Biol. Chem. Hoppe-Seyler Vol. 371, pp. 389-393, May 1990
Binding of the Bovine Basic Pancreatic Trypsin Inhibitor (Kunitz Inhibitor) to Human and Bovine Factor Xa AThermodynamic Study Paolo ASCENZI H , Massimo CoLETTAa, Gino AMICONI ;I , Martino BoLOGNESi b , Enea MENEGATTI C and Mario GUARNERI C ;
' C.N.R. Centre di Biologia Molecolare. Dipartimentodi Scienze Biochimiche, Universitä di Roma tl La Sapienza" Dipartimento di Genetica e Microbiologia, Sezione di Cristallografia, Universitä di Pavia c Dipartimento di Scienze Farmaceutiche. Universitä di Ferrara 11
(Received 27 November 1989/15 February 1990)
Summary: The effect of pH and temperature on the apparent association equilibrium constant (#a) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human and bovine factor Xa (Stuart-Prower factor; EC 3.4.21.6) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to human and bovine factor Xa are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0 °C) for BPTI binding to human and bovine factor Xa decrease, thus reflecting the acidic pK shift of the His57 catalytic residue from 7.1, in the free enzyme, to 5.2, in the proteinase-inhibitor complex. At pH 8.0, values of the apparent thermodynamic parameters for
BPTI binding to human and bovine factor Xa are: Ka = 2.1 x lO^'1 (at 21.0 °C), AG° = - 29.7 kJ/mol (at 21.0 °C), £° = + 161 entropy units (at 21.0 °C), and //° = + 17.6 kJ/mol (temperature-independent over the explored range, from 5.0 °C to 45.0 °C).Thermodynamics of BPTI binding to human and bovine factor Xa have been analysed in parallel with those of related serine (pro)enzyme/Kazal- and /Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human and bovine factor Xa was related to the inferred stereochemistry of the proteinase/inhibitor contact region.
Bindung des basalen Rinderpankreas-Trypsininhibitors (Kunitz-Inhibitor) an den Gerinnungsfaktor Xa von Mensch und Rind. Eine thermodynamische Untersuchung Zusammenfassung: Untersucht wurde der Einfluß von pH-Wert und Temperatur auf die apparente Assoziationsgleichgewichtskonstante (Ka) für die Bindung des basalen Rinderpankreas-Trypsininhibitors (BPTI, Kunitz-Inhibitor) an den Coagulationsfaktor
Xa (Stuart-Prower-Faktor, EC 3.4.21.6) von Mensch und Rind. Unter allen untersuchten Bedingungen waren die A:a-Werte für eine Bindung von BPTI an den menschlichen und den Rinder-Faktor Xa identisch. Senkung des pH-Wertes von 9.5 auf 4.5 bewirkte eine
Enzymes: Chymotrypsin (EC 3.4.21.1); Coagulation factor Xa, Stuart-Prower factor (EC 3.4.21.6); Glandular or tissue kallikreins (EC 3.4.21.35); Leukocyte elastase (EC 3.4.21.37); Plasma kallikreins (EC 3.4.21.34); Plasmin (EC 3.4.21.7); (Pro)thrombin (EC 3.4.21.5); Trypsin(ogen) (EC 3.4.21.4); Plasminogen activator (EC 3.4.21.31), also named urokinase. Abbreviations: BPTI, bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor); ZLysONp, A/"-benzyloxycarbonyl-L-lysine/?-nitrophenyl ester.
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390
P. Ascenzi, M. Coletta, G. Amiconi, M. Bolognesi, E. Menegatti and M. Guarneri
Erniedrigung der #a-Werte für die BPTI-Bindung an den Faktor Xa von Mensch und Rind. Diese Erniedrigung spiegelt die p/f-Verschiebung des katalytischen Restes His-57 ins Saure wider, nämlich von 7.1 im freien Enzym nach 5.2 im Proteinaseinhibitor-Komplex. Bei pH 8.0 wurden für die Bindung von BPTI an den Faktor Xa von Mensch und Rind die folgenden apparenten thermodynamischen Paramter gemessen: Ka = 2.1 lO5!^'1 (bei 21.0 °C), AG° = 29.7 kJ/mol (bei 21.0 °C), 5° = + 161 Entropie-Einheiten (bei 21.0 °C) und / = + 17.6 kJ/mol (temperaturunab-
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hängig im untersuchten Bereich von 5.0 bis 45.0 °C). Parallel zu den thermodynamischen Bestimmungen der Bindung von BPTI wurden entsprechende Messungen mit verwandten Serin-(Pro)enzym-Kazalund -Kunitz-Typ-Inhibitorsystemen durchgeführt. Unter Berücksichtigung der bekannten molekularen Modelle wurde das beobachtete Bindungsverhalten zur abgeleiteten stereochemischen Beschaffenheit der Proteinase-Inhibitor-Kontaktregion in Bezug gesetzt.
Key words: Human factor Xa, bovine factor Xa, bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor. BPTI), proteinase:inhibitor complex formation, thermodynamics (of proteinase:inhibitor complex formation), pH and temperature effects (on thermodynamics of proteinaserinhibitor complex formation).
Factor Xa (Stuart-Prower factor; EC 3.4.21.6) is a trypsin-like serine proteinase which plays a central role in blood coagulation, catalysing the conversion of prothrombin to thrombin11'31. Factor Xa action is modulated by the interaction with plasma serine proteinase inhibitors, such as «rproteinase inhibitor, a2antiplasmin, antithrombin, the a2-macroglobulin and protein C inhibitor[1~71. In addition, preliminary experiments indicated the ability of factor Xa to form adducts with the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor)' 81 . In view of the presence in human plasma of Kunitz-type proteinase inhibitors showing high homology with BPTI^91, and of the clinical use of BPTI[10J11, it appeared of interest to investigate, from the thermodynamic viewpoint, the inhibition of the enzymatic activity of human and bovine factor Xa by BPTI association. The human and bovine factor XaiBPTI complex formation has been analysed in parallel with the molecular and binding properties of related serine (pro)enzyme/Kazaland /Kunitz-type inhibitor systems'10·12·131.
