Biomedical subjects

A Baici

Publications and source records attributed to A Baici.

At least 19 recordsLinked to original sources

PubMed1997

Very rapid, ionic strength-dependent association and folding of a heterodimeric leucine zipper.

Leucine zippers (coiled coils) are dimerization motifs found in several DNA-binding transcription factors. A parallel leucine zipper composed of the acidic chain X1-EYQALEKEVAQLEAENX2-ALEKEVAQLEHEG-amide and the basic chain X1-EYQALKKKVAQLKAKNX2ALKKKVAQLKHKG-amide was designed to study the kinetics of folding of a heterodimeric leucine zipper and to investigate the role of electrostatic attraction between oppositely charged peptide chains to the folding reaction. Each peptide alone did not form a leucine zipper at ionic strength (mu) < 1 M because of electrostatic repulsion between like charges in a homodimer. Therefore, the formation of the heterodimeric leucine zipper could be investigated by simple mixing of acidic and basic chains. To monitor folding, a fluorescent label was located either at the N-terminus (X1 = fluorescein-GGG, X2 = Q) or in the center of the coiled coil (X1 = acetyl, X2 = W). Folding could be described by a simple two-state reaction involving the disordered monomers and the folded heterodimer. The same bimolecular rate constant (k(on)) was observed independent of the location of the fluorescent label, indicating that both fluorescence probes monitored the same reaction. Lowering of the ionic strength increased k(on) from 4 x 10(6) M-1 s-1 (mu = 525 mM) to about 5 x 10(7) M-1 s-1 (mu = 74 mM). When extrapolated to mu = O, k(on) was approximately 10(9) M-1 s-1, which is near the diffusion limit. In contrast, the rate of dissociation depended very weakly on ionic strength; k(off) decreased only by about 2-fold when mu was lowered from 525 to 74 mM. Equilibrium association constants (Ka) of the heterodimeric zippers measured directly and calculated from kinetic constants (Ka = k(on)/k(off) were in good agreement. The observed two-state mechanism, the strong dependence on ionic strength of k(on) but not of k(off), and the nearly diffusion-limited association rate at very low ionic strength point to a folding pathway in which the formation of an electrostatically stabilized dimeric intermediate may be rate-limiting and the subsequent folding to the final dimer is very rapid and follows a "down-hill" free energy landscape. The small increase of k(off) at increasing ionic strength indicates a minor contribution of electrostatics to the stability of the folded leucine zipper.

Amino Acid Sequence↗

PubMed1996

Antileukoprotease inhibits stratum corneum chymotryptic enzyme. Evidence for a regulative function in desquamation.

The stratum corneum chymotryptic enzyme (SCCE) has been previously purified from human stratum corneum and resembles a chymotryptic serine endopeptidase involved in physiological detachment of corneocytes from human stratum corneum. From human stratum corneum two inhibitory activities of SCCE could be extracted. These were due to serine protease inhibitors already known to be present in human epidermis, antileukoprotease (secretory leukocyte protease inhibitor) and elafin (skin-derived antileukoprotease). The Inhibition of SCCE by antileukoprotease shows a hyperbolic, mixed type inhibition with an equilibrium dissociation constant of 63 n. Antileukoprotease also inhibits detachment of corneocytes from human plantar callus in vitro almost completely (>96%). In addition, elafin was shown to be a weak inhibitor for SCCE activity, and elafin significantly reduces the shedding of corneocytes. Thus, antileukoprotease, which is known to be produced by human keratinocytes, is likely to be the major physiological inhibitor of SCCE in the epidermis. It seems to be involved in the regulation of desquamation under physiological and pathophysiological conditions.

Aprotinin↗

PubMed1996

pH-dependent hysteretic behaviour of human myeloblastin (leucocyte proteinase 3).

Human myeloblastin (leucocyte proteinase 3) showed a very slow approach to the steady-state velocity when the pH was rapidly increased from 3.2 to 7.0. The kinetic mechanism of this hysteretic process was interpreted as a slow conformational change of myeloblastin from an inactive form at acidic pH to the active form at neutral pH. The transition between the two enzyme forms could occur spontaneously in the absence of substrates with a first-order rate constant of 0.0033 s-1. In the presence of peptide substrates activation occurred more rapidly: the observed rate constant was linearly dependent upon the substrate concentration and contained a contribution of the spontaneous as well as of the substrate-dependent process, whose second-order rate constant was characteristic of the particular substrate. This pH-dependent phenomenon of hysteresis on the part of myeloblastin, that is not manifested by the closely related leucocyte elastase, may have a physiological control function during phagocytosis by damping the rate of interconversion between enzymically inactive and active enzyme conformations.

