Properties of chemically oxidized kininogens

Kininogens are multifunctional proteins involved in a variety of regulatory pro-cesses including the kinin-formation cascade, blood coagulation, fibrynolysis, inhibition of cysteine proteinases etc. A working hypothesis of this work was that the properties of kininogens may be altered by oxidation of their methionine residues by reactive oxygen species that are released at the inflammatory foci during phagocytosis of pathogen particles by recruited neutrophil cells. Two methionine-specific oxidizing reagents, N -chlorosuccinimide (NCS) and chloramine-T (CT), were used to oxidize the high molecular mass (HK) and low molecular mass (LK) forms of human kininogen. A nearly complete conversion of methionine residues to methionine sulfoxide residues in the modified proteins was determined by amino acid analysis. Production of kinins from oxidized kininogens by plasma and tissue kallikreins was significantly lower (by at least 70%) than that from native kininogens. This quenching effect on kinin release could primarily be assigned to the modification of the critical Met-361 residue adjacent to the internal kinin sequence in kininogen. However, virtually no kinin could be formed by human plasma kallikrein from NCS-modified HK. This observation sug-gests involvement of other structural effects detrimental for kinin production. In-deed, NCS-oxidized HK was unable to bind (pre)kallikrein, probably due to the modification of methionine and/or tryptophan residues at the region on the kininogen molecule responsible for the (pro)enzyme binding. Tests on papain inhibition by native and oxidized kininogens indicated that the inhibitory activity of kininogens against cysteine proteinases is essentially insensitive to oxidation.

