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Electrochemical activation of human factor XII (Hageman factor) immobilized on carbon electrodes
Anulyrica ckmica AC& 2J33(1993) 719-726 Elsevier Science Publishers B.V., Amsterdam
719
Electrochemical activation of human factor XII ( Hageman factor) immobilized on carbon electrodes Hyacinthe Randriamahazaka
and Jean-Max&e
Nigretto
Lboratoire d’Eiectrochimieet des Mathiau Appliquh, Universitti Cergy-Pontoise,B.P. 8428, 958M Cergy-PontoiseCedcx (France) (Received 12th March 1993; revised manuscript received 17th May 1993)
AhStIWt
Direct potential-mediated activation of purified human factor XII @XII) immobilized on carbon electrodes has been evidenced. This required first to find a procedure for the immobilization of FXII on the electrode with good reproducibility. The adsorption isotherms of this xymogen and those of the enxymatic form, FXIIa, were characterized and discussed. The catalytic activities were estimated under stirring with a specific substrate (S-2302) provided with chromogenic properties and dissolved in the solution. Pretreatment of the electrode with negative square-wave potential pulses resulted in changes in the adsorption capacity of the carbon surface towards FXIIa. When the untreated electrode was covered with FXII at saturation, application of these pulses generated catalytic activities according to first-order kinetics. Keyword: Enxymatic methods; UV-Visible spectrophotometry; Adsorption isotherms; Carbon electrodes; Electrochemical activation; Human factor XII; Immobiliiation
The contact system of human plasma consists of the zymogens factor XII (FXII; Hageman factor), prekallikrein (PK), factor XI and the nonenzymatic cofactor, high molecular weight kininogen @IMWK) [1,21. Upon contact of plasma with negatively charged surfaces, a series of reactions takes place in which surface-assembled zymogens are converted into active proteases. These events initiate coagulation [3], kinin release [4] and fibrinolysis [5]. Surface-bound FXII plays a central role in these reactions. Recently, it has been shown that the binding of FXII to a negatively charged surface occurs via a 20-residue sequence in the
Correspondence to: J.-M. Nigretto, Laboratoire d’Electrochimie et des Mat&iaux Appliques, Universitd Cergy-Pontoise, B.P. 8428,95806 Cergy Pontoise Cedex (France).
heavy-chain portion of FXII. The binding induces self-generated conformational changes in the zymogen molecule which thereby exhibits catalytic properties attributed to the active form FXIIa 161. Under physiological conditions, i.e. in human plasma, a kinetic loop of amplification then takes place. It begins with the FXIIa-catalyzed conversion of PK to kallikrein by cleavage of a single peptide bond [7]. The rate of this reaction is considerably increased by I-IMWK. In turn, kallikrein restrains the self-activation of the remaining surface-bound FXII by cleavage of a peptide bond in FXII via a second-order reaction. Under artificial conditions, however, the mechanism of FXII activation is different. It proceeds via a second-ordered autoactivation reaction, in which the zymogen form plays the role of the substrate with respect to its own enzyme
0003~2670/93/$06.00 0 1993 - Elsevier Science Publishers B.V. All rights reserved
720
H. Randriumahazakraand J.-M. Nig&to/A~l.
form. The role of the surface in the surface-assembly of the contact factors will receive considerable interest in the forthcoming literature. Conformational changes affecting the zymogen form are believed to coincide with an increased susceptibility to proteolytic cleavage of single-chain surface-bound FXII [8]. The extent of contact activation was examined in the presence of various negatively charged surfaces, a prerequisite to induce contact activation: urate crystals [9], kaolin, glass [lO,ll], ellagic acid [12,13], phospholipids [14,15], cholesterol sulphates [16], sulphatides (cerebrosides) 1171 or polysaccharide sulphates (namely dextran sulphate) [18]. Thus, under artificial conditions, it is not surprising that heparin, a powerful anticoagulant, may also display a procoagulant behaviour due to the polyanionic structure 1191. An explicit model for the second-order autoactivation of FXII in the presence of dextran sulphate of MW 500 000 was developed by Tankersley and Finlayson [201 and recently discussed by Silverberg et al. [19]. Both the rate constant and the extent of the biological modification of FXIIa were found to depend on the MW of the activating material [22]. It should, however, be stressed that though the kinetics of the main autoactivation reaction seem by now almost completely elucidated, much controversy remains concerning the origin of the initial traces of FXIIa. To explain the presence of these first traces, most reports attributed the triggering of the autoactivation to the presence of contaminating proteins, such as residual serine proteases or zymogens (e.g. PK) due to incomplete purification of FXII D71. Electrochemically modified carbon surfaces are known to promote irreversible binding of enzymes, generally with limited loss of activity. In this laboratory, we gained some experience in examining the catalytic behaviour of another blood serine protease, thrombin, which was also immobilized on the surface of the carbon-paste electrode. Catalytic activities could be characterized in the adsorbed state [23,24]. We shall show here that this technique can be successfully applied to characterize the early kinetic steps of FXII autoactivation.