Materials and Methods Materials Human and bovine factor Xa were obtained from Boehringer Mannheim GmbH, D-6800 Mannheim. BPTI was provided by Lepetit S.p.A., Milano, I. ZLysONp was obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. All the other products were from Merck AG, D-6100 Darmstadt. All chemicals were of analytical grade and used without further purification. The characterization of human and bovine factor Xa, BPTI and ZLysONp has been previously reported'10·14"18'. Determination of thermodynamic parameters for the human and bovine factor Xa:BPTl complex formation Thermodynamics for BPTI binding to human and bovine factor Xa has been obtained between pH 4.5 and 9.5 (acetate buffer, pH 4.5 to 6.0; phosphate buffer, pH 6.0 to 8.5; borate/glycine buffer, pH 8.5 to 9.5; all at / = O.lM; sodium salts), and between 5.0 °C and
45.0 °C. No specific ion effects were found using different buffers with overlapping pH values. Thermodynamic parameters for the human and bovine factor Xa:BPTI complex formation have been evaluated by the inhibitory effect on the enzymatic cleavage of ZLysONp; the proteinasecatalysed hydrolysis of ZLysONp was followed spectrophotometrically at 360 nm, the Ae.^mm value changing from 4500M'1 cm"1 at pH < 5.5 to 10200M'1 cnT1 at pH > 8.5 with a pK value of 7.1, at 20.0 °C'K'·17'. All the spectrophotometric measurements were carried out with double-beam spectrophotometers Jasco J-510 and Varian Cary 219 equipped with the Hi-Tech Scientific SFA-11 rapid kinetics accessory. The very short mixing time of the rapid kinetics accessory (^ 50 ms) allowed the determination of the initial rate for the proteinase-cataylsed hydrolysis of ZLysONp (from the initial 10% of the time course) with much higher accuracy with respect to the usual mixing of the reagents (i.e., the enzyme. BPTI and ZLysONp) into the classical spectrophotometric cuvette (mixing time 2: 10 s).Thus, the use of the rapid kinetics accessory is especially useful under conditions where the proteinase-catalysed hydrolysis of ZLysONp is fast (i.e., at high enzyme concentration, alkaline pH, and high temperature). At all pH values, initial rates were corrected for the acid and alkaline hydrolysis of the substrate'17·181. According to Ascenzi et al.'19'20', values of the apparent association equilibrium constant (Ka) for BPTI binding to human and bovine factor Xa were determined as follows.The value of the apparent intrinsic molar fraction of the BPTI-inhibited human and bovine factor Xa (Yo) was calculated, for every free inhibitor concentration, from the experimental value of the apparent molar fraction of the inhibited enzyme ( ) determined at a fixed substrate concentration (i.e., [ZLysONp]), taking into account the simple competition of BPTI with ZLysONp for the proteinase, according to the following expression: Y() = Y/{1 + Km x [ZLysONp]}
(1)
where Km represents the enzyme/substrate Michaelis constant (Y () = 1 refers to the completion of the process). Values of Km for the human and bovine factor Xa/ZLysONp systems range from 3.0 10"5M to 4.5 x 10"5M, and from 2.8 x 10"5M to 4.2 x 10"5M, respectively (from Robison et al.'16', and present study). The validity of Eqn 1 has been verified at different substrate concentrations; thus, at fixed BPTI concentration, values of YO turned out to be independent of [ZLysONp]. As expected for systems in rapid equilibrium, values of Y, and Y0, were independent of the proteinase/BPTI and proteinase/ZLysONp preincubätion time (ranging from 30 s_to 12 h_, and from 30 s to 1 h, respectively). Next, the same value of , and Y0, was obtained by mixing the proteinase:BPTI complex
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Vol. 371 (1990)
391
Binding of the Kunitz Inhibitor to Human and Bovine Factor Xa
with ZLysONp, the proteinase/ZLysONp system with BPTI, and the proteinase with a solution containing both BPTI and ZLysONp. Values of Ka were determined from the dependence of PO on the free inhibitor concentration (i.e., [BPTI]), according to the following expression: = [BPTI]/{[BPTI] + 1/ΚΛ}
(2)
The following free reagent concentrations were employed: human and bovine factor Xa concentration ranged between 1.0 x 10~'°M and 1.0 x 10"7M; BPTI concentration ranged between 1.0 x 10"7M and 1.0 x 10~2M; and ZLysONp concentration ranged between 2.0 x lCT 6 Mand2.0x KT3M. Values of the apparent free energy (AG°) for the human and bovine factor Xa:BPTI association were calculated, at 21.()°C. from values of Kj. according to the expression'21': AC 0 --2.3 x Rx Tx l
(3)
Values of the apparent enthalpy variation (Δ//°) accompanying the human and bovine factor Xa:BPTI complex formation were determined from the dependence of log Kaon l/Tby van't Hoff plots (see Fig. 1, inset), according to the expression'2'1: Δ//° = - 2.3 x R x [d log KJ(d\IT)]
(4)
Values of the apparent entropy variation (Δ5°) for the human and bovine factor Xa:BPTI complex formation were calculated, at 21.0 °C. from values of AG° and Δ//°. according to the expression' 21 ':
(5) Average error values of ± 8% (for Ka and ΔΟ° values) and ± 12% (for Δ//° and Δ5° values) were evaluated as the standard deviation"9·20". Data analysis was carried out on a Digital PDP 11/23 computer employing an iterative non-linear least-squares fitting procedure according to the Marquart algorithm.