Oxidation of proteins may occur in the organism wherever the concentration of suitable oxidants increases over the basal level. For example, at the inflammatory foci, a number of reactive oxygen species including superoxide anion and hydroxyl radicals, hydrogen peroxide, hypochlorous acid, etc. are released during fagocytosis of pathogen particles by recruited neutrophil cells (Babior, 1978;Li et al., 1996). On the protein molecules, the methionine and cysteine residues are particularly susceptible to oxidation by these destructive factors (Vogt, 1995). The oxidation of important regulatory proteins may have serious consequences for the homeostasis of the body. Spectacular examples of the effects exerted via protein methionine oxidation to methionine sulfoxide (MSO) include the inactivation of neutrophil elastase-controlling a 1 -proteinase inhibitor (Johnson & Travis, 1979;Ossanna et al., 1986), a 2 -macroglobulin (Reddy et al., 1994) and other plasma proteinase inhibitors (Swain & Pizzo, 1988), inactivation of numerous hormones including human growth hormone (Teh et al., 1987), ovine prolactin (Houghten & Li, 1976) or parathyroid hormone (Hong et al., 1986) but, on the other hand, activation of C5 component of human complement (Vogt et al., 1992), etc. A working hypothesis of this work was that mild oxidation of methionine residues might affect the properties of kininogens, multifunctional proteins involved in kinin formation, blood coagulation, fibrynolysis and inhibition of cysteine proteinases (Colman, 1994;Colman & Schmaier, 1997). Kinins (bradykinin and kallidin), universal mediators of inflammation, are bioactive peptides that are excised from kininogens by serine proteinases called kallikreins (Bhoola et al., 1992). Bradykinin (RPPGFSPFR) is produced from a high molecular mass form of human kininogen (HK) by plasma kallikrein (HPK). Formation of a tight complex of HK with prekallikrein (pre-HPK), a zymogen form of HPK, is essential for its activation (Colman & Schmaier, 1997;Joseph et al., 2001) and the active HPK remains tightly bound to HK. Another, low molecular mass form of kininogen (LK) cannot be processed by HPK but is a source of kallidin (KRPPGFSPFR) upon the action of a tissue-specific kallikrein (Margolius, 1998). In contrast to HK, LK does not posses any distinct kallikrein-binding domain.
In the primary structure of human kininogens, a methionine residue is adjacent to the N-terminus of the internal kallidin sequence. The hypothesis that the oxidation of this particular residue may affect the kinin production by kallikreins was recently tested with model peptides ISLMKRPPGFSPFRSSI and ISL(MSO)KRPPGFSPFRSSI (Kozik et al., 1998). Both types of kallikreins were essentially unable to process the kinin N-terminus in the MSO-containing peptide. It was additionally shown that HK that was oxidized with N-chlorosuccinimide (NCS) could hardly be processed by kallikreins. Unfortunately, no attempt to interpret that finding in terms of protein chemistry was made. Also, the effect of methionine oxidation in LK on kinin formation was not described, and the other possible influences of kininogen oxidation on the physiologically important properties of these multifunctional proteins were not characterized.
In this work we modified both forms of human kininogen with two methionine-oxidizing agents, NCS and chloramine-T (CT), determined the oxidation status of methionine-residues in the modified proteins and described in more detail the effects of kininogen oxidation on the kinin formation by kallikreins, on the complex formation between HK and HPK and on the cysteine proteinase (papain) inhibition by kininogens.
Chemical oxidation of kininogens. Kininogens (HK or LK) at a concentration of 2.5 mM in 0.05 M Hepes buffer pH 7.0, containing 0.15 M NaCl, were incubated with 0.625 mM NCS (added as a freshly prepared solution in the Hepes buffer) for 2 h at 37°C, with occasional stirring. Methionine (solution in the Hepes buffer) was then added to a concentration of 9 mM and the sample was further incubated for 0.5 h at 37°C. In some experiments, NCS concentration was varied (0.07 to 6.25 mM).
Modification of kininogens with CT (Schechter et al., 1975) was performed at the protein concentration of 2.5 mM in 0.05 M Tris/HCl buffer pH 8.5. CT (fresh solution in the same buffer) was added to a concentration of 0.57 mM (or varied within the range of 0.1-4 mM) and the sample was incubated for 20 min at room temperature with occasional stirring. Finally, the reaction was quenched with methionine (9 mM) for 10 min at room temperature.
Proteolytic processing of native or oxidized kininogens. Kininogens (1 mM) were treated with kallikreins (0.05 mM) in 0.05 M Hepes buffer pH 7.0, containing 0.15 M NaCl, at 37°C, with occasional stirring. After the desired time, aliquots of the incubation mixture were withdrawn, mixed with one-fifth volume of 1 M HCl and stored frozen until the HPLC analysis for kinin content.
In some experiment, NCS-modified HK was first pre-digested with HNE (0.02 mM) in the Hepes buffer for 2 h at 37°C before addition of HPK (0.05 mM) and further incubation at the same temperature.
Reversed-phase HPLC of kinins and related peptides. Bradykinin and kallidin released from kiniogens by kallikreins were determined by reversed-phase HPLC analysis on a Supelcosil LC-318 (ODS) column (4 mm2 50 mm) equipped with a Supelguard LC-318 cartridge precolumn (4 mm´20 mm), in a binary gradient developed between 0.1% TFA in water (solvent A) and 0.08% TFA in 80% acetonitrile (solvent B). The separations were performed at a flow rate of 1 ml/min at ambient temperature, with detection at 215 nm. The gradient applied was linear, starting from 10% B, with a slope of +1.5% B per minute. Solvents were filtered through 0.45 mm Nylon filters (Supelco) and degassed with helium. Samples were centrifuged for 2 min at 10 000 g at ambient temperature, and placed in autosampler vials from which, typically, 70-90 ml portions were withdrawn for injection. A Shimadzu liquid chromatograph series 10A VP was used that contained: (i) a high-pressure binary gradient forming solvent delivery system based on two LC-10AD VP pumps, (ii) a SIL-10AD VP autosampler, (iii) an SPD-10A VP two-channel variable wavelength spectrophotometric detector, (iv) an SCL-10A VP system controller, and (v) a Class VP (ver. 5.0) hardware/software package for system control and data acquisition/ analysis. Vol. 50 Chemical oxidation of kininogens Another reversed-phase HPLC system (Kozik et al., 1998) was applied for the identification of the peptide SL(MSO)KRPPGFSPFR in the samples of HK, sequentially digested with HNE and HPK. A LiChro-CART LiChrosphere 100 RP-18 250 mm´4 mm column (Merck) with a precolumn was applied. Solvent A was 0.1% TFA in water, solvent B was 0.1% TFA in 80% methanol. The separations were performed isocratically (53% B) at a flow rate of 1 ml/min at ambient temperature, with detection at 215 nm. The HPLC instrument used consisted of: (i) a Shimadzu DGU-14A solvent degasser, (ii) a Shimadzu low-pressure quaternary gradient solvent delivery unit (LC-9A HPLC pump and FCV-9AL proportioning valve), (iii) a Knauer model A0263 injector equipped with a 100 ml sample loop, (iv) a Merck-Hitachi L-4000A UV detector, and (v) Schimadzu Class VP (v. 4.0) hardware/software package for pump control, data acquisition and analysis.
Gel filtration of mixtures of pre-HPK with native or oxidized HK. Pre-HPK (1.5 mM) was incubated with native or oxidized HK (1 mM) in 0.05 M Hepes buffer pH 7.0, containing 0.15 M NaCl for 1 h and room temperature. Samples were filtered through Millex HV 0.45 mm filters with low protein binding Durapore membrane (Millipore, Bedford, U.S.A.) and subjected to high-resolution gel filtration on a Superdex-200 HR 10/30 column (Pharmacia, Uppsala, Sweden) eluted with 0.05 M sodium phosphate buffer pH 7.0 containing 0.15 M NaCl at a flow rate of 0.5 ml/min at ambient temperature. The chromatographic system used consisted of: (i) a Well Chrom K-1001 HPLC pump (Knauer, Germany), (ii) a Rheodyne 9725 injector equipped with a 200 ml sample loop, (iii) a UV-1 photometric monitor (Pharmacia), and (iv) Chromed PC software (Poznañ, Poland) for data acquisition and analysis. The column was calibrated with a Bio-Rad molecular mass standard mixture containing thyroglobulin (670 kDa), g-globulin (158 kDa), ovalbumin (44 kDa) and myoglobin (17 kDa).