Chim. Acta 283 (1993) 719-726
EXPERIMENTAL
Chemicals and materials For the buffer solutions (Tris buffer (50 mM, Carlo Erba); NaCl (125 r&I), pH 8.5 (HCI)) Millipore Bioblock filtered ultra-pure water was used. Purified FXII was prepared according to a described procedure [25]. FXIIa was generated from FXII stock solutions after achieving full autoactivation with an excess of dextran sulphate in ca. 10 min [183. Factor XII-deficient plasma as well as diagnostic kits for AP’IT (activated partial prothrombin time) measurements were purchased from Diagnostica Stago (France). Lyophilized kallikrein (0.1 unit/ mg) was from Sigma (France). The chromogenic substrate used for the spectrophotometric assay of FXII or kalhkrein (H-DPro-Phe-Arg-p-nitroaniline 2 HCl, denoted S2302) was from Kabi-Vitrum AB (Sweden). T500 (MW 500000 D) dextran sulphate (DS) used as the activator of FXII was from Pharmacia (Sweden). All other chemicals were analyticalgrade reagents. Graphite carbon powder (Ref. 9900 207-16594) was kindly provided by So&C Le Carbone Lorraine, Gennevilliers, France. The carbon paste was made of 15 g of graphite mixed with 4,5 ml of nujol (Merck). The homogeneous paste was compacted in a Teflon@ cavity and smoothened mechanically on a sheet of paper. Electrochemical measurements Electrochemical measurements were performed at 25°C using a potentio-dynamic set-up in a three-electrode configuration, including a PAR (Princeton Applied Research) galvanostat/ potentiostat Model 173, driven by a PAR 175 wave generator. The one-compartment Teflon (to limit loss of enzyme through adsorption that would occur on glass) electrochemical cell supported the carbon-paste electrode (CPE), a saturated calomel electrode (SCE) and a platinum auxiliary electrode. The electrochemical area of the CPE was measured in 0.51 M H,SO, by chronoamperometric treatment (following the Cottrell equation) of the oxidation signal of a known redox system, paminophenol, which involves an exchange of two
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H. Randriamahazah and&M. Nigretto /Anal. Chim. Acta 283 (1993) 719-726
Dextran sulphate was unavoidably present in the FXIIa stock solution. Accordingly, a possible interaction of this activating surface in the adsorption process could be suspected. To check this point, we enzymatically activated FXII with a low amount of the physiologic enzyme of FXII, i.e. kallikrein, instead of DS. The concentration was adjusted so as to prevent S-2302 from significant hydrolysis as compared to that due to FXIIa (the substrate is also sensitive to kallikrein). Moreover, the reaction time was lengthened enough to ensure total conversion of the initial amount of FXII to FXIIa. Under those conditions, results showed that the saturation plateau was enhanced up to about 3.5 f 0.2 X lo-i3 mol cm-‘. In other experiments, the effect of the prepolarization of the CPE prior to the adsorption of FXIIa was studied in the presence of DS. Direct measurement of the rest potential (the equilibrium potential taken by the modified electrode dipped in the buffer solution in absence of any current) was first made to know the initial value of the potentials to be applied further. Thereafter, the procedure consisted of applying during
0 E (V. vs SCE) Fig. 2. Potential-mediated adsorption isotherm of FXIIa on the pretreated carbon paste electrode. The treatment consisted of square-wave pulses of increasing negative potentials (vs. SCE) applied during 10 min to the CPE and prior to adsorption.
10 min one square-wave potential signal, from the rest potential potential (- 0.27 V vs. SCE) of the modified electrode to values ranging up to - 1.0 V (the cathodic discharge potential). Results presented in Fig. 2 indicate that negative polarization favored adsorption. Adsorption of FXII on different CPE electrode surfaces Experiments conducted likewise with unactivated FXII led to an irreversible adsorbtion in 30 minofamaximumof7.5f0.5X10-‘3molcm-2 of zymogen on untreated or prepolarized surfaces. At this time, it was important to point out that in any case the immobilized FXII was totally exempted from residual catalytic activity. Heterogeneous activation of immobilized FXII could only be revealed with DS present in the solution.
Time (min.) Fig. 1. Adsorption isotherm (n = 3) of preactivated FXIIa on the carbon paste electrode. FXIIa activities were determined by monitoring during 10 min the release in the solution of p-nitroaniline split by the immobilized enzyme from a specific substrate (see Experimental). Rates of the absorbance changes per minute were converted into FXIIa concentrations from the standardization of FXIIa in homogeneous solution.
