Effect of disulfide bonds on the structure, function, and stability of the trypsin/tPA inhibitor from Erythrina caffra: site-directed mutagenesis, expression, and physiochemical characterization.

Erythrina trypsin/tPA inhibitor (ETI) from the seeds of Erythrina caffra retains its native structure and inhibitory function after reducing its two disulfide bonds. In order to elucidate the specific role of these crosslinks, alanine residues were substituted for cysteines after cloning the gene in Escherichia coli. Expression of the recombinant inhibitor and the substitution mutants, C83A, CC39, 83AA, and CC132, 139AA, led to inclusion bodies. After solubilization in guanidinium-chloride (GdmCl)/dithiothreitol and oxidation in glutathione buffer, activity could be recovered at yields up to 80%. The mutant proteins exhibit full inhibitory function without detectable alterations of their native structure. However, their stability is reduced: at acid pH, where the oxidized natural inhibitor retains its native structure, the reduced wildtype protein and the mutants undergo at least partial denaturation, reflected by decreased pH ranges of stability: pH 5-7 for the reduced inhibitor, pH 2.5-8.5 for CC132, 139AA, and pH 3.5-8.5 for C83A and CC39, 83AA. Urea and GdmCl denaturation at pH 7 show hysteresis for both the oxidized inhibitor and the double mutant CC132, 139AA. In contrast, the reduced protein and the other mutants exhibit true equilibrium transitions at pH 7, with urea half-concentrations of 0.9 M and 1.9 M and GdmCl half-concentrations of 0.5 M and 1.0 M, respectively. The stability of Erythrina trypsin/tPA inhibitor follows the sequence: oxidized ETI > CC132, 139AA > CC39, 83AA and C83A > reduced ETI.

The 2.3 A crystal structure of the catalytic domain of recombinant two-chain human tissue-type plasminogen activator.

Tissue-type plasminogen activator (t-PA), a multidomainal serine proteinase of the trypsin-family, catalyses the rate-limiting step in fibrinolysis, the activation of plasminogen to the fibrin-degrading proteinase plasmin. Trigonal crystals have been obtained of the recombinant catalytic domain of human-two-chain t-PA, consisting of a 17 residue A chain and the 252 residue B chain. Its X-ray crystal structure has been solved applying Patterson and isomorphous replacement methods, and has been crystallographically refined to an R-value of 0.184 at 2.3 A resolution. The chain fold, active-site geometry and Ile276-Asp477 salt bridge are similar to that observed for trypsin. A few surface-located insertion loops differ significantly, however. The disulfide bridge Cys315-Cys384, practically unique to the plasminogen activators, is incorporated without drastic conformational changes as the insertion loop preceding Cys384 makes a bulge on the molecular surface. The unique basic insertion loop Lys296-Arg304 flanking the primed subsites, which has been shown to be of importance for PAI-1 binding and for fibrin specificity, is partially disordered; it can therefore freely adapt to proteins docking to the active site. The S1 pocket of t-PA is almost identical to that of trypsin, whereas the S2 site is considerably reduced in size by the imposing Tyr368 side-chain, in agreement with the measured preference for P1 Arg and P2 Gly residues. The neighbouring S3-S4 hydrophobic groove is mainly hydrophobic in nature. The structure of the proteinase domain of two-chain t-PA suggests that the formation of a salt bridge between Lys429 and Asp477 may contribute to the unusually high catalytic activity of single-chain t-PA, thus stabilizing the catalytically active conformation without unmasking the Ile276 amino terminus. Modeling studies show that the covalently bound kringle 2 domain in full-length t-PA could interact with an extended hydrophobic groove in the catalytic domain; in such a docking geometry its "lysine binding site" and the "fibrin binding patch" of the catalytic domain are in close proximity.

Mapping of the catalytic site of CHO-t-PA and the t-PA variant BM 06.022 by synthetic inhibitors and substrates.

BM 06.022 is a t-PA deletion variant that is produced as inactive inclusion bodies in Escherichia coli and transformed into the native form by an in vitro refolding process. Until now, no X-ray and NMR structures of BM 06.022 were available. Therefore a detailed kinetic analysis of the hydrolysis of peptide substrates and of the inhibition by several benzamidine-derived inhibitors was carried out in order to assess that the active site region of the protease domain of BM 06.022 is correctly structured in comparison with t-PA. Our data reveal that the single-chain as well as the two-chain form of BM 06.022 and native t-PA are similar in catalytic and in inhibitor binding properties. This indicates that the active site and the highly complex rearrangement of t-PA upon cleavage of the Arg275-Ile276 bond are maintained in BM 06.022.