Results and Discussion Under all the experimental conditions, BPTI binding to human and bovine factor Xa conforms to a simple equilibrium as indicated by the unitary value of the Hill coefficient (n = 1.00 ± 0.02). Moreover, Ka values were always independent of the enzyme concentration. As shown in Fig. 1, values of Ka for BPTI binding to human and bovine factor Xa are identical. This finding is in line with the undistinguishable catalytic properties of human and bovine factor Xa[22], as well as with the identity of their amino acid sequence around the catalytic site and the substrate/inhibitor recognition region(s)'23^. The pH dependent change in affinity of BPTI binding to human and bovine factor Xa shown in Fig. 1, is strictly reminescent of those obtained for the binding of Kazal- and/or Kunitz-type inhibitors to bovine aand /3-trypsin, bovine trypsinogen, bovine a-chymotrypsin, porcine pancreatic -kallikreins A and B, human urinary kallikrein, human Lys77-plasmin, human leukocyte elastase and human urokinase (33000 Mr and 54000 Mr species)[12'24'25], and thus it may be described with the same model.The decrease
σ> ο
3.2 3.4 3.6 1/Τχ103 (Κ'1)
5
6
7 pH
8
9
Fig. 1. pH dependence of the apparent association equilibrium constant (ΚΆ [M~']) for BPTI binding to human (O) and bovine (D) factor Xa, at 21.0 °C. The unbroken line was generated from Eqn 6 using C = 5.36, P/CUNL = 7.1 and pKUc — 5.2; the parameters were obtained with an iterative non-linear least-squares fitting procedure. An average error value of ± 12% was evaluated for /CUNL and Α^,ιο values, as the standard deviation, according to the fitting procedure. The inset shows the van't Hoff plot describing the temperature dependence, between 5.0 °C and 45.0 °C, of Ka [vT1] for the human (O) and bovine (D) factor Xa:BPTI complex formation at pH 8.O. From the slope of this plot, the apparent ΔΗ° value (= + 17.6 kJ/mol) was determined' 21 '. Next, the temperature independence of the apparent Δ//° value indicates that the apparent Δ(Γ£ value is very close to 0 kJ/mol'21'. For further details see text.
in Ka values (i.e., in affinity) for BPTI binding to human and bovine factor Xa, on lowering pH from 9.5 to 4.5, can be accounted for by the acidic ρ Κ shift of a single ionizing group upon proteinase:inhibitor complex formation. According to linkage relations{26], this model leads to the following expression: (6)
where C is a constant that corresponds to the alkaline asymptote of log Ka, and /MJNL and KUG are the apparent proton dissociation equilibrium constants for the BPTI-free and the BPTI-bound human and bovine factor Xa. Eqn 6 has been used to generate the unbroken line shown in Fig. 1; the agreement with the experimental data is fully satisfactory. Molecular data and amino-acid sequence homologles[i,23.27] anow to SUggest that the ionization process responsible for the observed pH effects refers only to the enzyme side. In fact, BPTI amino acid side chains, which might be expected to ionize over the explored pH range (i.e., between pH 4.5 and 9.5), do not lie near the proteinase/inhibitor contact area. Next, the pH profile for BPTI binding to human and bovine factor Xa is strongly similar to those published for the interaction of Kazal- and Kunitz-type inhibitors with different homologous serine proteinases Brought to you by | University of Arizona Authenticated Download Date | 5/31/15 10:19 PM
392
P. Ascenzi, M. Coletta, G. Amiconi, M. Bolognesi, E. Menegatti and M. Guarneri
and their zymogens, in which the only relevant ionizing group has been identified on the (pro)en-
zyme[12,13,24.25]_
Inspection of the different active site residues capable of affecting BPTI binding to human and bovine factor Xa suggests that only the His57 catalytic residue'23·271 has a pK comparable to the p/C UNL in the free proteinase (see Fig. 1). By analogy with what has been reported in related (pro)enzyme/inhibitor systems'12'24·251, the observed one-proton acidic pK shift could be interpreted as reflecting the burial of the enzyme active site residues upon the proteinase:BPTI complex formation with the concomitant strengthening of the proteinase Serl95 OG - His57 NE2 intramolecular hydrogen bond; this interaction is very weak, or absent, in the homologous inhibitor-free proteinasest13·23·271. At pH 8.0, values of the apparent thermodynamic parameters for BPTI binding to human and bovine factor Xa are: K.a = 2.1 x lOV (at 21.0 °C), AG° = -29.7kJ/mol(at21.0 0 C),A t $°= + 161 entropy units (at 21.0 °C), and Δ/¥° = + 17.6 kJ/mol (temperatureindependent over the explored range, from 5.0 °C to 45.0 °C) (see Fig. 1, inset). Both the apparent ΔΗ° and Δ5° values for BPTI binding to human and bovine factor Xa are comparable to those reported for the association of Kazal- and Kunitz-type inhibitors to serine proteinases and their zymogens, and indicate that the complex formation is an entropy-driven process. Next, the apparent positive AS10 values could reflect the removal of the proteinase- and/or inhibitorbound water molecules during complexation^12'13·281. By comparison with other plasma proteinases, the affinity of BPTI for human and bovine factor Xa is lower than that observed for the formation of the plasmin: and kallikrein:BPTI complexes by about three and four orders of magnitude, respectively. On the other hand, the affinity of BPTI for human and bovine factor Xa is about two orders of magnitude higher than that reported for the inhibitor association to thrombin[1(U2-2(K291. Such functional behaviour is likely to be related to the structural differences occurring at the plasma proteinase/BPTI contact area; in particular, regions 20-30, 45-54, 88-93, 139-152, 167-177, 190-198, 215-224 and 227-232 show sequence hypervariability, different dimensions and structures. Thus, such domains can be considered as potential sites exerting different levels of steric hindrance to the inhibitor access and (de)stabilization of the enzyme:inhibitor complex'13·23'27'29"311. Finally, from the physiophathological viewpoint, the reported data obtained in vitro (Ka~l = 4.8 x 10~6M; see above), together with the normal plasmatic levels of factor Xa (= 1.8 x 10~8M[2]) and of the Kunitz-type
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proteinase inhibitors (= 1.5 x 10 8 M [yl ), as well as of the therapeutical concentration of BPTI (~ 1 x 10~6Μ'10·η1) for a wide variety of pathophysiological states, thought to be associated with an increase of proteinase activity, do not favour the hypothesis of any interaction of human factor Xa with BPTI and BPTI-like inhibitors. On the other hand, such a behaviour appears to be of interest in relation to a rationale for the therapeutic action of BPTI.