Electrochemical activation of surface-bound FXIZ We found that the application of potentials below -0.27 V vs. SCE (mean zero current potential) during 10 min to FXII-modified electrodes developed catalytic activities at the solution/ modified electrode interface, as evidenced by the data given in Fig. 3. Maximum activity
H. Randriamahazah and J.-M. Nigretto /Anal
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E (V. vs SCE) Fig. 3. Potential-mediated activation of FXII immobilized on the untreated CPE. The signals consisted of square-wave pulses of increasing amplitudes in the negative range.
occurred at ca. -0.5 V vs. SCE, before decreasing upon a cathodic shift of the potential. The effect of the polarization time is represented in Fig. 4. The maximum amount of electro-activatable FXII was attained after ca. 30 min of contact. Separate experiments aimed at examining the possibility for Faradaic exchanges to occur between the electrode and the enzyme or zymogen
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Time (min.) Fig. 4. Kinetics of the electrogeneration of FJXIIaon the CPE. (a) Variation of the superficial density of FXIIa as a function of time. (b) Integration according to first order kinetics.
723
layer were undertaken. Cyclic voltammograms, conducted with the FXII-modified electrode over the pertinent potential range, showed, as expected due to the modification of the surface, increases in the charging current but no apparent Faradaic current as compared to curves obtained with a bare surface [2S]. Otherwise, we noted that the zero current potential remained unaffected whether the surface was modified or not. The shape of the time-dependent activation curve of FXII (Fig. 4, curve a) prompted us to analyze it according to a first-order formalism (Fig. 4, curve b). The resulting linear relationship led to calculate under these conditions an apparent heterogeneous rate constant of 0.04 mm-‘. Under the condition of a heterogeneous firstorder activation, there is no collision between the surface and the surface-bound zymogen since the process occurs locally. The dimension of the rate constant should therefore be expressed adequately without surface units. The data also showed that the maximal amount of FXIIa which was formed in 30 min under the appropriate conditions tended to be 5.0 f 0.1 x lo-l3 mol cm-’ since the maximal adsorption ability of the surface with respect to FXII was estimated to be 7.5 f 0.1 x lo-l3 mol cm-‘. The yield of the electrochemically-induced catalytic activity was accordingly estimated to be ca. 66%. Biological functionality of electro-activated FxIIa The existence of specific activity towards synthetic substrates does not unambiguously ascertain the biological integrity of the electrogenerated FXIIa since the distance from the catalytic site (in the a-FXIIa form) to the residues involved in adsorption is expectably long. We therefore tested the functional integrity of this adsorbed enzyme with biological substrates, i.e. in the presence of human plasma. This was achieved by measurement of the AFTI clotting time (see Experimental) produced by the surface-bound FXIIa in normal plasma samples. Since the amount of the electro-activated FXIIa on the electrode was rather low as compared to that involved in the conventional assay (ca. 0.3 PM), contact with plasma was prolonged up to 30
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H. Randriamahazaka and J.-M. Nigretto /Anal. 0th. Acta 283 (1993) 719-726
min. Past this duration, the normal clotting time value of 50 s was recovered. By using FXII-deficient instead of normal plasma, the same value was obtained, thus confirming the biological functionality of the electrogenerated enzyme.
DISCUSSION AND CONCLUSION
Adsorption of the proteins on carbon surfaces The criteria for irreversible adsorption of a protein on a surface has been the subject of controversy in the literature. For some workers, an adsorption is considered to be irreversible if there is a change in protein conformation [28]. For others, adsorption is irreversible only when desorption is negligible or absent [29]. In the present instance, as desorption of either fixed FXIIa or FXII did not occur after adsorption and subsequent treatment of the modified electrode, it was concluded that these proteins were both irreversibly fixed to the carbon surfaces. The fact that the catalytic behaviour remained unaltered following adsorption suggested that the active site of the enzyme does not exhibit too strong interactions with the carbon surface. This observation corroborated the pattern of FXII adsorption on negatively charged surfaces, which was known to occur preferentially via the histidine-rich region of the carboxylate end, thus keeping the catalytic end free. As a result, the affinity for the proteins to adsorb would be higher on the carbon particles dispersed on the surface than for the hydrophobic nujol phase, since otherwise the potential-induced modification of the surface would remain ineffective. This behaviour paralleled that of thrombin, another serine protease involved in coagulation [23]. With regard to the enhanced capacity of the surface to adsorb proteins in the presence of DS, it cannot be excluded that the polyelectrolyte also binds the carbon surface. In homogeneous solutions, dextran sulphate-mediated auto-activation of FXII requires the formation of DS-FXII and DS-FXIIa complexes [21]. The extent of activation is both enzyme- and polymer-dependent. In the latter event, it may even be limited by exceed-