Papain inhibition assay.
Papain (Sigma type III), supplied as a solution of 25 mg/ml in 0.05 M sodium acetate buffer pH 4.5, containing 0.01% thymol, was diluted 20-fold into 0.05 M sodium phosphate buffer pH 6.8, containing 10 mM EDTA and 5 mM cysteine. The sample was incubated for 30 min at 37°C for enzyme activation. The activated papain solution (5 ml), 2.5 mM native or oxidized kininogen solution (3-20 ml) and 0.05 M sodium phosphate buffer pH 6.8 with 10 mM EDTA and 5 mM cysteine added to the final volume of 200 ml were mixed in the wells of a 96-well flat-bottom NUNCLON cell culture microplate (Nalge Nunc, Denmark). The plate was incubated for 30 min at 37°C and then placed in the thermostated chamber of a Bio-Tek PowerWave X-Select scanning microplate reader (Winooski, VE, U.S.A.) set at the same temperature. After 5 min, 4 ml portions of BAPNA solution (50 mM in Me 2 SO) were added to each well and a continuous monitoring of absorbance at 410 nm was started. The mean reaction rate was calculated from the linear part of the time course of absorbance increase and was used as a measure of the enzyme activity. Amino acid analysis. Amino acid analysis was performed according to protocols described by Cohen et al. (1989). Briefly, protein samples were subjected to gas-phase hydrolysis in 6 M HCl at 115°C for 24 h and the amino acids released were converted to phenylthiocarbamyl (PTC) derivatives. PTC-amino acids were then analysed on a PicoTag 3.9´150 mm column (Waters, U.S.A.) using a Waters HPLC system equipped with a 490E-type UV/VIS detector and two 501-type pumps.
Modifications of the hydrolysis protocol were applied for the analysis of MSO and tryptophan contents. For determination of MSO, the samples were hydrolyzed in liquid phase using 4 M NaOH at 115°C for 24 h. For tryptophan analysis, the samples were hydrolyzed in liquid phase using 3 M mercaptoethane sulfonic acid at 115°C for 24 h. After hydrolysis the acid was neutralized by addi-tion of equal volume of 4 M NaOH. In both cases the resulting solutions were then evaporated on a vacuum concentrator, derivatized and analyzed as above.