Humans↗

PubMed1996

Human myeloblastin (leukocyte proteinase 3): reactions with substrates, inactivators and activators in comparison with leukocyte elastase.

Human myeloblastin (leukocyte proteinase 3) shares many biochemical properties with leukocyte elastase, but rapidly loses enzymatic activity when raising the pH and/or the ionic strength of an acidic solution or when handled in glass vessels. This poses limits to kinetic experiments requiring long incubation times. After purification, myeloblastin was conveniently stored in a glycine/HCl buffer at pH 3.2, while assays were performed in sodium/potassium phosphate buffer at pH 7.0, ionic strength 0.11, in the presence of 0.05% w/v Triton X-100 and taking care to avoid any contact with glass surfaces. The kinetic parameters of leukocyte elastase and myeloblastin with peptide substrates, irreversible inactivators and glycosaminoglycans were compared under these conditions. MeO-succinyl-Lys(2-picolinoyl)Ala-Pro-Val-4-nitroanilide, an excellent substrate for leukocyte elastase, also proved to be a good substrate for myeloblastin (Km = 16 microM, kcat/Km = 30,600 M(-1)s(-1)). Inactivation of myeloblastin by 3,4-dichloroisocoumarin (ki/Ki = 6,389 M(-1)s(-1)) and MeO-Suc-Ala-Ala-Pro-Val-chloromethane (ki/Ki = 579 M(-1) S(-1)) occurred via a two-step, irreversible complexing mechanism with potencies one-half and one-fifth that of leukocyte elastase, respectively. Glycosaminoglycans such as chondroitin sulfate, dermatan sulfate and a chondroitin polysulfate, interacted with myeloblastin as non-essential activators in the presence of peptide substrates (activation up to a 6.7-fold factor) and as partial inhibitors (about 50% inhibition at saturation) in the presence of elastin. This property distinguishes myeloblastin from leukocyte elastase, which is always inhibited by glycosaminoglycans, independently of the substrate.

Enzyme Activation↗

PubMed1995

Kinetics of folding of leucine zipper domains.

Leucine zippers are short coiled coils frequently found in transcription factors where they serve as dimerization domains. The basic features contributing to the thermodynamic stability of leucine zippers are well understood, but very little is known about their folding kinetics. Leucine zippers have a simple and well defined structure and are, therefore, excellent models for the study of the concerted folding and assembly of polypeptide chains. Here we report on a fluorescence stopped flow investigation of the kinetics of association and dissociation of a series of model leucine zippers based on the common sequence Xzero-EYEALEKKLAAX1EAKX2QALEKKLEALEHG-amide (Xzero = N alpha-acetyl, N alpha-fluorescein-GGG, or N alpha-dimethylaminocoumarin-GGG; Xl = Leu or Ala; X2 = Leu, Ala, or Asn). When Xzero is fluorescein, self-quenching between adjacent fluorophores leads to a decrease in fluorescence emission intensity whereas unfolding of the coiled coil leads to an increase. In a heteromeric coiled coil containing both fluorophores, resonance energy transfer between the donor coumarin and the acceptor fluorescein is observed, and the mixing of labeled and nonlabeled peptides allows the measurement of the rates of strand exchange between leucine zippers. Exchange rates do not depend on peptide concentration, indicating that strand exchange is governed by the rate of dissociation of the coiled coil. Strand exchange between leucine zippers with X1 and X2 = Leu occurs with a half-time of approximately 30 min. A single Leu/Ala substitution at X1 or X2 decreases the half-time to approximately 1 s. Folding was also studied in a relaxation experiment in which a preexisting equilibrium between monomeric chains and coiled coils was rapidly disturbed by dilution with buffer, and the relaxation to the new equilibrium was followed by the increase in fluorescence. In peptides with X1, X2 = Ala or X1 = Ala, X2 = Asn the folding process can be described by a simple two-state monomer<-->dimer equilibrium with k(on) approximately 4 x 10(6) M-1 s-1 and k(off) approximately 10 s-1. Kd = k(off)/k(on) approximately 2.5 microM is in good agreement with the value of Kd obtained from equilibrium measurements. The peptides with a single Ala at X1 or X2 exhibit biphasic folding kinetics. One phase is concentration dependent and the other apparently concentration independent. This behavior can be interpreted as a monomer<-->dimer equilibrium coupled to an equilibrium between different conformational isomers. Leu to Ala and Leu to Asn substitutions in the hydrophobic core alter the folding kinetics in a position-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1995

Comparison of chondroitin sulphate composition of femoral head articular cartilage from patients with femoral neck fractures and osteoarthritis and controls.