ing concentrations of polymer [30]. Inasmuch as these protein-polyelectrolyte assemblies are also effective on the surface by means of adsorption, there would be some propensity for the high molecular weight DS to develop locally electrostatic interactions and steric hindrance. This might explain why the mode of activation of the adsorbed FXII depended on the nature of the activator. When DS was used, it would be reasonable to assume that the negative charge density surrounding the FXIIa complexes was higher as compared to kallikrein. This hypothesis may account for the observed enhancement in the superficial density of protein, from 2.0 x lo-l3 to 3.5 x lo-l3 mol cm-’ whether the activation of FXII was carried out without kallikrein or with other DS-bound FXIIa molecules. The finding that electrochemical modification of the carbon surface affects the adsorption capacity of the electrode was not surprising. In the current literature, a great deal of reports have evidenced the generation of negatively charged groups on carbon surfaces following cathodic polarization [31]. Since our treatment was applied in the presence of air, the formation of negatively charged or of hydrophilic surface groups was straightforward, particularly beyond -0.5 V vs. SCE, which corresponds with the reduction potential of dissolved oxygen. The gradual (Fig. 2) or bell-shaped (Fig. 3) variation of the plateau observed in the FXII and FXIIa adsorption isotherms should, at least partially, reflect such modifications. Indeed, attributing the isotherm variations solely to these effects seems questionable since changes in the hydrophilic/ hydrophobic balance of the surface may also affect the surface properties of FXIIa. Electrogeneration of FXZZa from surface-bound FXZZ The most prominent finding in this study was
that negative potentials to immobilized FXII generated appreciable catalytic activity via time-dependent and potential-controlled processes. The absence of any Faradaic current upon negative potential scanning suggested that hypothetical reduction of disulphide bonds was not of concern. The potential-induced catalytic activities should
H. Randriamahazaka and J.-M. N&tto /Anal. Chitn. Acta 283 (1993) 719-7.26
rather account for other kinds of mechanisms. Being subjected to the high electric field prevailing at the electrode/solution interface, the immobilized FXII molecules might exhibit local conformational rearrangements or even rupture of electro-inactive hydrogen bonds [321. Also, eiectrocatalytic breaks of peptide bonds were supposed to explain the biomimetic activation of prothrombin on platinum electrodes [33]. Kinetic studies carried out during the compression or the expansion of protein monolayers indicated that the intramolecular moves of the chemical bonds followed a first order mechanism [34]. The behaviour of FXII induced by negative potentials may also be explained on theses basis. Indeed, it is known that the biological mechanism accounting for FXII activation in the alpha-form proceeds via the break of arginin-valin peptide bonds [35,36], irrespective of the activator which is used, kallikrein or FXIIa [37,38]. These breaks do not require electrons and the amino acids are devoid of electrochemical properties. Therefore and as observed, changes should not affect the traces of the cyclic voltammograms run with the modified electrode. We also noted a decrease in FXIIa catalytic activity when potentials less than - 0.5 V vs. SCE were applied to the FXII-modified electrode. Since the optimal activity coincided with the reduction half-wave potential of oxygen as evoked above, the decrease may be attributed to the electrogeneration of oxygen radicals on the electrode surface, which would damage the catalytic sites. Finally, we have shown that the electro-activation of FXII is a first-order reaction (Fig. 41, thus reinforcing the assumption of a heterogeneous mechanism of activation. To our knowledge, the description of the early stages of surface-bound FXII activation in the absence of an initiator had never been reported before. The reason for this might be that under homogeneous conditions the time lag during which the initial amounts of activated FXIIa have to be assayed is too short to afford precise determinations. However, under our conditions, the kinetics are considerably slowed down due to the heterogeneous state in which the proteins evolve.
725
This work was supported by Contrat Exteme No. 89 9012 from INSERM and from MRT-GBM Pale Grand Ouest.
REFERENCES 1 C.G. Gxhrane and J.H. Griffin, Adv. Immunol., 33 (1982) 241. A.P. Kaplan and M. Silverberg, Blood, 70 (1987) 1. O.D. Ratnoff, Thromb. Haemost., 43 (1980) 95. J. Margolis, J. Physiol., 144 (1958) 1. S. Niewiarowski and 0. Prou-Watelle, Thromb. Diath. Haemorrh., 3 (1959) 593. 6 R.A. Pbdey, L.G. Stumpo, K. Birkmeyer, L. Silver and R.W. Cohnan, J. Biol. Chem., 262 (1987) 10140. 7 B.N. Bouma, L.A. Miles, G. Beretta and J.H. Griffin, Biochemistry, 19 (1980) 1151. 8 J.H. Griffin, Proc. Natl. Acad. Sci. USA, 75 (1978) 1998. 9 M.H. Ginsberg, B. Jaques, C.G. Cochrane and J.H. Griifin, J. Lab. Clin. Med., 93 (1980) 497. 10 A.P. Kaplan, Prog. Hemost. Thromb., 4 (1979) 127. 11 J.H. Griffin and C.G. Cochrane, Semin. Thromb. Hemost., 4 (1979) 254. 12 O.D. Ratnoff and J.D. Crum, J. Lab. Clin. Med., 63 (1964) 359. 13 P.E. Block, K.R. Srinivasan and J.D. Shore, Biochemistry, 20 (1981) 7258. 14 M.A. Griep, K. Fujukawa and G.L. Nelsestuen, Biochemistry, 25 (1986) 6688. 15 I. Schousboe, Int. J. B&hem., 20 (1988) 309. 16 T. Shimada, H. Kato, S. Iwanaga, M. Iwamori and Y. Nagai, Thromb. Res., 38 (1985) 21. 17 G. Tans, J. Rosing and J.H. Griffin, J. Biol. Chem., 258 (1983) 8215. 18 C. Kluft, J. Lab. Clin. Med., 91 (1978) 83. 19 M. Silverberg and S.V. Diehl, Biochem. J., 248 (1987) 715. 20 D.L. Tankersley and J.S. Finlayson, Biochemistry, 23 (1984) 273. 21 M. Samuel, R.A. Piiey, M.A. Villanueva, R.W. Colman and G.B. Villanueva, J. Biol. Chem., 267 (1992) 1%91. 22 S. Tazi, G. Tans, H.C. Hemker and J.M. Nigretto, Thromb. Res., 67 (1992) 665 23 H. Randriamahazaka and J.M. Nigretto, Anal. Chim. Acta, 257 (1992) 247. 24 H. Randriamahazaka and J.M. Nigretto, Electroanalysis, 5 (1993) 231. 25 S. Tazi, PhD Thesis, University of Tours, 1989. 26 R.N. Adams, Electrochemistry at Solid Electrodes, Marcel Dekker, New York, 2nd edn., 1969, p. 220. 27 J. Caen, M.J. Larrieu and M. Samama, in L’Hhmostase. MCthodes d’Exploitation et Diagnostique Pratique, 2nd edn., Expansion Scientifique FranGaise, Paris, 1975, p. 339. 28 W. Norde, F. MacRitchie, G. Now&a and J. Lyklema, J. Colloid. Interface Sci., 112, (1986) 447.
