Trypsin-kallikrein Inhibitor Research Articles

The bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor): a milestone protein.

The pancreatic Kunitz inhibitor, also known as aprotinin, bovine basic pancreatic trypsin inhibitor (BPTI), and trypsin-kallikrein inhibitor, is one of the most extensively studied globular proteins. It has proved to be a particularly attractive and powerful tool for studying protein conformation as well as molecular bases of protein/protein interaction(s) and (macro)molecular recognition. BPTI has a relatively broad specificity, inhibiting trypsin- as well as chymotrypsin- and elastase-like serine (pro)enzymes endowed with very different primary specificity. BPTI reacts rapidly with serine proteases to form stable complexes, but the enzyme: inhibitor complex formation may involve several intermediates corresponding to discrete reaction steps. Moreover, BPTI inhibits the nitric oxide synthase type-I and -II action and impairs K+ transport by Ca2+-activated K+ channels. Clinically, the use of BPTI in selected surgical interventions, such as cardiopulmonary surgery and orthotopic liver transplantation, is advised, as it significantly reduces hemorrhagic complications and thus blood-transfusion requirements. Here, the structural, inhibition, and bio-medical aspects of BPTI are reported.

Permeation of Proteins through a Biodegradable Hydrogel

The permeation of proteins of different molecular weights into a biodegradable hydrogel, a model system for controlled release of biologically active macromolecules, was investigated. The hydrogels were prepared by a crosslinking copolymerization from a biodegradable synthetic polypeptide, poly[N5-(2-hydroxyethyl)-L-glutamine] (PHEG) with methacryloylated side chains and acrylamide. The permeation of sodium azide, trypsin-kallikrein inhibitor (TKI), trypsin, and albumin into the gels with network densities between 0.04 and 0.226 mmol/mL were determined. The permeation of sodium azide was not influenced by the gel network density. The permeability of T[and trypsin was controlled by gels with permeability coefficients ranging from 2.8 x 10−9 to 4.0 x 10−10 mmol/mL. Gels with different network densities controlled the diffusion of peptides and proteins with molecular weights between 6,000 and 60,000. The combined effect of diffusion and biodegradation of the gel on the permeation rate of macromolecules was determined for poly-α,β-[N-(2-hydroxyethyl)-DL-aspartamide]. A decrease in the gel cross-linking density due to the degradation efficiently increased the permeability to the model macromolecule.

Detection of antigen immunologically related to basic pancreatic inhibitor (kunitz) in porcine blood plasma by immunoaffinity chromatography

Rabbit antiserum against trypsin-kallikrein inhibitor (TKI) was prepared. Purified immunoglobulin G (IgG) fraction was bound to Sepharose 4B. An antigen immunologically related to TKI was obtained from porcine blood plasma by adsorbing it onto the immunosorbent column. Its immunoreactivity with TKI antibodies was confirmed by immunoelectrophoresis. The antigen was an inhibitor of trypsin and acrosin.

Carbohydrate-Containing Derivatives of the Trypsin-Kallikrein Inhibitor Aprotinin from Bovine Organs. II. Inhibitor Coupled to the (Carboxymethyl)dextran Derivatives of D-Galactose

The trypsin-kallikrein inhibitor aprotinin was coupled to (carboxymethyl)dextran derivatives of D-galactose. The conjugates contained 14 and 38 D-galactose residues/mol of protein, respectively. The apparent dissociation constants Ki of the complexes between trypsin and modified aprotinins proved to be one order of magnitude higher than the respective values for native aprotinin. The distribution of the modified aprotinins in rat organs after endocardial injection has been studied. The conjugates of aprotinin with (carboxymethyl)dextran derivatives of D-galactose were characterized by decreased clearance rates; they accumulated in the active form in liver. The accumulation was 2.5-10 times higher than native aprotinin for the time of observation (5 min-2 h).

Carbohydrate-containing derivatives of the trypsin-kallikrein inhibitor aprotinin from bovine organs. I. Modification with lactose, characterization and behaviour of the preparation in vivo.

The trypsin-kallikrein inhibitor aprotinin was modified with lactose. The influence of reactant concentrations, temperature, reaction time and sodium borohydride on the carbohydrate residue content and the inhibiting activity of glycated aprotinin were studied. Glycation of aprotinin neither shifts the pH optimum of the inhibitor-trypsin association reaction nor does it alter the apparent dissociation constant Ki of the complex measured at pH optimum. Glycation by lactose stabilizes aprotinin against denaturation by increased temperature. The distribution of native and modified aprotinin in rat organs after endocardiac injection was studied. Fixation of glycated aprotinin increases 2.5- to 3-fold in liver and decreases 2-fold in kidneys during the observation time (5 min-2 h) compared to native aprotinin.