The glycosaminoglycan (GAG) and uronic acid (UA) composition of human hip articular cartilage from patients with femoral neck fractures [assumed osteoporosis (OP); n = 12], from patients with osteoarthritis (OA; n = 12) and from normal controls (n = 9) was determined. Full depth tissue samples from the control and OP groups were analysed from the superior, inferior, anterior and posterior regions, while the OA tissue was from cystic (tissue growing on top of cystic bone lesions) and osteophytic regions, from normal and fibrillated resident cartilage and from regions immediately adjacent to eburnated bone. The total sulphated GAG and UA content was reduced in the inferior region of control cartilage compared to the other regions and the values of all regions of the assumed OP group. Cystic regions and OA cartilage adjacent to the bone also showed lower GAG and UA levels than the other regions. The ratios of chondroitin 6-sulphate (C6S) to chondroitin 4-sulphate (C4S) indicated a similar pattern in the different regions of controls and the patient group with femoral neck fracture (OP group). The cystic and osteophytic cartilage of the OA group exhibited lower C6S/C4S ratios than any other region. The levels of dermatan sulphate (DS) in the cartilage of all regions of the OP and control groups were very similar and low, while the tissues of the OA group contained significantly higher amounts, particularly the cartilage from osteophytes. The previously presumed compositional similarity between normal aged and osteoporotic articular hip cartilage was essentially confirmed in a comparative analysis. Significant changes in GAG and UA composition of OA cartilage from distinct regions was also recorded.

Aged↗

PubMed1995

Cathepsin B in osteoarthritis: zonal variation of enzyme activity in human femoral head cartilage.

OBJECTIVES: To determine the quantitative topographical distribution of cathepsin B in human femoral head cartilage by measuring the zonal variation of enzyme activity in specimens taken from various anatomical regions of normal and osteoarthritic (OA) tissues, and to correlate this parameter with the severity of the OA lesions. METHODS: OA articular cartilage was obtained at surgery for total hip replacement and control cartilage obtained at postmortem. Cylinders of full thickness cartilage with underlying bone were retrieved with a biopsy trephine. Sections of cartilage were produced by cryocutting the tissue as slices parallel to the articular surface and assayed for cathepsin B with a specific, highly sensitive fluorogenic substrate. The severity of the OA lesions was graded according to the histopathological-histochemical method of Mankin. RESULTS: Zonal cathepsin B activity of normal cartilage was uniform and low in all regions of the femoral head. In apparently intact OA cartilage and in severely degraded tissue the zonal distribution and the amounts of enzyme were similar to control values. At sites with active disease, cathepsin B activity was much greater than in controls and its irregular zonal distribution correlated with tissue degeneration, hypercellularity, or cloning of chondrocytes as determined histochemically. Particularly high enzyme levels were observed at sites with regenerating cartilage, where some zonal peaks attained 20-fold activity with respect to controls. CONCLUSION: Cathepsin B may play a role in sustaining the chronicity of OA, not as an initiator, but rather as a perpetuator of the disease and as an antagonist of regeneration.

Adult↗

PubMed1995

Cathepsin B in osteoarthritis: cytochemical and histochemical analysis of human femoral head cartilage.