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29 B.M.C. Chan and J.L. Brash, J. Colloid Interface Sci., 82 (1981) 217. 30 C. Loiseau, H. Randriamahaaaka and J.M. Nigretto, unpublished results. 31 K. Kinoshita, in Carbon. Electrochemical and Physicochemical Properties, Wiley Interscience, New York, 1987, p. 359. 32 F. Mac Ritchie, Adv. Protein Chem., 32 (1978) 283. 33 H. Durliat, C. Davet and M. Comtat, J. Electrochem. Sot., 132 (1985) 2310.
34 F. Mac Ritehie, J. Colloid Sci., 18 (1963) 555. 35 K. Fujikawa, KA. Walsh and E.W. Davie, Biochemistry, 16 (1977) 2270. 36 K. Fujikawa, K. Kuraehi and E.W. Davie, Biochemistry, 16 (1977) 4182 37 K Fujikawa, R.L. Heinmark, K Kurachi and E.W. Davie, Biochemistry, 19 (1980) 1322. 38 G. Tans and J. Rosing, Semin. Thromb. Haemost., 13 (1987) 1.
719
Electrochemical activation of human factor XII ( Hageman factor) immobilized on carbon electrodes Hyacinthe Randriamahazaka
and Jean-Max&e
Nigretto
Lboratoire d’Eiectrochimieet des Mathiau Appliquh, Universitti Cergy-Pontoise,B.P. 8428, 958M Cergy-PontoiseCedcx (France) (Received 12th March 1993; revised manuscript received 17th May 1993)
AhStIWt
Direct potential-mediated activation of purified human factor XII @XII) immobilized on carbon electrodes has been evidenced. This required first to find a procedure for the immobilization of FXII on the electrode with good reproducibility. The adsorption isotherms of this xymogen and those of the enxymatic form, FXIIa, were characterized and discussed. The catalytic activities were estimated under stirring with a specific substrate (S-2302) provided with chromogenic properties and dissolved in the solution. Pretreatment of the electrode with negative square-wave potential pulses resulted in changes in the adsorption capacity of the carbon surface towards FXIIa. When the untreated electrode was covered with FXII at saturation, application of these pulses generated catalytic activities according to first-order kinetics. Keyword: Enxymatic methods; UV-Visible spectrophotometry; Adsorption isotherms; Carbon electrodes; Electrochemical activation; Human factor XII; Immobiliiation
The contact system of human plasma consists of the zymogens factor XII (FXII; Hageman factor), prekallikrein (PK), factor XI and the nonenzymatic cofactor, high molecular weight kininogen @IMWK) [1,21. Upon contact of plasma with negatively charged surfaces, a series of reactions takes place in which surface-assembled zymogens are converted into active proteases. These events initiate coagulation [3], kinin release [4] and fibrinolysis [5]. Surface-bound FXII plays a central role in these reactions. Recently, it has been shown that the binding of FXII to a negatively charged surface occurs via a 20-residue sequence in the
Correspondence to: J.-M. Nigretto, Laboratoire d’Electrochimie et des Mat&iaux Appliques, Universitd Cergy-Pontoise, B.P. 8428,95806 Cergy Pontoise Cedex (France).