High molecular weight derivative of trypsin‐kallikrein inhibitor for potential medical use, 1. Preparation and characterization

AbstractBovine trypsin‐kallikrein inhibitor (TKI) was bound on three different water‐soluble hydrophilic carriers derived from poly[N‐(2‐hydroxyethyl)‐D,L‐aspartamide] and containing a binding group (aromatic amine) and an additional modifying group (anionic or hydrophobic). The binding was performed by coupling of diazonium salts formed on the carrier with tyrosines in TKI. The product was characterized by means of sedimentation measurement, viscometry, electrophoresis, amino acid analysis, GPC, and antiproteolytic activity. TKI was found to be attached to the carrier by one or two tyrosines, the latter case being more frequent than it would correspond to independent random reactivity of the accessible tyrosines and the carrier molecules are crosslinked by the attached TKI. This is due to a fraction of two point attached TKI, as estimated according to a tree‐like model. All three samples preserved good activity toward trypsin and chymotrypsin.

A high-molecular mass derivative of trypsin-kallikrein inhibitor for potential medical use. II. Study of inhibitory activity.

Three derivatives of poly[alpha-N beta-(2-hydroxyethyl)-DL-asparagine, beta-N alpha-(2-hydroxyethyl)-DL-asparagine] were used as soluble carrier polymers for binding the trypsin-kallikrein inhibitor (TKI). In addition to the parent polymer an anionic derivative with 19.2 mol of carboxyl groups/100 mol of residues and a hydrophobized derivative with 18.0 mol of butyl groups/100 mol of residues were also used. All carriers contained 6.6 mol of 2-(4-aminobenzamido)ethyl residues/100 mol which were used for the binding after diazotation. All derivatives retained a high inhibitory activity toward trypsin and chymotrypsin whereas the effect on kallikrein was substantially reduced. The kinetic constants were estimated under the simplifying assumption, that classical kinetic equations for the enzyme-inhibitor interaction may be applied. The equilibrium dissociation constants (5 X 10(-9) mol/l) and association rate constants (1 X 10(5) l X mol-1 X s-1) with respect to trypsin are unaffected by the two chemical modifications of the carrier. The increase in the equilibrium dissociation constant (compared to native TKI) is probably a result of the decreasing strength of the enzyme-inhibitor complex rather than a manifestation of reduced rate of diffusion of the enzyme to the active center of the polymer-bound inhibitor.

Effect of Modification of Reactive Lysine on Antitryptic and Antichymotryptic Activity of Proteinase Inhibitor from Cow Colostrum

40% of the primary structure of the cow colostrum proteinase inhibitor (CTI) is homologous with the structure of the trypsin kallikrein inhibitor (TKI) from bovine organs; the positions of the reactive lysine residues are also the same in both inhibitors. Both CTI and TKI were modified by carbamoylation and the fully labeled derivatives were isolated by ion-exchange chromatography. The effect of the modification on the antitryptic and antichymotryptic activity of both inhibitors was investigated. The antichymotryptic activity of both inhibitors is not decreased after the modification. The antitryptic activity of modified TKI is retained, yet the dissociation constant of the complex of the modified inhibitor with trypsin is considerably increased; nevertheless, modified TKI is a good trypsin inhibitor. The antitryptic activity of modified CTI is hardly detectable. We explain this difference in the behaviour of both inhibitors by a replacement of basic residues Arg-17 and Arg-39 in TKI by neutral amino acids Ala-20 and Gln-42 in CTI.

Kinetics of the interaction of alpha-chymotrypsin with trypsin kallikrein inhibitor (Kunitz) in which the reactive-site peptide bond Lys-15--Ala-16 is split.

Modified trypsin kallikrein inhibitor (I*), with the reactive-site peptide bond Lys-15--Ala-16 split, reacts with alpha-chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C:E + I in equilibrium X leads to C. Formation X constitutes a fast pre-equilibrium (equilibrium constant Kx = 7 X 10(-5) M, association rate constant kx = 4 X 10(3)M-1s-1) to the slow reaction X leads to C (rate constant kc = 2 X 10(-3) s-1), all values at pH 7.5. No intermediate X is observed when alpha-chymotrypsin reacts with I*-OMe in which the carboxyl group of Lys-15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*-OMe indicate tha formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with beta-trypsin and I* or I*-OMe.