OBJECTIVE: To localise the cysteine endopeptidase cathepsin B in chondrocytes and cartilage from normal and osteoarthritic (OA) human femoral heads in order to provide qualitative information on its cellular expression and distribution at possible sites of action. METHODS: OA articular cartilage was obtained at surgery for total hip replacement; control cartilage was obtained at postmortem. Chondrocytes were isolated by sequential enzymatic digestion and cathepsin B analysed by immunocytochemistry and activity staining with a fluorogenic substrate. Lysosomes were visualised by fluorescence microscopy after staining of living cells with acridine orange. Using a histochemical reaction, enzyme activity was measured in cryosections of full thickness cartilage. RESULTS: Chondrocytes from normal cartilage contained very few lysosomes and only a minor cell population was cathepsin B positive. A high proportion of chondrocytes from active OA cartilage contained a large number of lysosomes and an excess of cathepsin B in intracellular organelles; the enzyme was stored in an active form. In this respect, OA chondrocytes closely resembled normal cells that had been phenotypically modulated by serial subcultures. No cathepsin B activity could be detected by histochemistry in either chondrocytes or matrix of normal cartilage. While apparently intact and severely degraded OA cartilage was also cathepsin B negative, tissue at sites of active destruction and, particularly, at repair sites was highly positive. CONCLUSION: The presence and the particular distribution of active cathepsin B in OA cartilage at 'more involved' sites suggest a pathological role for this enzyme in sustaining and perpetuating cartilage degradation. While other stimuli may also be responsible for cathepsin B expression in OA chondrocytes, the similarity with artificially modulated cells indicates fibroblastic metaplasia as a plausible mechanism.

Adult↗

PubMed1994

Cephem sulfones as inactivators of human leukocyte elastase. 5. 7 alpha-Methoxy- and 7 alpha-chloro-1,1-dioxocephem 4-ketones.

Studies on cephem sulfones as inhibitors of human leukocyte elastase (HLE) have been extended to the new class of cephem 4-ketones. tert-Butyl and phenyl ketones were prepared from 4-carboxycephem derivatives, at either the sulfide or sulfone oxidation level, by chemoselective Grignard reaction. Obtained products were functionalized with heterocyclothio and acyloxy substituents at C-3', C-2, or both positions. tert-Butyl ketones of the 7 alpha-chlorocephem series were in general at least as potent as the corresponding esters at inhibiting the enzyme, but improvements in hydrolytic stability were only marginal. On the other hand, tert-butyl ketones of the 7 alpha-methoxycephem series combined potent biochemical activity with acceptable hydrolytic stability, thus overstepping the esters, thiolesters, and amides reported previously. In particular, the tert-butyl ketones possessing a heterocyclothio group at C-3' or C-2 were at least as active as the corresponding tert-butyl esters but 1 order of magnitude more stable in physiologic buffers (pH 7.4, 37 degrees C). Introduction of acyloxy groups at C-2 delivered the most potent HLE inhibitors of the cephem class ever reported, with inhibition parameters often outside the determination limits of our standard protocol (second-order rate constant kon > 2,000,000 M-1 s-1; Ki at steady state < 2 nM). Keto-enol tautomerism was found to depress activity and boost hydrolytic stability. Thus, double substitution with heterocyclic thiols produced compounds with diverging properties, according to the extent of enolate formation at the investigated pH (7.4): the weakly acidic tert-butyl ketones (pKa > or = 5.8) proved to be potent inhibitors (kon over 10(4) M-1 s-1) with reasonable hydrolytic stability (t1/2 = 30-75 h), while the phenyl ketones (pKa < 4) were fair inhibitors (kon over 10(3) M-1 s-1; Ki at steady state approximately 50 nM) with hydrolytic half-lives exceeding 1000 h. Selected compounds efficiently inhibited the degradation of insoluble bovine neck elastin by HLE in a concentration-dependent manner. Intracellular HLE of polymorphonuclear leukocytes was in general unaffected; however, a lipophilic cephem sulfone apparently able to inactivate the enzyme in living cells was identified.

Cells, Cultured↗

PubMed1994

Kinetics of molecular chaperone action.

Molecular chaperones of the Hsp70 type transiently sequester unfolded segments of proteins and promote their correct folding. Target peptides were labeled with an environmentally sensitive fluorophore so that their binding to the molecular chaperone DnaK of Escherichia coli could be followed in real time. The two-step process was characterized by relaxation times of 27 seconds and 200 seconds with 2 microM DnaK and 0.1 microM ligand at 25 degrees C. In the presence of adenosine triphosphate, the formation of the complex was greatly accelerated and appeared to be a single-exponential process with a relaxation time of 0.4 second. The binding-release cycle of DnaK thus occurs in the time range of polypeptide chain elongation and folding and is too fast to be stoichiometrically coupled to the adenosine triphosphatase activity of the chaperone (turnover number, 0.13 per minute at 30 degrees C).