heavy-chain portion of FXII. The binding induces self-generated conformational changes in the zymogen molecule which thereby exhibits catalytic properties attributed to the active form FXIIa 161. Under physiological conditions, i.e. in human plasma, a kinetic loop of amplification then takes place. It begins with the FXIIa-catalyzed conversion of PK to kallikrein by cleavage of a single peptide bond [7]. The rate of this reaction is considerably increased by I-IMWK. In turn, kallikrein restrains the self-activation of the remaining surface-bound FXII by cleavage of a peptide bond in FXII via a second-order reaction. Under artificial conditions, however, the mechanism of FXII activation is different. It proceeds via a second-ordered autoactivation reaction, in which the zymogen form plays the role of the substrate with respect to its own enzyme
0003~2670/93/$06.00 0 1993 - Elsevier Science Publishers B.V. All rights reserved
720
H. Randriumahazakraand J.-M. Nig&to/A~l.
form. The role of the surface in the surface-assembly of the contact factors will receive considerable interest in the forthcoming literature. Conformational changes affecting the zymogen form are believed to coincide with an increased susceptibility to proteolytic cleavage of single-chain surface-bound FXII [8]. The extent of contact activation was examined in the presence of various negatively charged surfaces, a prerequisite to induce contact activation: urate crystals [9], kaolin, glass [lO,ll], ellagic acid [12,13], phospholipids [14,15], cholesterol sulphates [16], sulphatides (cerebrosides) 1171 or polysaccharide sulphates (namely dextran sulphate) [18]. Thus, under artificial conditions, it is not surprising that heparin, a powerful anticoagulant, may also display a procoagulant behaviour due to the polyanionic structure 1191. An explicit model for the second-order autoactivation of FXII in the presence of dextran sulphate of MW 500 000 was developed by Tankersley and Finlayson [201 and recently discussed by Silverberg et al. [19]. Both the rate constant and the extent of the biological modification of FXIIa were found to depend on the MW of the activating material [22]. It should, however, be stressed that though the kinetics of the main autoactivation reaction seem by now almost completely elucidated, much controversy remains concerning the origin of the initial traces of FXIIa. To explain the presence of these first traces, most reports attributed the triggering of the autoactivation to the presence of contaminating proteins, such as residual serine proteases or zymogens (e.g. PK) due to incomplete purification of FXII D71. Electrochemically modified carbon surfaces are known to promote irreversible binding of enzymes, generally with limited loss of activity. In this laboratory, we gained some experience in examining the catalytic behaviour of another blood serine protease, thrombin, which was also immobilized on the surface of the carbon-paste electrode. Catalytic activities could be characterized in the adsorbed state [23,24]. We shall show here that this technique can be successfully applied to characterize the early kinetic steps of FXII autoactivation.
Chim. Acta 283 (1993) 719-726
EXPERIMENTAL
Chemicals and materials For the buffer solutions (Tris buffer (50 mM, Carlo Erba); NaCl (125 r&I), pH 8.5 (HCI)) Millipore Bioblock filtered ultra-pure water was used. Purified FXII was prepared according to a described procedure [25]. FXIIa was generated from FXII stock solutions after achieving full autoactivation with an excess of dextran sulphate in ca. 10 min [183. Factor XII-deficient plasma as well as diagnostic kits for AP’IT (activated partial prothrombin time) measurements were purchased from Diagnostica Stago (France). Lyophilized kallikrein (0.1 unit/ mg) was from Sigma (France). The chromogenic substrate used for the spectrophotometric assay of FXII or kalhkrein (H-DPro-Phe-Arg-p-nitroaniline 2 HCl, denoted S2302) was from Kabi-Vitrum AB (Sweden). T500 (MW 500000 D) dextran sulphate (DS) used as the activator of FXII was from Pharmacia (Sweden). All other chemicals were analyticalgrade reagents. Graphite carbon powder (Ref. 9900 207-16594) was kindly provided by So&C Le Carbone Lorraine, Gennevilliers, France. The carbon paste was made of 15 g of graphite mixed with 4,5 ml of nujol (Merck). The homogeneous paste was compacted in a Teflon@ cavity and smoothened mechanically on a sheet of paper. Electrochemical measurements Electrochemical measurements were performed at 25°C using a potentio-dynamic set-up in a three-electrode configuration, including a PAR (Princeton Applied Research) galvanostat/ potentiostat Model 173, driven by a PAR 175 wave generator. The one-compartment Teflon (to limit loss of enzyme through adsorption that would occur on glass) electrochemical cell supported the carbon-paste electrode (CPE), a saturated calomel electrode (SCE) and a platinum auxiliary electrode. The electrochemical area of the CPE was measured in 0.51 M H,SO, by chronoamperometric treatment (following the Cottrell equation) of the oxidation signal of a known redox system, paminophenol, which involves an exchange of two
722
H. Randriamahazah and&M. Nigretto /Anal. Chim. Acta 283 (1993) 719-726
Dextran sulphate was unavoidably present in the FXIIa stock solution. Accordingly, a possible interaction of this activating surface in the adsorption process could be suspected. To check this point, we enzymatically activated FXII with a low amount of the physiologic enzyme of FXII, i.e. kallikrein, instead of DS. The concentration was adjusted so as to prevent S-2302 from significant hydrolysis as compared to that due to FXIIa (the substrate is also sensitive to kallikrein). Moreover, the reaction time was lengthened enough to ensure total conversion of the initial amount of FXII to FXIIa. Under those conditions, results showed that the saturation plateau was enhanced up to about 3.5 f 0.2 X lo-i3 mol cm-‘. In other experiments, the effect of the prepolarization of the CPE prior to the adsorption of FXIIa was studied in the presence of DS. Direct measurement of the rest potential (the equilibrium potential taken by the modified electrode dipped in the buffer solution in absence of any current) was first made to know the initial value of the potentials to be applied further. Thereafter, the procedure consisted of applying during
0 E (V. vs SCE) Fig. 2. Potential-mediated adsorption isotherm of FXIIa on the pretreated carbon paste electrode. The treatment consisted of square-wave pulses of increasing negative potentials (vs. SCE) applied during 10 min to the CPE and prior to adsorption.
