. Such con- formational changes during complex formation have been shown using immunochemical analyses, for example on az- antiplasmin [26] and antithrombin I11 [27 -291.

If such immunochemical data are found out when a,-anti- chymotrypsin reacts with cathepsin G, one must take care not to misinterpret the results of immunochemical measure-

111

ments, which can be obtained in biological fluids where cathepsin G is able to react with this inhibitor, since ai- antichymotrypsin can be in at least three forms: native, complexed or modified. Immunochemical measurements have to be carried out using the appropriate standards and exploited very carefully.

The skilful assistance of Mrs M. P. Ducourouble and M. Lohez is gratefully acknowledged. This work is supported by Institut National de la Santd et de la Recherche MPdicale (CRL no. 825044).

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A. Laine, M. Davril, and A. Hayem, Unite de Recherches sur la Biochimie des Proteines Normales et Pathologiques (Unite 16 de l’lnstitut National de la SantC et de la Recherche Mtdicale), 2 Place de Verdun, F-59045 Lille-Cedex, Nord, France