References 1 2 3 4 5 6 7 8
9 10 11 12
13
14 15 16 17 18 19
Jackson, C.M. & Nemerson, Y. (1980) Annu. Rev. Biochem. 49,765-811. Furie, B. & Furie, B.C. (1988) Cell S3, 505-518. Mann, K.G., Jenny, R.J. & Krishnaswamy, S. (1988) Annu. Rev. Biochem. 57, 915-956. Heidtmann, H. &Travis, J. (1986) in Proteinase Inhibitors (Barrett, A.J. & Salvesen, G., eds.) pp. 441-456, Elsevier, Amsterdam, New York, Oxford. Lijnen, H.R. & Collen, D. (1986) in Proteinase Inhibitors (Barrett, A.J. & Salvesen, G., eds.) pp. 457-476, Elsevier, Amsterdam, New York, Oxford. Bj rk, I. & Danielsson, A. (1986) in Proteinase Inhibitors (Barrett, A.J. & Salvesen, G., eds.) pp. 489-513, Elsevier, Amsterdam, New York, Oxford. Cawston, T.E. (1986) in Proteinase Inhibitors (Barrett, A.J. & Salvesen, G., eds.) pp. 589-610, Elsevier, Amsterdam, New York, Oxford. Ascenzi, P., Coletta, M., Amiconi, G., De Cristofaro, R., Bolognesi, M., Guarneri, M. & Menegatti, E. (1988) in Proteine '88, (Balestrieri, C., Brunori, M., DAlessio, G., Di Frisco, G., Fontana, A., Ronchi, S. & Rotilio, G., eds.) pp. 154-155, Poligrafica F.lli Ariello, Editori s.a.s., Napoli. Fioretti, E., Angeletti, M., Citro, G., Barra, D. & Ascoli, F. (1987) J. Biol. Chem. 262, 3586-3589. Gebhard, W., Tschesche, H. & Fritz, H. (1986) in Proteinase Inhibitors (Barrett, A.J. & Salvesen, G., eds.) pp. 375-388, Elsevier, Amsterdam, New York, Oxford. Schnebli, H.-P. & Braun, N.J. (1986) in Proteinase Inhibitors (Barrett, A.J. & Salvesen, G., eds.) pp. 613-627, Elsevier, Amsterdam, New York, Oxford. Amiconi, G., Ascenzi, P., Bolognesi, M., Menegatti, E. & Guarneri, M. (1988) in Macromolecular Biorecognition: Principles and Methods (Chaiken, I., Chiancone, E., Fontana, A. & Neri, P., eds.) pp. 117-130,The Humana Press, Clifton. Bolognesi, M., Ascenzi, P., Amiconi, G., Menegatti, E. & Guarneri, M. (1988) in Macromolecular Biorecognition: Principles and Methods (Chaiken, I., Chiancone, E., Fontana, A. & Neri, P., eds.) pp. 81-100,The Humana Press, Clifton. Fujikawa, K. & Davie, E.W. (1976) Methods Enzymol. 45, 89-95. Jesty, J. & Nemerson, Y. (1976) Methods Enzymol. 45, 95-107. Robison, D.J., Furie, B., Furie, B.C. & Bing, D.H. (1980) J. Biol. Chem. 255, 2014-2021. Ascenzi, P., Bertollini, A.,Verzili, D., Brunori, M. & Antonini, E. (19SO) Anal. Biochem. 103,235-239. Ascenzi, P., Sleiter, G. & Antonini, E. (1982) Gazz. Chim. Ital. 112, 307-317. Ascenzi, P., Coletta, M., Amiconi, G., Bolognesi, M., Guarneri, M. & Menegatti, E. (1988)7. Mol. Recognition 1, 130-137.
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Vol. 371 (1990)
Binding of the Kunitz Inhibitor to Human and Bovine Factor Xa
20 Ascenzi, P., Coletta, M., Amiconi, G., De Cristofaro, R., Bolognesi, M., Guarneri, M. & Menegatti, E. (1988) Biochim. Biophys. Acta 956, 156-161. 21 Keleti,T. (1983) Biochem. J. 209, 277-280. 22 Lottenberg, R., Christensen, U., Jackson, C.M. & Coleman, P.L. (1981) Methods Enzymol. 80, 341-361. 23 Fung, M.R., Hay, C.W. & MacGillavray, R.T.A. (1985) Proc. Nail. Acad. Sei. U.S.A. 82, 3591-3595. 24 Fioretti, ., Angeletti, M., Passed, D. & Ascoli, F. (1989) J. Protein Chem. 8, 51-60. 25 Ascenzi, P., Amiconi, G., Bolognesi, M., Menegatti, E. & Guarneri, M. (1990)7. Enzyme Inhibition, in press.
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26 Wyman, J. (1964) Adv. Protein Chem. 19, 223-286. 27 Furie, B., Bing, D.H., Feldmann, R.J., Robison, D.J., Burnier, J.P. & Furie, B.C. (1982) J. Biol. Chem. 257, 3875-3882. 28 Amiconi, G., Ascenzi, P., Bolognesi, M., Guarneri, M. & Menegatti, E. (1987) Adv. Biosciences65, 177-180. 29 Berliner, L.J., Birktoft, J.J., Miller, T.L., Musci, G., Scheffler, J.E., Shen,Y.Y. & Sugawara,Y. (l9S6)Ann. NY Acad. Sei. 485, 80-95. 30 Chen, Z. & Bode,W. (1983)7. Mol. Biol. 164, 283-311. 31 Creighton,T.E. & Darby, N.J. (1989) Trends Biochem. Sei. 14, 319-324.
Paolo Ascenzi*, Massimo Coletta and Gino Amiconi, C.N.R. Centre di Biologia Molecolare, Dipartimento di Scienze Biochimiche, Universitä di Roma "La Sapienza", Piazzale Aldo Moro 5,1-00185 Roma, Italia. Martino Bolognesi, Dipartimento di Genetica e Microbiologia, Sezione di Cristallografia, Universitä di Pavia, ViaTaramelli 16,1-27100 Pavia, Italia. Enea Menegatti and Mario Guarneri, Dipartimento di Scienze Farmaceutiche, Universitä di Ferrara, Via Scandiana 21,1-44100 Ferrara, Italia. 'f To whom correspondence should be sent.