10 min one square-wave potential signal, from the rest potential potential (- 0.27 V vs. SCE) of the modified electrode to values ranging up to - 1.0 V (the cathodic discharge potential). Results presented in Fig. 2 indicate that negative polarization favored adsorption. Adsorption of FXII on different CPE electrode surfaces Experiments conducted likewise with unactivated FXII led to an irreversible adsorbtion in 30 minofamaximumof7.5f0.5X10-‘3molcm-2 of zymogen on untreated or prepolarized surfaces. At this time, it was important to point out that in any case the immobilized FXII was totally exempted from residual catalytic activity. Heterogeneous activation of immobilized FXII could only be revealed with DS present in the solution.
Time (min.) Fig. 1. Adsorption isotherm (n = 3) of preactivated FXIIa on the carbon paste electrode. FXIIa activities were determined by monitoring during 10 min the release in the solution of p-nitroaniline split by the immobilized enzyme from a specific substrate (see Experimental). Rates of the absorbance changes per minute were converted into FXIIa concentrations from the standardization of FXIIa in homogeneous solution.
Electrochemical activation of surface-bound FXIZ We found that the application of potentials below -0.27 V vs. SCE (mean zero current potential) during 10 min to FXII-modified electrodes developed catalytic activities at the solution/ modified electrode interface, as evidenced by the data given in Fig. 3. Maximum activity
H. Randriamahazah and J.-M. Nigretto /Anal
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E (V. vs SCE) Fig. 3. Potential-mediated activation of FXII immobilized on the untreated CPE. The signals consisted of square-wave pulses of increasing amplitudes in the negative range.
occurred at ca. -0.5 V vs. SCE, before decreasing upon a cathodic shift of the potential. The effect of the polarization time is represented in Fig. 4. The maximum amount of electro-activatable FXII was attained after ca. 30 min of contact. Separate experiments aimed at examining the possibility for Faradaic exchanges to occur between the electrode and the enzyme or zymogen
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723
layer were undertaken. Cyclic voltammograms, conducted with the FXII-modified electrode over the pertinent potential range, showed, as expected due to the modification of the surface, increases in the charging current but no apparent Faradaic current as compared to curves obtained with a bare surface [2S]. Otherwise, we noted that the zero current potential remained unaffected whether the surface was modified or not. The shape of the time-dependent activation curve of FXII (Fig. 4, curve a) prompted us to analyze it according to a first-order formalism (Fig. 4, curve b). The resulting linear relationship led to calculate under these conditions an apparent heterogeneous rate constant of 0.04 mm-‘. Under the condition of a heterogeneous firstorder activation, there is no collision between the surface and the surface-bound zymogen since the process occurs locally. The dimension of the rate constant should therefore be expressed adequately without surface units. The data also showed that the maximal amount of FXIIa which was formed in 30 min under the appropriate conditions tended to be 5.0 f 0.1 x lo-l3 mol cm-’ since the maximal adsorption ability of the surface with respect to FXII was estimated to be 7.5 f 0.1 x lo-l3 mol cm-‘. The yield of the electrochemically-induced catalytic activity was accordingly estimated to be ca. 66%. Biological functionality of electro-activated FxIIa The existence of specific activity towards synthetic substrates does not unambiguously ascertain the biological integrity of the electrogenerated FXIIa since the distance from the catalytic site (in the a-FXIIa form) to the residues involved in adsorption is expectably long. We therefore tested the functional integrity of this adsorbed enzyme with biological substrates, i.e. in the presence of human plasma. This was achieved by measurement of the AFTI clotting time (see Experimental) produced by the surface-bound FXIIa in normal plasma samples. Since the amount of the electro-activated FXIIa on the electrode was rather low as compared to that involved in the conventional assay (ca. 0.3 PM), contact with plasma was prolonged up to 30
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H. Randriamahazaka and J.-M. Nigretto /Anal. 0th. Acta 283 (1993) 719-726
min. Past this duration, the normal clotting time value of 50 s was recovered. By using FXII-deficient instead of normal plasma, the same value was obtained, thus confirming the biological functionality of the electrogenerated enzyme.