Comparison of the recombinant Escherichia coli-produced protease domain of tissue-type plasminogen activator with alteplase, reteplase and streptokinase in a canine model of coronary artery thrombolysis.

Recent in vitro studies have shown that although recombinant Escherichia coli-produced protease domain of tissue-type plasminogen activator (t-PA) has no appreciable fibrin binding and less plasminforming activity compared to the wild-type, it is nevertheless an effective fibrinolytic agent in a dynamic in vitro plasma clot lysis system. The purpose of the present study was to evaluate the pharmacological profile of the protease in a canine model of coronary artery thrombosis. The effects of a single i.v. bolus injection of 1 mg/kg protease were compared with those of alteplase, reteplase and streptokinase at clinically relevant doses and dosing regimens in eight dogs per group. The protease rapidly restored coronary blood flow at 12 +/- 1 min in all treated dogs with a significantly higher maximal coronary blood flow than in the reference groups, but was associated with short cycles of reocclusion in 4/8 animals. Overall, the coronary blood flow quality of the protease was not significantly different from that of the reference thrombolytics. Although fibrinogen was nearly completely degraded during protease treatment, the bleeding time was not significantly more prolonged than in reference groups. In conclusion, the protease domain is a rapidly acting, effective, bolus-injectable thrombolytic agent associated with a systemic lytic state and does not appear to cause significantly more bleeding than the reference thrombolytic agents.

Thrombolysis with an Escherichia coli-produced recombinant plasminogen activator (BM 06.022) in the rabbit model of jugular vein thrombosis.

The recombinant plasminogen activator BM 06.022 consists of the kringle 2 and the protease domains of human t-PA and is unglycosylated because of the expression in Escherichia coli. The thrombolytic and pharmacokinetic properties as well as the hemostasis effects of BM 06.022 were investigated in the rabbit model of jugular vein thrombosis. The thrombi were 125I-fibrin labeled. Intravenous bolus injection of 50, 100, 200, and 400 kU/kg BM 06.022 or 400, 800, and 1600 kU/kg alteplase over 15 s to six rabbits/dose produced a dose-dependent increase of thrombolysis determined 2 h post injection. The dose-response curve of BM 06.022 was located left compared with that of alteplase. The effective dose of 50% thrombolysis (ED50) obtained by half-logarithmic regression analysis was 163 kU/kg (= 0.28 mg/kg) for BM 06.022 and 871 kU/kg (= 1.09 mg/kg) for alteplase. At equipotent doses (50% thrombolysis), the residual concentration of fibrinogen was 74.2% and 76.5%, that of plasminogen 66.7% and 69.4%, and that of alpha 2-antiplasmin 47.3% and 46% for BM 06.022 and alteplase, respectively. Pharmacokinetic analysis for plasma activity at a dose of 400 kU/kg revealed a half-life of 18.9 +/- 1.5 min for BM 06.022, whereas alteplase was distributed with a half-life of 2.1 +/- 0.1 min, accounting for 86.7 +/- 1.9% of the total AUC, followed by a beta-phase with a half-life of 13.8 +/- 0.9 min. Plasma clearance of BM 06.022 was 4.7 +/- 0.7 ml min-1 kg-1 compared with 20 +/- 1.2 ml min-1 kg-1 for alteplase.(ABSTRACT TRUNCATED AT 250 WORDS)

The spectroscopic analysis, inhibition and binding studies demonstrate the equivalence of Erythrina caffra trypsin inhibitor and the recombinant substitution variant recSerETI.

A recombinant substitution mutant (recSerETI) of the Erythrina caffra trypsin inhibitor, with the N-terminal valine residue substituted by serine, was produced in E. coli and compared to the wildtype protein (wtETI) with respect to physicochemical and functional properties. The spectral properties, including UV absorbance, fluorescence emission and circular dichroism, were indistinguishable. Furthermore, the inhibitory activities of the two proteins regarding the inhibition of trypsin, chymotrypsin, tissue plasmininogen activator (t-PA) and reteplase (BM 06.022, t-PA deletion variant comprising the kringle 2 and the protease domains, isolated from transformed E. coli cells) and the affinity of the immobilized inhibitors for reteplase were closely similar. Five repetitive cycles of guanidinium chloride (GdmCl)-induced denaturation-renaturation yield the native mutant protein with its inhibitory activity fully restored. The only difference between the wildtype and the mutant protein refers to the intrinsic stability. Comparing the pH- and GdmCl-dependent transitions, as well as the thermal denaturation, recSerETI exhibits decreased stability compared to the wildtype protein. The pH range of stability is shifted from pH 1-9.5, for wtETI, to pH 2-9, for recSerETI; similarly the GdmCl-induced denaturation is found to occur at a GdmCl half concentration of 3.7 M instead of 4.5 M; in both cases the renaturation exhibits strong hysteresis. The mid-point of the thermal unfolding transition of the mutant protein is at approximately 65 degrees C, as compared to approximately 75 degrees C for the wildtype protein.