2-Naphthylamine↗

PubMed1993

Inhibition of the human leukocyte endopeptidases elastase and cathepsin G and of porcine pancreatic elastase by N-oleoyl derivatives of heparin.

N-oleoyl-heparin derivatives differing in their oleic acid and sulfate contents were synthesized and studied for their abilities to inhibit human leukocyte elastase (HLE), human leukocyte cathepsin G (CatG) and porcine pancreatic elastase (PPE) at pH 8.0, ionic strength 0.05 M and 37 degrees. Heparin (Hep) as well as N-oleoyl-heparins behaved as tight-binding, hyperbolic noncompetitive inhibitors of HLE (KiHep = 75 pM) and CatG (KiHep < 25 pM). The main driving force for the interaction between enzymes and glycosaminoglycans was electrostatic in nature. Under the condition [enzyme] >> Ki, the stoichiometries of the interaction with Hep were 1:2 (Hep:HLE) and 1:4 (Hep:CatG). Coupling one oleic acid residue to three disaccharide units of partially N-desulfated Hep, Ol1:3Hep, lowered HLE inhibition (Ki = 0.3 nM) and the stoichiometry of binding was reduced to 1:1. Re-N-sulfation of a similar derivative, Ol1:5Hep(SO4), containing one fatty acid residue for five disaccharide units, led to a substance with similar HLE inhibitory characteristics as Hep (Ki = 92 pM) and stoichiometry 1:2. Ol1:5Hep(SO4) was also a more efficient inhibitor of CatG (Ki < 33 pM) than Ol1:3Hep (Ki = 9.5 nM). The residual activities of N-oleoyl-Hep complexes with CatG were much lower than the corresponding activities in the presence of Hep. While oleate and Hep could not inhibit PPE, N-oleoyl-Hep, independently of fatty acid substitution and sulfate content, could inhibit this enzyme with Ki congruent to 60 nM and low residual activity. The efficient endopeptidase inhibitory characteristics of N-oleoyl-Hep derivatives, together with their non-anticoagulant properties and their capacity to interact with elastin, may be therapeutically useful in connective tissue degenerative diseases.

Amino Acid Sequence↗

PubMed1992

Analysis of glycosaminoglycans in human serum after oral administration of chondroitin sulfate.

Chondroitin sulfate was administered orally to six healthy volunteers, six patients with rheumatoid arthritis and six patients with osteoarthritis. Blood was collected at intervals before and after treatment and the glycosaminoglycan concentration was analyzed in serum using a sensitive assay based on the metachromatic reaction with 1,9-dimethylmethylene blue. The glycosaminoglycan concentration in serum before and after ingestion of chondroitin sulfate was statistically unchanged in all of the subjects studied. We suggest that chondroprotection by orally administered chondroitin sulfate is a biologically and pharmacologically unfounded theory. Any possible benefit to osteoarthritic patients after ingestion of chondroitin sulfate should be sought at the gastrointestinal rather than at the plasmatic or articular cartilage level.

Administration, Oral↗

PubMed1991

[Arthritis from a biochemical aspect].

The dynamic processes of remodeling and cartilage destruction occurring in osteoarthritis are associated with an altered metabolism of the chondrocytes. Two mechanisms can be made responsible for chondrocyte activation: Stimulation by cytokines (mainly interleukin-1) and phenotypic modulation. Remodeling of osteoarthritic joint structures is associated with cell proliferation and neovascularization, while erosion of the articular cartilage is promoted by a variety of endopeptidases of chondrocytic origin. These aspects are briefly reviewed in the light of recent findings.

Cartilage, Articular↗

PubMed1990

Effect of interleukin-1 beta on the production of cathepsin B by rabbit articular chondrocytes.

Rabbit articular chondrocytes in monolayer culture were stimulated with human recombinant interleukin-1 beta. Under the influence of the cytokine the intracellular pool of the cysteine endopeptidase cathepsin B was increased by a 2-4-fold factor, while enzyme secretion was not stimulated at a significant level. Under the same conditions, the secretion of collagenase, measured as an internal control, was stimulated about 6-fold. The effects of interleukin-1 beta were compared to those caused by phenotypic modulation. Chondrocytes modulated by serial subcultures in monolayer secreted more cathepsin B, but less collagenase than differentiated cells (cultured within collagen gels). Thus, interleukin-1 beta and phenotypic modulation affected differently two endopeptidases which are relevant in the pathogenesis of osteoarthritis.