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Binding of the Bovine Basic Pancreatic Trypsin Inhibitor (Kunitz Inhibitor) to Human and Bovine Factor Xa AThermodynamic Study Paolo ASCENZI H , Massimo CoLETTAa, Gino AMICONI ;I , Martino BoLOGNESi b , Enea MENEGATTI C and Mario GUARNERI C ;
' C.N.R. Centre di Biologia Molecolare. Dipartimentodi Scienze Biochimiche, Universitä di Roma tl La Sapienza" Dipartimento di Genetica e Microbiologia, Sezione di Cristallografia, Universitä di Pavia c Dipartimento di Scienze Farmaceutiche. Universitä di Ferrara 11
(Received 27 November 1989/15 February 1990)
Summary: The effect of pH and temperature on the apparent association equilibrium constant (#a) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human and bovine factor Xa (Stuart-Prower factor; EC 3.4.21.6) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to human and bovine factor Xa are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0 °C) for BPTI binding to human and bovine factor Xa decrease, thus reflecting the acidic pK shift of the His57 catalytic residue from 7.1, in the free enzyme, to 5.2, in the proteinase-inhibitor complex. At pH 8.0, values of the apparent thermodynamic parameters for
BPTI binding to human and bovine factor Xa are: Ka = 2.1 x lO^'1 (at 21.0 °C), AG° = - 29.7 kJ/mol (at 21.0 °C), £° = + 161 entropy units (at 21.0 °C), and //° = + 17.6 kJ/mol (temperature-independent over the explored range, from 5.0 °C to 45.0 °C).Thermodynamics of BPTI binding to human and bovine factor Xa have been analysed in parallel with those of related serine (pro)enzyme/Kazal- and /Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human and bovine factor Xa was related to the inferred stereochemistry of the proteinase/inhibitor contact region.
Bindung des basalen Rinderpankreas-Trypsininhibitors (Kunitz-Inhibitor) an den Gerinnungsfaktor Xa von Mensch und Rind. Eine thermodynamische Untersuchung Zusammenfassung: Untersucht wurde der Einfluß von pH-Wert und Temperatur auf die apparente Assoziationsgleichgewichtskonstante (Ka) für die Bindung des basalen Rinderpankreas-Trypsininhibitors (BPTI, Kunitz-Inhibitor) an den Coagulationsfaktor
Xa (Stuart-Prower-Faktor, EC 3.4.21.6) von Mensch und Rind. Unter allen untersuchten Bedingungen waren die A:a-Werte für eine Bindung von BPTI an den menschlichen und den Rinder-Faktor Xa identisch. Senkung des pH-Wertes von 9.5 auf 4.5 bewirkte eine
Enzymes: Chymotrypsin (EC 3.4.21.1); Coagulation factor Xa, Stuart-Prower factor (EC 3.4.21.6); Glandular or tissue kallikreins (EC 3.4.21.35); Leukocyte elastase (EC 3.4.21.37); Plasma kallikreins (EC 3.4.21.34); Plasmin (EC 3.4.21.7); (Pro)thrombin (EC 3.4.21.5); Trypsin(ogen) (EC 3.4.21.4); Plasminogen activator (EC 3.4.21.31), also named urokinase. Abbreviations: BPTI, bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor); ZLysONp, A/"-benzyloxycarbonyl-L-lysine/?-nitrophenyl ester.
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390
P. Ascenzi, M. Coletta, G. Amiconi, M. Bolognesi, E. Menegatti and M. Guarneri
Erniedrigung der #a-Werte für die BPTI-Bindung an den Faktor Xa von Mensch und Rind. Diese Erniedrigung spiegelt die p/f-Verschiebung des katalytischen Restes His-57 ins Saure wider, nämlich von 7.1 im freien Enzym nach 5.2 im Proteinaseinhibitor-Komplex. Bei pH 8.0 wurden für die Bindung von BPTI an den Faktor Xa von Mensch und Rind die folgenden apparenten thermodynamischen Paramter gemessen: Ka = 2.1 lO5!^'1 (bei 21.0 °C), AG° = 29.7 kJ/mol (bei 21.0 °C), 5° = + 161 Entropie-Einheiten (bei 21.0 °C) und / = + 17.6 kJ/mol (temperaturunab-
Vol. 371(1990)
hängig im untersuchten Bereich von 5.0 bis 45.0 °C). Parallel zu den thermodynamischen Bestimmungen der Bindung von BPTI wurden entsprechende Messungen mit verwandten Serin-(Pro)enzym-Kazalund -Kunitz-Typ-Inhibitorsystemen durchgeführt. Unter Berücksichtigung der bekannten molekularen Modelle wurde das beobachtete Bindungsverhalten zur abgeleiteten stereochemischen Beschaffenheit der Proteinase-Inhibitor-Kontaktregion in Bezug gesetzt.
Key words: Human factor Xa, bovine factor Xa, bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor. BPTI), proteinase:inhibitor complex formation, thermodynamics (of proteinase:inhibitor complex formation), pH and temperature effects (on thermodynamics of proteinaserinhibitor complex formation).
Factor Xa (Stuart-Prower factor; EC 3.4.21.6) is a trypsin-like serine proteinase which plays a central role in blood coagulation, catalysing the conversion of prothrombin to thrombin11'31. Factor Xa action is modulated by the interaction with plasma serine proteinase inhibitors, such as «rproteinase inhibitor, a2antiplasmin, antithrombin, the a2-macroglobulin and protein C inhibitor[1~71. In addition, preliminary experiments indicated the ability of factor Xa to form adducts with the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor)' 81 . In view of the presence in human plasma of Kunitz-type proteinase inhibitors showing high homology with BPTI^91, and of the clinical use of BPTI[10J11, it appeared of interest to investigate, from the thermodynamic viewpoint, the inhibition of the enzymatic activity of human and bovine factor Xa by BPTI association. The human and bovine factor XaiBPTI complex formation has been analysed in parallel with the molecular and binding properties of related serine (pro)enzyme/Kazaland /Kunitz-type inhibitor systems'10·12·131.