Pharmacokinetic properties of an Escherichia-coli-produced recombinant plasminogen activator (BM 06.022) in rabbits.

The recombinant plasminogen activator BM 06.022 consists of the kringle 2 and the protease domains of human t-PA and is unglycosylated because of its expression in Escherichia coli. The pharmacokinetic properties of BM 06.022 following intravenous injection over 1 min were characterized in anesthetized male New Zealand white rabbits. BM 06.022 was injected at doses of 50, 100, 200, and 400 kU/kg bw (n = 5-6/dose). Activity concentrations in plasma were determined using an indirect spectrophotometric assay. The maximum plasma concentration and the area under the plasma concentration vs. time curve (AUC0-00) of BM 06.022 increased linearly with dose. The systemic clearance ranged from 2.5 to 3.0 ml.min-1.kg-1 and did not show dose-dependency, in contrast to alteplase which was studied at doses of 200, 400, 800, and 1600 kU/kg. A direct comparison of clearance rates of BM 06.022 and alteplase at doses of 200 and 400 kU/kg each revealed a 8.5-fold slower clearance rate of BM 06.022. The majority (18/23) of rabbits with BM 06.022 injection showed a pharmacokinetic profile which was best characterized by a one-compartment model in contrast to alteplase (10/23). The dose-groups of BM 06.022 showed an average dominant half-life ranging from 11.6 to 15.4 min, which was about five-times longer than the dominant half-life values of alteplase (2.3 to 4.5 min). Assuming a two-compartment model in the remaining animals, the initial alpha-phase of BM 06.022 accounted for 40.1 +/- 13.2% (n = 5) of the total AUC, whereas the alpha-phase of alteplase accounted for 82.7 +/- 3% (n = 13) of the total AUC.

Binding of recombinant variants of human tissue-type plasminogen activator (t-PA) to human umbilical vein endothelial cells.

Endothelial cells synthesize and secrete hemostatic components like tissue-type plasminogen activator (t-PA) which is thought to be the major determinant of fibrinolytic activity in the blood. Most recently, a receptor protein for t-PA on human umbilical vein endothelial cells (HUVEC) in culture has been described (1); there are, however, in addition low affinity binding sites for t-PA on HUVEC. The sites of binding are of particular interest, because they are potential regulators of t-PA activity and clearance. We analysed the low affinity binding of recombinant t-PA (rt-PA) to normal diploid HUVEC and to the permanent human cell lines Jurkat, Daudi, HL 60 and K562 by flow cytometry applying t-PA specific monoclonal antibodies. Using this test system binding of both recombinant glycosylated human t-PA produced in Chinese hamster ovary cells (CHO-t-PA) and of nonglycosylated t-PA, produced in E. coli (BM 06.021) was investigated. Analysis of the binding pattern to HUVEC and other cell lines revealed that deglycosylation of full length rt-PA increases non-specific binding. Additionally, we investigated the binding properties of an unglycosylated t-PA deletion variant which comprises the kringle 2 and the protease domains (BM 06.022). Data obtained show that deletion of these domains most drastically reduces non-specific binding to HUVEC and other human cell lines.

Pharmacokinetic and thrombolytic properties of unglycosylated recombinant tissue-type plasminogen activator (BM 06.021) produced in Escherichia coli.