Reaction of ozone with protease inhibitors from bovine pancreas, egg white, and human serum

Ozone inactivates the bovine pancreas trypsin-kallikrein inhibitor. Tyrosine and methionine residues are oxidized. When methionine is completely oxidized and 0.5 of the four tyrosine residues is oxidized, 90% of the inhibitor capacity is retained. When both methionine and tyrosine residues are completely oxidized, 37% of the inhibitor capacity is retained. For 50% loss of anti-trypsin activity, 62 moles of 0 3/mole of protein were required. Inhibition of the chicken ovomucoid proteinase inhibitor by ozone is accompanied by oxidation of tyrosine, histidine, and methionine residues. For 50% loss of anti-trypsin activity, 32 moles of O 3/mole of protein were required. Anti-trypsin activity of diluted human serum was only slightly inhibited by ozone. Purified α-1-antiprotease from human serum was 50% inactivated by 88 moles of O 3/mole of protein. The oxidation of free tyrosine by ozone proceeds by way of dihydroxyphenylalanine and dihydroxyphenylalanine quinone, finally yielding colored polymeric material.

ARGININE MODIFICATION IN KUNITZ BOVINE TRYPSIN INHIBITOR THROUGH 1, 2‐CYCLOHEXANEDIONE*

Arginine residues (5.5 out of 6) of the trypsin-kallikrein inhibitor from bovine organs (Kunitz inhibitor) were selectively modified by reaction with 1, 2-cyclohexanedione in sodium borate buffer, pH 9.0. The modified inhibitor is still highly active in inhibiting trypsin and chymotrypsin at 1:1 inhibitor: enzyme molar ratio and full inhibition was achieved at slightly higher molar ratio. The extent of correct refolding, upon reoxidation, of the reduced, arginine-modified inhibitor is diminished and regeneration of two arginines occurred under the reduction conditions. The stability constants and the standard-free energies of binding of the complexes between trypsin, or chymotrypsin, and the native, the arginine-modified and the reduced and reoxidized arginine-modified inhibitor have been determined from inhibitory assays.

Effect of Serum Proteinase Inhibitors on The Fertilizing Capacity of Rabbit Spermatozoa

The in vivo effect of purified human alpha1-antitrypsin and inter-alpha-trypsin inhibitor on the fertilizing ability of capacitated rabbit spermatozoa was investigated and compared with that of the trypsin-kallikrein inhibitor from bovine organs (Trasylol). Only 250 mug of alpha1-antitrypsin/5 X 10(4) sperm in 0.05 ml inhibited fertilization by more than 50%. Lower concentrations of alpha1-antitrypsin (50 mug/5 X 10(4) sperm in 0.05 mo) and inter-alpha-trypsin inhibitor in amounts of 250 mug/5 X 10(4) sperm and less had no significant effect on the fertilization rate. Comparative experiments with Trasylol in conentrations of 50 mug/5 X 10(4) sperm resulted in more than 50% fertilization inhibition, whereas 100 mug/5 X 10(4) sperm decreased the fertilization rate by approximately 90%. The differences in the antifertility effect of these acrosin inhibitors toward capacitated spermatozoa may be due to differences in molecular weight. The effective concentrations required for fertilization inhibition appear to be relatively high under the experimental conditions used.

67] Trypsin-kallikrein inhibitors from snails (Helix pomatia)

Proteinase inhibitors have long been known in the species of Annelida. Investigations on another androgyne species from the pedigree of Mollusca led to the discovery of proteinase inhibitors in snails (Helix pomatia), in cuttlefish (Loligo vulgaris) and in mussels (Mytilus edulis ).The snail (Helix pomatia) produces two inhibitory proteins differing in molecular size and properties. A group of low-molecular-weight, acid- and heat-stable trypsin–kallikrein isoinhibitors of broad specificity is synthesized from the epidermal parts of the body walls and is excreted into the mucus. An acid- and heat-labile inhibitor of higher molecular weight is produced and secreted from the albumin gland. Easy separation of both inhibitor proteins obtained from extracts of undissected snails can be achieved by a single step of ammonium sulfate fractionation. In this chapter a comparison of the epidermal (or mucous) inhibitors are related in structure and properties to the bovine trypsin–kallikrein inhibitor (Kunitz), the cow colostrum inhibitor, the turtle egg white inhibitor, the Russell's viper venom inhibitor, and the toxins I and K from black mamba venom. It also elaborates on Trypsin–Kallikrein inhibitor purification procedure.

Replacement of lysine by arginine, phenylalanine and tryptophan in the reactive site of the bovine trypsin-kallikrein inhibitor (Kunitz) and change of the inhibitory properties.