Materials and Methods Materials Human and bovine factor Xa were obtained from Boehringer Mannheim GmbH, D-6800 Mannheim. BPTI was provided by Lepetit S.p.A., Milano, I. ZLysONp was obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. All the other products were from Merck AG, D-6100 Darmstadt. All chemicals were of analytical grade and used without further purification. The characterization of human and bovine factor Xa, BPTI and ZLysONp has been previously reported'10·14"18'. Determination of thermodynamic parameters for the human and bovine factor Xa:BPTl complex formation Thermodynamics for BPTI binding to human and bovine factor Xa has been obtained between pH 4.5 and 9.5 (acetate buffer, pH 4.5 to 6.0; phosphate buffer, pH 6.0 to 8.5; borate/glycine buffer, pH 8.5 to 9.5; all at / = O.lM; sodium salts), and between 5.0 °C and
45.0 °C. No specific ion effects were found using different buffers with overlapping pH values. Thermodynamic parameters for the human and bovine factor Xa:BPTI complex formation have been evaluated by the inhibitory effect on the enzymatic cleavage of ZLysONp; the proteinasecatalysed hydrolysis of ZLysONp was followed spectrophotometrically at 360 nm, the Ae.^mm value changing from 4500M'1 cm"1 at pH < 5.5 to 10200M'1 cnT1 at pH > 8.5 with a pK value of 7.1, at 20.0 °C'K'·17'. All the spectrophotometric measurements were carried out with double-beam spectrophotometers Jasco J-510 and Varian Cary 219 equipped with the Hi-Tech Scientific SFA-11 rapid kinetics accessory. The very short mixing time of the rapid kinetics accessory (^ 50 ms) allowed the determination of the initial rate for the proteinase-cataylsed hydrolysis of ZLysONp (from the initial 10% of the time course) with much higher accuracy with respect to the usual mixing of the reagents (i.e., the enzyme. BPTI and ZLysONp) into the classical spectrophotometric cuvette (mixing time 2: 10 s).Thus, the use of the rapid kinetics accessory is especially useful under conditions where the proteinase-catalysed hydrolysis of ZLysONp is fast (i.e., at high enzyme concentration, alkaline pH, and high temperature). At all pH values, initial rates were corrected for the acid and alkaline hydrolysis of the substrate'17·181. According to Ascenzi et al.'19'20', values of the apparent association equilibrium constant (Ka) for BPTI binding to human and bovine factor Xa were determined as follows.The value of the apparent intrinsic molar fraction of the BPTI-inhibited human and bovine factor Xa (Yo) was calculated, for every free inhibitor concentration, from the experimental value of the apparent molar fraction of the inhibited enzyme ( ) determined at a fixed substrate concentration (i.e., [ZLysONp]), taking into account the simple competition of BPTI with ZLysONp for the proteinase, according to the following expression: Y() = Y/{1 + Km x [ZLysONp]}
(1)
where Km represents the enzyme/substrate Michaelis constant (Y () = 1 refers to the completion of the process). Values of Km for the human and bovine factor Xa/ZLysONp systems range from 3.0 10"5M to 4.5 x 10"5M, and from 2.8 x 10"5M to 4.2 x 10"5M, respectively (from Robison et al.'16', and present study). The validity of Eqn 1 has been verified at different substrate concentrations; thus, at fixed BPTI concentration, values of YO turned out to be independent of [ZLysONp]. As expected for systems in rapid equilibrium, values of Y, and Y0, were independent of the proteinase/BPTI and proteinase/ZLysONp preincubätion time (ranging from 30 s_to 12 h_, and from 30 s to 1 h, respectively). Next, the same value of , and Y0, was obtained by mixing the proteinase:BPTI complex
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Binding of the Kunitz Inhibitor to Human and Bovine Factor Xa
with ZLysONp, the proteinase/ZLysONp system with BPTI, and the proteinase with a solution containing both BPTI and ZLysONp. Values of Ka were determined from the dependence of PO on the free inhibitor concentration (i.e., [BPTI]), according to the following expression: = [BPTI]/{[BPTI] + 1/ΚΛ}
(2)
The following free reagent concentrations were employed: human and bovine factor Xa concentration ranged between 1.0 x 10~'°M and 1.0 x 10"7M; BPTI concentration ranged between 1.0 x 10"7M and 1.0 x 10~2M; and ZLysONp concentration ranged between 2.0 x lCT 6 Mand2.0x KT3M. Values of the apparent free energy (AG°) for the human and bovine factor Xa:BPTI association were calculated, at 21.()°C. from values of Kj. according to the expression'21': AC 0 --2.3 x Rx Tx l
(3)
Values of the apparent enthalpy variation (Δ//°) accompanying the human and bovine factor Xa:BPTI complex formation were determined from the dependence of log Kaon l/Tby van't Hoff plots (see Fig. 1, inset), according to the expression'2'1: Δ//° = - 2.3 x R x [d log KJ(d\IT)]
(4)
Values of the apparent entropy variation (Δ5°) for the human and bovine factor Xa:BPTI complex formation were calculated, at 21.0 °C. from values of AG° and Δ//°. according to the expression' 21 ':
(5) Average error values of ± 8% (for Ka and ΔΟ° values) and ± 12% (for Δ//° and Δ5° values) were evaluated as the standard deviation"9·20". Data analysis was carried out on a Digital PDP 11/23 computer employing an iterative non-linear least-squares fitting procedure according to the Marquart algorithm.