Recombinant tissue-type plasminogen activator (rt-PA) was produced in Escherichia coli cells in order to obtain an unglycosylated rt-PA (BM 06.021) with increased thrombolytic potency due to altered pharmacokinetic properties. The pharmacokinetics were studied in rabbits upon intravenous infusion of 200 kU/kg over 30 min. The thrombolytic dose-response effects were evaluated in a rabbit model with 125I-labeled venous thrombi upon intravenous infusion over 4 h. The thrombolytic effects after intravenous bolus injection of 200 kU/kg BM 06.021 were investigated in a canine model of coronary artery thrombosis. All studies were performed comparing BM 06.021 with glycosylated rt-PA (alteplase). BM 06.021 demonstrated a longer (p less than 0.05) half-life (5.6 +/- 2.6 vs. 2.1 +/- 0.3 min) and a lower (p less than 0.05) clearance rate (7.5 +/- 0.8 vs. 22.2 +/- 3.1 ml.min-1.kg-1) than alteplase in rabbits upon intravenous infusion. The dose-response curve of BM 06.021 for thrombolysis in a rabbit model of jugular vein thrombosis was located to the left of that for alteplase with a 2.1-fold lower effective dose of 50% thrombolysis (ED50) of BM 06.021 (207 vs. 436 kU/kg). Intravenous bolus injection of 200 kU/kg BM 06.021 induced the same reperfusion rate (4/6) as intravenous infusion of 800 kU/kg alteplase over 90 min in a canine model of coronary artery thrombosis. The residual thrombus wet weight did not significantly differ between BM 06.021 and alteplase (5.7 +/- 1.8 vs. 6.3 +/- 1.1 mg). The results indicate that unglycosylated rt-PA (BM 06.021) has a higher in vivo thrombolytic potency than glycosylated rt-PA (alteplase).(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction of recombinant tissue type plasminogen activator and recombinant plasminogen activator (r-PA/BM 06.022) with human endothelial cells.

The Escherichia coli-expressed recombinant plasminogen activator (r-PA) comprising the kringle 2 and protease domains of human tissue-type plasminogen activator (t-PA) has a four-fold longer half-life time in the circulation than t-PA, possibly resulting in an increased opportunity for r-PA to interact with the endothelial lining. In the present study we investigated the interaction of r-PA and t-PA with human umbilical vein endothelial cells (HUVEC). Specific binding of 125I-t-PA and 125I-r-PA were similar at 4 degrees C (Kd 6 nmol/l; Bmax about 120 fmol/mg cell protein). About half of the specific binding sites were shared by t-PA and r-PA, because unlabeled t-PA and r-PA competed equally with 125I-labeled t-PA and r-PA for binding to HUVEC. The low affinity interaction of 125I-t-PA was several-fold higher than that of 125I-r-PA. When PA binding was studied at 37 degrees C, HUVEC bound more t-PA than r-PA to both specific and non-specific binding sites. Both t-PA and r-PA were internalized and degraded, but t-PA internalization proceeded more efficiently than that of r-PA. In the presence of 100 microM chloroquine, the degradation of t-PA and r-PA was inhibited by 75% and 40%, respectively, indicating lysosomal degradation. When the active sites of t-PA and r-PA were blocked by PPACK, part of the cell association and most of the degradation of both t-PA and r-PA were inhibited. This points to plasminogen activator inhibitor-1 (PAI-1) as one of the specific binding sites. A possible role of LDL-receptor related protein (LRP) or related members of this receptor family was investigated by using the 39 kD receptor associated protein (RAP) which prevents interaction of ligands with these receptors. RAP reduced the association of 125I-t-PA by 25% and the degradation of 125I-t-PA and 125I-r-PA by 65% and 50%, respectively. Our data show that both t-PA and r-PA bind to HUVEC and can subsequently be internalized and degraded. However, r-PA interacts less effectively with HUVEC than t-PA. This indicates that binding to the endothelium does not prevent the clearance of r-PA and is not the cause of its long half-life.

The effects of Tween 20 and sucrose on the stability of anti-L-selectin during lyophilization and reconstitution.

We have chosen an anti-L-selectin antibody as a model protein to investigate the effects of sucrose and/or Tween 20 on protein stability during lyophilization and reconstitution. Native anti-L-selectin secondary structure is substantially retained during lyophilization in the presence of sucrose (1 or 0.125%). However, aggregation of the protein during reconstitution of lyophilized protein powders prepared without sucrose is not reduced by the presence of sucrose in the reconstitution medium. Aggregate formation upon reconstitution is completely inhibited by freeze drying the protein with sucrose and reconstituting with a 0.1% Tween 20 solution. Tween 20 (0.1%) also partially inhibits loss of native anti-L-selectin secondary structure during lyophilization. However, upon reconstitution the formulations lyophilized with Tween 20 contain the highest levels of aggregates. The presence of Tween in only the reconstitution solution appears to inhibit the transition from dimers to higher order oligomers. Potential mechanism(s) for the Tween 20 effects were investigated. However, no evidence of thermodynamic stabilization of anti-L-selectin conformation (e.g., by Tween 20 binding) could be detected.