Determination of the oxidation state of amino-acid residues in chemically oxidized kininogens
The results obtained from amino acid analysis of the samples of HK that were oxidized with 0.625 mM NCS (250 molar excess over protein) and then were subjected to hydrolysis under various conditions, are presented in Table 1. After standard acid hydrolysis, the amino-acid composition of oxidized HK was insignificantly different from that of the native protein. However, this method does not allow one to quantify the content of MSO that is converted with a variable yield to methionine during acid hydrolysis. Hence, samples of NCS-oxidized HK were also subjected to alkaline hydrolysis to separately analyze for MSO and methionine contents. In oxidized HK, the determined amount of MSO was at least 86% of the methionine content of native HK. No residual methionine was detected in oxidized HK. Similar results were obtained upon analysis of oxidized LK (not shown). These data suggest a nearly full conversion of methionine to MSO upon kininogen oxidation with NCS. Only slightly lower methionine oxidation extent (at least 70%) was determined in CT-oxidized HK (not shown). Another method of hydrolysis (in 3 M mercaptoethane sulfonic Vol. 50 Chemical oxidation of kininogens 757 acid) was applied to determine the content of tryptophan in native and NCS-modified kininogens. About 32% of Trp residues seemed to undergo NCS-mediated modification.
Although the results of amino acid analysis suggested complete oxidation of kininogen methionine residues by NSC, additional analysis was performed to confirm the oxidation state of Met-361, one adjacent to the N-terminus of the internal kallidin sequence in kininogen. A sample of NCS-oxidized HK was first digested with human neutrophil elastase (HNE) that had previously been reported to cleave the peptide bond of kininogen after Ile-358 (Sato & Nagasawa, 1988;Imamura et al., 2002). After further action of HPK that should expose the kinin C-terminus, the sample was analyzed in a specifically dedicated reversed-phase HPLC system. On the chromatograms obtained (Fig. 1) a species is visible with the retention time identical to that of the peptide SL(MSO)KRPPGFSPFR that is generated upon a short treatment of the peptide SL(MSO)KRPPGFSPFRSSRI with HPK. These data suggest, at least qualitatively, that Met-361 is oxidized in NCS-oxidized HK.

Kinin formation from chemically oxidized kininogens by plasma-and tissue kallikreins
Time courses for bradykinin production from native and NCS-oxidized HK by HPK and for kallidin formation from native and NCS-oxidized kininogens (HK and LK) by a tissue kallikrein (PPK) are shown in Fig. 2. Kinin production was strongly quenched after substrate oxidation. However, this effect was rather moderate in the tissue kallikrein-dependent system as compared to the nearly complete abolishment of bradykinin production by HPK from NCS-oxidized HK. As shown in Fig. 3A, quite a low molar excess of the oxidizing agent (28-fold over kininogen, i.e. 3.5-fold over methionine residues) was sufficient to lower the kinin yield by about 50%. At these low NCS concentrations no difference between the actions of plasma and tissue kallikreins on the oxidized substrate was detectable. As NCS excess increased, the kallidin yield in the tissue-kallikrein system seemed to stabilize at the level of about 30% of that for native kininogen (HK or LK); however, the bradykinin formation from HK by HPK fell down to a really residual yield at NCS concentration higher than 2 mM. When CT was used for kininogen oxidation, the dependence of kinin yield on the oxidant concentration (Fig. 3B) was quantitatively similar between the HK-HPK and LK-PPK systems. Moreover, it essentially matched the characteristics obtained for the tissue kallikrein-dependent processing of NCS-modified kininogens; at the highest CT concentration the kinin production reached 30% of that from native substrate. Oxidized kininogen (1 mM) was incubated in 0.05 M Hepes buffer pH 7.0, containing 0.15 M NaCl, with HNE (0.02 mM) for 2 h at 37°C and then with HPK (0.05 mM) at the same temperature. After specified time of kallikrein treatment, the reaction was stopped with 1 M HCl (one-fifth volume) and samples were analyzed by reversed-phase HPLC in isocratic water/methanol/TFA system (42% methanol). Dotted/dashed line, sample just after HNE treatment; dashed and solid lines, sample treated with HNE and then with HPK for 15 min and 60 min, respectively; dashed line, the peptide SL(MSO)KRPPGFSPFRSSRI (5 mM), treated with HPK (0.05 mM) in 0.05 M Hepes buffer pH 7.0, containing 0.15 M NaCl, for 5 min at 37°C.

Pre-HPK binding to native and oxidized HK
The results of high-performance gel filtration of mixtures of pre-HPK with native or oxidized HK are presented in Fig. 4. Pre-HPK (molecular mass 85 kDa) was applied in a 1.5 molar excess over kininogen (molecular mass 120 kDa). Analysis of the samples containing pre-HPK with native HK revealed two peaks with apparent molecular masses of 220 kDa and 85 kDa. The first one seemed to represent a complex of both components, the second was of the prekallikrein excess. Essentially the same elution profile was obtained for the mixtures of pre-HPK with CT-oxidized HK, clearly indicating that the modified kininogen retained full capability of pre-HPK binding. In sharp contrast, an insignificant amount of the 220 kDa species could be detected upon analysis of mixtures of pre-HPK with NCS-oxidized HK. Thus, NCS-modified HK seemed to be unable to form a stable complex with pre-HPK.