DISCUSSION AND CONCLUSION
Adsorption of the proteins on carbon surfaces The criteria for irreversible adsorption of a protein on a surface has been the subject of controversy in the literature. For some workers, an adsorption is considered to be irreversible if there is a change in protein conformation [28]. For others, adsorption is irreversible only when desorption is negligible or absent [29]. In the present instance, as desorption of either fixed FXIIa or FXII did not occur after adsorption and subsequent treatment of the modified electrode, it was concluded that these proteins were both irreversibly fixed to the carbon surfaces. The fact that the catalytic behaviour remained unaltered following adsorption suggested that the active site of the enzyme does not exhibit too strong interactions with the carbon surface. This observation corroborated the pattern of FXII adsorption on negatively charged surfaces, which was known to occur preferentially via the histidine-rich region of the carboxylate end, thus keeping the catalytic end free. As a result, the affinity for the proteins to adsorb would be higher on the carbon particles dispersed on the surface than for the hydrophobic nujol phase, since otherwise the potential-induced modification of the surface would remain ineffective. This behaviour paralleled that of thrombin, another serine protease involved in coagulation [23]. With regard to the enhanced capacity of the surface to adsorb proteins in the presence of DS, it cannot be excluded that the polyelectrolyte also binds the carbon surface. In homogeneous solutions, dextran sulphate-mediated auto-activation of FXII requires the formation of DS-FXII and DS-FXIIa complexes [21]. The extent of activation is both enzyme- and polymer-dependent. In the latter event, it may even be limited by exceed-
ing concentrations of polymer [30]. Inasmuch as these protein-polyelectrolyte assemblies are also effective on the surface by means of adsorption, there would be some propensity for the high molecular weight DS to develop locally electrostatic interactions and steric hindrance. This might explain why the mode of activation of the adsorbed FXII depended on the nature of the activator. When DS was used, it would be reasonable to assume that the negative charge density surrounding the FXIIa complexes was higher as compared to kallikrein. This hypothesis may account for the observed enhancement in the superficial density of protein, from 2.0 x lo-l3 to 3.5 x lo-l3 mol cm-’ whether the activation of FXII was carried out without kallikrein or with other DS-bound FXIIa molecules. The finding that electrochemical modification of the carbon surface affects the adsorption capacity of the electrode was not surprising. In the current literature, a great deal of reports have evidenced the generation of negatively charged groups on carbon surfaces following cathodic polarization [31]. Since our treatment was applied in the presence of air, the formation of negatively charged or of hydrophilic surface groups was straightforward, particularly beyond -0.5 V vs. SCE, which corresponds with the reduction potential of dissolved oxygen. The gradual (Fig. 2) or bell-shaped (Fig. 3) variation of the plateau observed in the FXII and FXIIa adsorption isotherms should, at least partially, reflect such modifications. Indeed, attributing the isotherm variations solely to these effects seems questionable since changes in the hydrophilic/ hydrophobic balance of the surface may also affect the surface properties of FXIIa. Electrogeneration of FXZZa from surface-bound FXZZ The most prominent finding in this study was
that negative potentials to immobilized FXII generated appreciable catalytic activity via time-dependent and potential-controlled processes. The absence of any Faradaic current upon negative potential scanning suggested that hypothetical reduction of disulphide bonds was not of concern. The potential-induced catalytic activities should
H. Randriamahazaka and J.-M. N&tto /Anal. Chitn. Acta 283 (1993) 719-7.26
rather account for other kinds of mechanisms. Being subjected to the high electric field prevailing at the electrode/solution interface, the immobilized FXII molecules might exhibit local conformational rearrangements or even rupture of electro-inactive hydrogen bonds [321. Also, eiectrocatalytic breaks of peptide bonds were supposed to explain the biomimetic activation of prothrombin on platinum electrodes [33]. Kinetic studies carried out during the compression or the expansion of protein monolayers indicated that the intramolecular moves of the chemical bonds followed a first order mechanism [34]. The behaviour of FXII induced by negative potentials may also be explained on theses basis. Indeed, it is known that the biological mechanism accounting for FXII activation in the alpha-form proceeds via the break of arginin-valin peptide bonds [35,36], irrespective of the activator which is used, kallikrein or FXIIa [37,38]. These breaks do not require electrons and the amino acids are devoid of electrochemical properties. Therefore and as observed, changes should not affect the traces of the cyclic voltammograms run with the modified electrode. We also noted a decrease in FXIIa catalytic activity when potentials less than - 0.5 V vs. SCE were applied to the FXII-modified electrode. Since the optimal activity coincided with the reduction half-wave potential of oxygen as evoked above, the decrease may be attributed to the electrogeneration of oxygen radicals on the electrode surface, which would damage the catalytic sites. Finally, we have shown that the electro-activation of FXII is a first-order reaction (Fig. 41, thus reinforcing the assumption of a heterogeneous mechanism of activation. To our knowledge, the description of the early stages of surface-bound FXII activation in the absence of an initiator had never been reported before. The reason for this might be that under homogeneous conditions the time lag during which the initial amounts of activated FXIIa have to be assayed is too short to afford precise determinations. However, under our conditions, the kinetics are considerably slowed down due to the heterogeneous state in which the proteins evolve.
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This work was supported by Contrat Exteme No. 89 9012 from INSERM and from MRT-GBM Pale Grand Ouest.
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