Matrix degrading proteinases from human granulocytes: type I, II, III collagenase, gelatinase and type IV, V-collagenase. A survey of recent findings and inhibition by gamma-anticollagenase.

Three human matrix degrading leukocyte proteinases, type I collagenase, gelatinase and a new type IV collagenase were isolated in latent and active form. Activation of all three latent enzymes could be achieved by treatment with either organomercurials or with trypsin. In addition the 90 kDa latent type I-collagenase could be activated by disulfides, while a newly discovered 70 kDa latent form could be activated with organomercurials or with trypsin. The active type I collagenase was inhibited by gamma-anticollagenase from human serum (and the leukocyte type I collagenase inhibitor, while the newly found type IV collagenase was inhibited only partially. The complexes formed from gamma-anticollagenase with type I collagenase, i. e. latent enzyme, are not reactive site associated complexes. The binding is not of a substrate-like and competitive manner. After inhibition of the enzyme though inactive against its natural substrates it is still hydrolyzing the synthetic low molecular weight octapeptide DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH.

Active site labeling with dansyl-glutamyl-glycyl-arginyl chloromethyl ketone demonstrates the full activity of the refolded and purified tissue-type plasminogen activator variant BM 06.022.

BM 06.022 is a tissue-type plasminogen activator deletion variant that is comprised of the kringle 2 and the protease domain of the native molecule. BM 06.022 is expressed as inactive inclusion bodies in E. coli and transferred into the active enzyme by an in vitro folding process. Active site labeling with dansyl-glutamyl-glycyl-arginyl chloromethyl ketone provides evidence that the purified BM 06.022 is fully active and that misfolded species are completely removed by affinity chromatography on ETI-Sepharose. The comparison of the kinetics of the inhibition of BM 06.022 with that of CHO-t-PA indicates that the active centers of both enzymes are rather similar. The further evaluation of the site of interaction of BM 06.022 and DnsEGRck by mass spectroscopy and amino acid sequence analysis revealed that the inhibitor is bound selectively to His322, which is part of the catalytic triad of this serine protease.

Determination of LDL cholesterol and LDL apolipoprotein B following precipitation of VLDL in blood serum with phosphotungstic acid/MgCl2.

A method is described for the selective precipitation of VLDL in blood serum using phosphotungstic acid/MgCl2. The method allows for the calculation of LDL apolipoprotein B as well as for the calculation of LDL cholesterol (following the additional determination of HDL cholesterol). Dependent on the triglyceride and the cholesterol content of the serum, three different procedures were developed using phosphotungstic acid and MgCl2 in different concentrations in the precipitation assay. Within the tested range of 3-10 mmol/l total cholesterol and 1-4 mmol/l triglyceride in blood serum the VLDL were nearly completely precipitated with negligible coprecipitation of LDL and HDL, but 40-50% coprecipitation of Lp(a). Regression analysis of the cholesterol values obtained by precipitation with phosphotungstic acid/MgCl2 (= serum cholesterol - LDL cholesterol), and the cholesterol values obtained by ultracentrifugation (d greater than 1.006 kg/l) revealed a good measure of agreement (r = 0.97, y = 0.93 X + 0.35, n = 76). An equally good measure of agreement was found for the corresponding apolipoprotein B values (r = 0.96, y = 1.03 X - 0.2, n = 61). In the determination of LDL cholesterol a variation coefficient of 4.3% (n = 20) was found in relation to the precision in the series, and a variation coefficient of 4.8% (n = 25) in relation to day to day precision.

Formulation of proteins in vacuum-dried glasses. II. Process and storage stability in sugar-free amino acid systems.