Inhibition of papain by native and oxidized kininogens
The characteristics of papain inhibition (Fig. 5) were essentially indistinguishable between native kininogens (HK or LK) and kininogens subjected to oxidation with NCS at submillimolar concentrations. The inhibitory capability was slightly impaired only at the NCS concentrations higher than 6 mM, i.e. exceeding 10-fold the levels used in all experiments described in the previous sections. Similarly, a complete resistance of the papain-inhibitory activity of kininogens toward oxidation by CT was determined (not shown). In control experiments it was shown that under Vol. 50 Chemical oxidation of kininogens 759 Kininogens (1 mM) were treated with kallikreins (0.05 mM) in 0.05 M Hepes buffer pH 7.0, containing 0.15 M NaCl, at 37°C. After specified time, aliquots of the incubation mixtures were withdrawn, mixed with one-fifth volume of 1 M HCl to stop the reaction, and then were analyzed by reversed-phase HPLC for bradykinin or kallidin contents. Empty symbols, native kininogens; filled symbols, oxidized kininogens; squares, HK processed with HPK; triangles, HK processed with PPK; circles, LK processed with PPK.

Figure 3. Dependence of kinin formation from oxidized kininogens upon the concentration of oxidizing agents: NCS (A) or CT (B).
Native or oxidized kininogens (1 mM) were treated with kallikreins (0.05 mM) for 15 min at 37°C and then samples were analyzed by reversed-phase HPLC for kinin content. Squares, HK treated with HPK; triangles, HK treated with PPK; circles, LK treated with PPK.
conditions required for papain activation (5 mM cysteine, 37°C, 1 h) the model peptide SL(MSO)KRPPGFSPFRSSRI could not be converted to SLMKRPPGFSPFRSSRI in any significant yield. Hence, the possibility of a reversal of kininogen oxidation during the papain inhibition assay was definitely excluded.

DISCUSSION
Reactive oxygen species that may occur in the living organism have an oxidative potential sufficient to convert methionine residues to MSO (Vogt, 1995). Further oxidation to methionine sulfone is unlikely to proceed in vivo. Of the chemicals commonly used for protein oxidation for the purposes of structural studies, NCS and CT were reported to be satisfactorily specific toward methionine (Schecht-er et al., 1975) and to be effective at submillimolar concentrations that might be sufficiently non-destructive to the protein native conformation. Both reagents oxidize methionine to MSO; hence, in terms of the product formed, the modified protein may have some biological relevance. Moreover, chemical properties of CT resemble to some degree those of numerous chloramines that may be formed in vivo by the action of hypochlorous acid on biological amines. Obviously, both NCS and CT also oxidize cysteine residues if present in the protein being modified. However, all cysteine residues of kininogens are involved in disulfide bridges so that no free sulfhydryl group occurs in the protein molecule (Colman, 1994). Another, although physiologically perhaps less relevant, side reaction of NCS is the modification of tryptophan residues (Schechter et al., 1975). This type of mod- In the wells of a standard microplate, 5 ml of papain solution (1.25 mg/ml in 0.05 M sodium phosphate buffer pH 6.8, containing 10 mM EDTA and 5 mM cysteine) were mixed with 3-20 ml native or NCS-oxidized HK (2.5 mM) and the phosphate buffer in a total 200 ml volume. After incubation for 30 min at 37°C, 4 ml of BAPNA solution (50 mM in Me 2 SO) was added and the increase of absorbance at 410 nm was continuously followed. Empty squares, dotted line, native HK; filled squares, solid line, HK oxidized with 0.625 mM NSC (i.e. at a 250 molar excess of the oxidizing agent over kininogen); circles, dashed line, HK oxidized with 2 mM NCS; triangles, dashed/dotted line, HK oxidized with 6.25 mM NCS. observed in HK oxidized with exceptionally high NCS concentration. Although under those conditions Trp-204 might be modified, non-specific denaturation of some kininogen molecules was more likely. Taken together, our results indicate that mild oxidation of kininogens, via conversion of methionine residues to MSO residues, results in a selective quenching effect on kininprecursor properties of these multifunctional proteins. Other biologically essential kininogen functions, interaction of HK with plasma prekallikrein (and probably the entire pro-coagulation function of HK) and inhibition of cysteine proteinases by both HK and LK, may be preserved. A biological significance of those findings remains yet to be established. It is possible that quenching of kinin release at the inflammatory foci due to the action of reactive oxygen species released by phagocyting neutrophils may help to stop kinin production at late stages of inflammatory reaction and to initiate the tissue restoration.