Animals↗

PubMed1990

Interaction of human leukocyte elastase with soluble and insoluble protein substrates. A practical kinetic approach.

A progress-curve kinetic method was developed to investigate the interaction between human leukocyte elastase and macromolecular substrates, such as insoluble elastin and soluble plasma proteins. A fluorogenic, synthetic peptide (reporter substrate) was incubated in the presence of finely powdered elastin and enzyme under continuous stirring. The progress curves, which corresponded to the release of product from the reporter substrate, were very sensitive to the presence of various amounts of the macromolecular substrate. The kinetic parameters for the interaction between elastase and elastin were calculated using a pre-steady-state approach characteristic of slow-binding inhibitors. The interaction of elastase with the soluble protein substrates was studied with similar techniques, but formally treating the substrates as classical, fully competitive inhibitors. The adsorption of elastase on insoluble elastin was a time-dependent process consisting of at least three observable phases: The first step was a rapid formation of an encounter complex followed by a very slow step lasting several minutes, and the third step consisted of a steady-state release of products. On the contrary, elastase very rapidly formed productive complexes with bovine serum albumin and a human monoclonal immunoglobulin G. The progress-curve method was also suitable for analyzing the behavior of inhibitors in the presence of protein substrates. The kinetic parameters which characterize the interaction between elastase and protein substrates represent a practical tool to formulate hypotheses on the efficiency of inhibitors in vivo.

Amino Acid Sequence↗

PubMed1990

The kinetic mechanism of inhibition of human leukocyte elastase by MR889, a new cyclic thiolic compound.

The cyclic thiolic compound 2-[3-thiophencarboxythio]-N-[dihydro-2(3H)-thiophenone-3-il] - propionamide (MR889) was investigated as inhibitor of endopeptidases. The activity of bovine pancreatic alpha-chymotrypsin, human leukocyte cathepsin G and rabbit liver cathepsin B was not affected by MR889, whereas porcine pancreatic elastase and human leukocyte elastase were inhibited. The kinetic mechanism of inhibition of human leukocyte elastase was of the reversible, slow-binding, fully competitive type. The rate constants for complex formation between MR889 and leukocyte elastase, determined by pre-steady-state kinetic analysis in the presence of a tetrapeptide substrate at 37 degrees and pH 7.40, were kon = 2363 +/- 15 M-1 sec-1, koff = 3.01 +/- 0.34 x 10(-3) sec-1. The inhibition equilibrium constant was Ki = koff/kon = 1.27 +/- 0.15 microM. Ki, calculated from steady-state kinetic experiments, was 1.38 microM. MR889 also inhibited the elastolytic activity of leukocyte elastase, as determined with insoluble elastin as the substrate.

Cathepsin B↗

PubMed1990

Cathepsin B secretion by rabbit articular chondrocytes: modulation by cycloheximide and glycosaminoglycans.

Rabbit articular chondrocytes in monolayer culture are modulated away from their differentiated state and undergo morphological and biochemical changes. One of the characteristics of the modulated state is an abnormally high production of the cysteine endopeptidase cathepsin B. Addition to chondrocyte cultures of the protein biosynthesis inhibitor, cycloheximide, resulted in a concentration-dependent reduction of cathepsin B secretion, which was fully restored after removal of cycloheximide. Glycosaminoglycans added to the culture medium of modulated chondrocytes partially reduced the rate of secretion of cathepsin B, this effect being dependent on their structure, the degree of sulfation, and concentration. The age of the chondrocytes and the duration of the treatment also influenced this response. The switching off of cathepsin B release was apparently best favored by a high concentration of negatively charged sulfate groups attached to a polymeric glycosaminoglycan chain; this simulates the natural environment of the chondrocytes in articular cartilage.

Animals↗

PubMed1988

Interaction of site specific hirudin variants with alpha-thrombin.

The kinetics of complex formation between recombinant hirudin or recombinant hirudin mutants with thrombin were analyzed. In order to elucidate the inhibitor's reactive site peptide bond predetermined amino acid substitutions were introduced at positions of basic amino acid residues by means of site-directed mutagenesis of a hirudin gene. In comparison to recombinant hirudin (Ki = 19 pM) only those mutant inhibitors which were modified at amino acid position Lys47 showed a higher Ki value for their complexes with thrombin. The observed effects are mainly due to increased koff rate constants.

Algorithms↗