The purpose of this research was to investigate the freeze- and vacuum-drying behavior of L-amino acids of current/potential use as adjuvants for formulating proteins. The analytical methods used were wide-angle x-ray diffraction, differential scanning calorimetry, and scanning electron microscopy. Protein analysis was performed either as an activity assay (lactate dehydrogenase [LDH]) or by size-exclusion chromatography (granulocyte colony-stimulating factor [rhG-CSF]). After samples were freeze-dried, only the four basic amino acids (arginine, lysine, histidine, and citrulline) formed amorphous solids, which, however, were partially crystalline. The remaining amino acids all formed fully crystalline solids. After samples were vacuum-dried, (20 degrees C, 0.1 mbar, 1 ml fill volume in 2-ml vials) fully crystalline solids were formed by all of the amino acids. For arginine, the addition of either HCl, H3PO4, or H2SO4 sufficient to form the respective salt produced amorphous solids after vacuum-drying, but they had high residual water contents and low glass transition temperatures (Tg). Addition of phenylalanine to arginine base inhibited crystallization of the latter at low concentrations during vacuum-drying procedure, leading to formation of a pure rubbery solid. At higher concentrations the phenylalanine crystallized, producing dry products with glass transition temperatures of > 60 degrees C. The process and storage stability of LDH and rhG-CSF in the vacuum-dried phenylalanine/arginine glasses was greatly improved at temperatures up to 40 degrees C compared with the unprotected proteins. Uptake of moisture during storage was, however, a complicating factor, reducing Tg, promoting crystallization, and leading to decreased protein stability. The PO4 salt of arginine produced especially high glass transition temperatures after it was vacuum-dried. These sugar-free amino acid formulations thus are potential stabilizes for proteins.

Coronary thrombolytic properties of a novel recombinant plasminogen activator (BM 06.022) in a canine model.

We studied the thrombolytic dose-response relationship of a recombinant plasminogen activator (rPA) (BM 06.022) compared with alteplase in a canine model of coronary artery thrombosis. BM 06.022 consists of the kringle 2 and protease domains of human tissue PA (tPA) and lacks oligosaccharide side chains because of its expression in Escherichia coli. Thrombus formation in anesthetized, open-chest dogs was induced by electrical injury to the intimal surface of the left circumflex coronary artery in the presence of a critical stenosis. Intravenous bolus injection of BM 06.022 (50, 100, 140, and 200 kU/kg) or of alteplase (200, 800, 1,130, and 1,600 kU/kg) 30 min after coronary occlusion to six heparinized dogs per group achieved a dose-dependent increase in reperfusion rate and decrease in residual thrombus wet weight. Vehicle-treated dogs did not reperfuse. Semilogarithmic regression analysis showed that the effective dose that produced 50% reperfusion of BM 06.022 (83 kU/kg) was 11.6-fold lower than that of alteplase (951 kU/kg). Comparison with infusion experiments showed that intravenous bolus injection of 140 kU/kg of BM 06.022 was equieffective to a 90-min infusion of 800 kU/kg (= 1 mg/kg) of alteplase as a standard treatment regarding reperfusion rate (66%) and time to reperfusion (15 +/- 6 vs. 18 +/- 8 min). Pharmacokinetic analysis for functionally active BM 06.022 or alteplase in plasma revealed a total plasma clearance of 4.1-6.6 ml/min/kg for BM 06.022 and of 12.6-42.3 ml/min/kg for alteplase.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue-type plasminogen activator domain-deletion mutant BM 06.022: modular stability, inhibitor binding, and activation cleavage.

Recombinant BM 06.022 (M(r) 39,589) is a domain-deletion mutant of the human tissue-type plasminogen activator (tPA) structured by the kringle 2 and protease modules. Unfolding under various conditions was investigated via 1H-NMR spectroscopy by monitoring the well-resolved high-field methyl resonances at approximately -0.97 ppm (kringle 2) and approximately -0.29 and -0.54 ppm (protease). Reversible acid/base unfolding is manifest under low pH (< 4.8) conditions. It is observed that, relative to the protease, the kringle exhibits higher overall stability at low pH. At pH 4.6, BM 06.022 undergoes two distinct thermal melting transitions, at approximately 334 and approximately 352 K, assigned to an irreversible denaturation of the protease and a reversible unfolding of the kringle 2, respectively. Under the same conditions, the protease reacted with the active site inhibitor 1,5 dansyl-L-glutamylglycyl-L-arginine chloromethyl ketone (EGRck) exhibits a higher (approximately 10 K) thermal stability than the inhibitor-free protease. Upon acidification, the EGRck-modified protease unfolds irreversibly around pH 3.4. As exemplified by BM 06.022, a single-chain protein, as defined by continuity of the polypeptide backbone, can exhibit simultaneous folding reversibility and irreversibility for autonomous segments of the sequence. Conversion of the isolated (single-chain) protease or intact BM 06.022 to their catalytically active two-chain forms via plasminolytic cleavage of the Arg275-Ile276 peptide bond leaves the kringle 2 spectrum unaffected while perturbing the resolved high-field methyl resonances stemming from the protease. The latter also shift when the protease is reacted with EGRck, indicating that these signals are sensitive to events at the binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)