Results and Discussion Under all the experimental conditions, BPTI binding to human and bovine factor Xa conforms to a simple equilibrium as indicated by the unitary value of the Hill coefficient (n = 1.00 ± 0.02). Moreover, Ka values were always independent of the enzyme concentration. As shown in Fig. 1, values of Ka for BPTI binding to human and bovine factor Xa are identical. This finding is in line with the undistinguishable catalytic properties of human and bovine factor Xa[22], as well as with the identity of their amino acid sequence around the catalytic site and the substrate/inhibitor recognition region(s)'23^. The pH dependent change in affinity of BPTI binding to human and bovine factor Xa shown in Fig. 1, is strictly reminescent of those obtained for the binding of Kazal- and/or Kunitz-type inhibitors to bovine aand /3-trypsin, bovine trypsinogen, bovine a-chymotrypsin, porcine pancreatic -kallikreins A and B, human urinary kallikrein, human Lys77-plasmin, human leukocyte elastase and human urokinase (33000 Mr and 54000 Mr species)[12'24'25], and thus it may be described with the same model.The decrease
σ> ο
3.2 3.4 3.6 1/Τχ103 (Κ'1)
5
6
7 pH
8
9
Fig. 1. pH dependence of the apparent association equilibrium constant (ΚΆ [M~']) for BPTI binding to human (O) and bovine (D) factor Xa, at 21.0 °C. The unbroken line was generated from Eqn 6 using C = 5.36, P/CUNL = 7.1 and pKUc — 5.2; the parameters were obtained with an iterative non-linear least-squares fitting procedure. An average error value of ± 12% was evaluated for /CUNL and Α^,ιο values, as the standard deviation, according to the fitting procedure. The inset shows the van't Hoff plot describing the temperature dependence, between 5.0 °C and 45.0 °C, of Ka [vT1] for the human (O) and bovine (D) factor Xa:BPTI complex formation at pH 8.O. From the slope of this plot, the apparent ΔΗ° value (= + 17.6 kJ/mol) was determined' 21 '. Next, the temperature independence of the apparent Δ//° value indicates that the apparent Δ(Γ£ value is very close to 0 kJ/mol'21'. For further details see text.
in Ka values (i.e., in affinity) for BPTI binding to human and bovine factor Xa, on lowering pH from 9.5 to 4.5, can be accounted for by the acidic ρ Κ shift of a single ionizing group upon proteinase:inhibitor complex formation. According to linkage relations{26], this model leads to the following expression: (6)
where C is a constant that corresponds to the alkaline asymptote of log Ka, and /MJNL and KUG are the apparent proton dissociation equilibrium constants for the BPTI-free and the BPTI-bound human and bovine factor Xa. Eqn 6 has been used to generate the unbroken line shown in Fig. 1; the agreement with the experimental data is fully satisfactory. Molecular data and amino-acid sequence homologles[i,23.27] anow to SUggest that the ionization process responsible for the observed pH effects refers only to the enzyme side. In fact, BPTI amino acid side chains, which might be expected to ionize over the explored pH range (i.e., between pH 4.5 and 9.5), do not lie near the proteinase/inhibitor contact area. Next, the pH profile for BPTI binding to human and bovine factor Xa is strongly similar to those published for the interaction of Kazal- and Kunitz-type inhibitors with different homologous serine proteinases Brought to you by | University of Arizona Authenticated Download Date | 5/31/15 10:19 PM
392
P. Ascenzi, M. Coletta, G. Amiconi, M. Bolognesi, E. Menegatti and M. Guarneri
and their zymogens, in which the only relevant ionizing group has been identified on the (pro)en-
zyme[12,13,24.25]_
Inspection of the different active site residues capable of affecting BPTI binding to human and bovine factor Xa suggests that only the His57 catalytic residue'23·271 has a pK comparable to the p/C UNL in the free proteinase (see Fig. 1). By analogy with what has been reported in related (pro)enzyme/inhibitor systems'12'24·251, the observed one-proton acidic pK shift could be interpreted as reflecting the burial of the enzyme active site residues upon the proteinase:BPTI complex formation with the concomitant strengthening of the proteinase Serl95 OG - His57 NE2 intramolecular hydrogen bond; this interaction is very weak, or absent, in the homologous inhibitor-free proteinasest13·23·271. At pH 8.0, values of the apparent thermodynamic parameters for BPTI binding to human and bovine factor Xa are: K.a = 2.1 x lOV (at 21.0 °C), AG° = -29.7kJ/mol(at21.0 0 C),A t $°= + 161 entropy units (at 21.0 °C), and Δ/¥° = + 17.6 kJ/mol (temperatureindependent over the explored range, from 5.0 °C to 45.0 °C) (see Fig. 1, inset). Both the apparent ΔΗ° and Δ5° values for BPTI binding to human and bovine factor Xa are comparable to those reported for the association of Kazal- and Kunitz-type inhibitors to serine proteinases and their zymogens, and indicate that the complex formation is an entropy-driven process. Next, the apparent positive AS10 values could reflect the removal of the proteinase- and/or inhibitorbound water molecules during complexation^12'13·281. By comparison with other plasma proteinases, the affinity of BPTI for human and bovine factor Xa is lower than that observed for the formation of the plasmin: and kallikrein:BPTI complexes by about three and four orders of magnitude, respectively. On the other hand, the affinity of BPTI for human and bovine factor Xa is about two orders of magnitude higher than that reported for the inhibitor association to thrombin[1(U2-2(K291. Such functional behaviour is likely to be related to the structural differences occurring at the plasma proteinase/BPTI contact area; in particular, regions 20-30, 45-54, 88-93, 139-152, 167-177, 190-198, 215-224 and 227-232 show sequence hypervariability, different dimensions and structures. Thus, such domains can be considered as potential sites exerting different levels of steric hindrance to the inhibitor access and (de)stabilization of the enzyme:inhibitor complex'13·23'27'29"311. Finally, from the physiophathological viewpoint, the reported data obtained in vitro (Ka~l = 4.8 x 10~6M; see above), together with the normal plasmatic levels of factor Xa (= 1.8 x 10~8M[2]) and of the Kunitz-type
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proteinase inhibitors (= 1.5 x 10 8 M [yl ), as well as of the therapeutical concentration of BPTI (~ 1 x 10~6Μ'10·η1) for a wide variety of pathophysiological states, thought to be associated with an increase of proteinase activity, do not favour the hypothesis of any interaction of human factor Xa with BPTI and BPTI-like inhibitors. On the other hand, such a behaviour appears to be of interest in relation to a rationale for the therapeutic action of BPTI.
References 1 2 3 4 5 6 7 8
9 10 11 12
13
14 15 16 17 18 19
Jackson, C.M. & Nemerson, Y. (1980) Annu. Rev. Biochem. 49,765-811. Furie, B. & Furie, B.C. (1988) Cell S3, 505-518. Mann, K.G., Jenny, R.J. & Krishnaswamy, S. (1988) Annu. Rev. Biochem. 57, 915-956. Heidtmann, H. &Travis, J. (1986) in Proteinase Inhibitors (Barrett, A.J. & Salvesen, G., eds.) pp. 441-456, Elsevier, Amsterdam, New York, Oxford. Lijnen, H.R. & Collen, D. (1986) in Proteinase Inhibitors (Barrett, A.J. & Salvesen, G., eds.) pp. 457-476, Elsevier, Amsterdam, New York, Oxford. Bj rk, I. & Danielsson, A. (1986) in Proteinase Inhibitors (Barrett, A.J. & Salvesen, G., eds.) pp. 489-513, Elsevier, Amsterdam, New York, Oxford. Cawston, T.E. (1986) in Proteinase Inhibitors (Barrett, A.J. & Salvesen, G., eds.) pp. 589-610, Elsevier, Amsterdam, New York, Oxford. Ascenzi, P., Coletta, M., Amiconi, G., De Cristofaro, R., Bolognesi, M., Guarneri, M. & Menegatti, E. (1988) in Proteine '88, (Balestrieri, C., Brunori, M., DAlessio, G., Di Frisco, G., Fontana, A., Ronchi, S. & Rotilio, G., eds.) pp. 154-155, Poligrafica F.lli Ariello, Editori s.a.s., Napoli. Fioretti, E., Angeletti, M., Citro, G., Barra, D. & Ascoli, F. (1987) J. Biol. Chem. 262, 3586-3589. Gebhard, W., Tschesche, H. & Fritz, H. (1986) in Proteinase Inhibitors (Barrett, A.J. & Salvesen, G., eds.) pp. 375-388, Elsevier, Amsterdam, New York, Oxford. Schnebli, H.-P. & Braun, N.J. (1986) in Proteinase Inhibitors (Barrett, A.J. & Salvesen, G., eds.) pp. 613-627, Elsevier, Amsterdam, New York, Oxford. Amiconi, G., Ascenzi, P., Bolognesi, M., Menegatti, E. & Guarneri, M. (1988) in Macromolecular Biorecognition: Principles and Methods (Chaiken, I., Chiancone, E., Fontana, A. & Neri, P., eds.) pp. 117-130,The Humana Press, Clifton. Bolognesi, M., Ascenzi, P., Amiconi, G., Menegatti, E. & Guarneri, M. (1988) in Macromolecular Biorecognition: Principles and Methods (Chaiken, I., Chiancone, E., Fontana, A. & Neri, P., eds.) pp. 81-100,The Humana Press, Clifton. Fujikawa, K. & Davie, E.W. (1976) Methods Enzymol. 45, 89-95. Jesty, J. & Nemerson, Y. (1976) Methods Enzymol. 45, 95-107. Robison, D.J., Furie, B., Furie, B.C. & Bing, D.H. (1980) J. Biol. Chem. 255, 2014-2021. Ascenzi, P., Bertollini, A.,Verzili, D., Brunori, M. & Antonini, E. (19SO) Anal. Biochem. 103,235-239. Ascenzi, P., Sleiter, G. & Antonini, E. (1982) Gazz. Chim. Ital. 112, 307-317. Ascenzi, P., Coletta, M., Amiconi, G., Bolognesi, M., Guarneri, M. & Menegatti, E. (1988)7. Mol. Recognition 1, 130-137.
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Binding of the Kunitz Inhibitor to Human and Bovine Factor Xa
20 Ascenzi, P., Coletta, M., Amiconi, G., De Cristofaro, R., Bolognesi, M., Guarneri, M. & Menegatti, E. (1988) Biochim. Biophys. Acta 956, 156-161. 21 Keleti,T. (1983) Biochem. J. 209, 277-280. 22 Lottenberg, R., Christensen, U., Jackson, C.M. & Coleman, P.L. (1981) Methods Enzymol. 80, 341-361. 23 Fung, M.R., Hay, C.W. & MacGillavray, R.T.A. (1985) Proc. Nail. Acad. Sei. U.S.A. 82, 3591-3595. 24 Fioretti, ., Angeletti, M., Passed, D. & Ascoli, F. (1989) J. Protein Chem. 8, 51-60. 25 Ascenzi, P., Amiconi, G., Bolognesi, M., Menegatti, E. & Guarneri, M. (1990)7. Enzyme Inhibition, in press.
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26 Wyman, J. (1964) Adv. Protein Chem. 19, 223-286. 27 Furie, B., Bing, D.H., Feldmann, R.J., Robison, D.J., Burnier, J.P. & Furie, B.C. (1982) J. Biol. Chem. 257, 3875-3882. 28 Amiconi, G., Ascenzi, P., Bolognesi, M., Guarneri, M. & Menegatti, E. (1987) Adv. Biosciences65, 177-180. 29 Berliner, L.J., Birktoft, J.J., Miller, T.L., Musci, G., Scheffler, J.E., Shen,Y.Y. & Sugawara,Y. (l9S6)Ann. NY Acad. Sei. 485, 80-95. 30 Chen, Z. & Bode,W. (1983)7. Mol. Biol. 164, 283-311. 31 Creighton,T.E. & Darby, N.J. (1989) Trends Biochem. Sei. 14, 319-324.
Paolo Ascenzi*, Massimo Coletta and Gino Amiconi, C.N.R. Centre di Biologia Molecolare, Dipartimento di Scienze Biochimiche, Universitä di Roma "La Sapienza", Piazzale Aldo Moro 5,1-00185 Roma, Italia. Martino Bolognesi, Dipartimento di Genetica e Microbiologia, Sezione di Cristallografia, Universitä di Pavia, ViaTaramelli 16,1-27100 Pavia, Italia. Enea Menegatti and Mario Guarneri, Dipartimento di Scienze Farmaceutiche, Universitä di Ferrara, Via Scandiana 21,1-44100 Ferrara, Italia. 'f To whom correspondence should be sent.
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