The reactive-site sequence of a proteinase inhibitor can be written as . . . -P3-P2-P1-P'1-P'2-P'3- . . . , where-P1-P'1-denotes the reactive site. Three semisynthetic homologues have been synthesized of the bovine trypsin-kallikrein inhibitor (Kunitz) with either arginine, phenylalanine or tryptophan in place of the reactive-site residue P1, lysine-15. These homologues correspond to gene products after mutation of the lysine 15 DNA codon to an arginine, phenylalanine or tryptophan DNA codon. Starting from native (virgin) inhibitor, reactive-site hydrolyzed, still active (modified) inhibitor was prepared by chemical and enzymic reactions. Modified inhibitor was then converted into inactive des-Lys15-inhibitor by reaction with carboxypeptidase B. Inactive des-Lys15-inhibitor was reactivated by enzymic replacement of the P1 residue according to Leary and Laskowski, Jr. The introduction of arginine was catalyzed by an inverse reaction with carboxypeptidase B, while phenylalanine or tryptophan were replaced by carboxypeptidase A. The reactivated semisynthetic inhibitors were trapped by complex formation with either trypsin or chymotrypsin. The enzyme - inhibitor complexes were subjected to kinetic-control dissociation, and the semisynthetic virgin inhibitors were isolated. The inhibitory properties of the semisynthetic inhibitors have been investigated against bovine trypsin and chymotrypsin and against porcine pancreatic kallikrein and plasmin. The homologues with either lysine or arginine in the P1 position are equally good inhibitors of trypsin, plasmin and kallikrein. The Arg-15-homologue is a slightly more effective kallikrein inhibitor than the Lys15-inhibitor. The semisynthetic phenylalanine and tryptophan homologues, however, are weak inhibitors of trypsin and still weaker inhibitors of kallikrein, but are excellent inhibitors of chymotrypsin. Their association constant with chymotrypsin is at least ten times higher than that of native Lys-15-inhibitor. A dramatic specificity change is observed with the phenylalanine and tryptophan homologues, which in contrast to the native inhibitor do not at all inhibit porcine plasmin. Thus, the nature of the P1 residue strongly influences the primary inhibitory specificity of the bovine inhibitor (Kunitz).

Preparation and characterization of the active derivative bovine trypsin-kallikrein inhibitor (Kunitz) with the reactive site lysine-15 -- alanine-16 hydrolyzed.

The derivative of the trypsin-kallikrein inhibitor (Kunitz), TKI+, was prepared with the reactive-site peptide bond Lys-15-Ala-16 hydrolyzed. This was achieved by selective borohydride reduction of the Cys-14-Cys-38 disulfide bond, followed by tryptic cleavage of the reactive-site peptide bond, air reoxidation of the half-cystine residues, and purification by ion-exchange chromatography. The derivative corresponds to the hypothetical 'modified' inhibitor TKI+, which so far could not be obtained from virgin inhibitor by a direct modification reaction (partial proteolysis). The derivative isolated was homogeneous as revealed by amino acid analysis, disc electrophoresis, inactivation by carboxypeptidase B, and inactivation by sodium borohydride reduction. The inhibitory activity of the sodium-borohydride-reduced inhibitor was fully recovered after air reoxidation. The site of cleavage in the inhibitor was confirmed by performic acid oxidation and subsequent isolation of the two corresponding peptides containing residues 1-15 and 16-58 of the entire polypeptide chain. From several aminopeptidases tested only aminopeptidase K rapidly cleaved Ala-16 and Arg-17 from the modified inhibitor and at a reduced rate Ile-18. Des-(Ala16,Arg17)-inhibitor and des-Ala16-inhibitor are both lacking a strong inhibitory activity against bovine trypsin. This indicates a decrease in the association constant by factor of at least 10(8)-10(10). The reactive-site-modified inhibitor is not subject to further enzymic breakdown and therefore is a permanent inhibitor of trypsin. However, the modified inhibitor forms the inactive complex much slower than virgin inhibitor. In the modified inhibitor the hydrolyzed peptide bone was resynthesized to yield virgin inhibitor by forming the complex with trypsin and subjecting the complex to kinetic control dissociation. This proves that the bond Lys-15--Ala-16 is at the reactive site of this inhibitor. Preparation of a modified and still active inhibitor (Kunitz) is in agreement with the general model proposed for the interaction of proteinase-inhibitor--proteinase interactions. This presents new evidence that this model is generally applicable.

The amino-acid sequence of isoinhibitor K form snails (Helix pomatia). A sequence determination by automated Edman degradation and mass-spectral identification of the phenylthiohydantoins.

Performic-acid-oxidized isoinhibitor K of snails (Helix pomatia) was subjected to arginine-directed tryptic proteolysis. Six peptide fragments including one overlap peptide from limited cleavage of the Arg-3-Pro-4 bond were purified to homogeneity. Four arginine peptides and the C-terminal peptide were sequenced by automatic Edman degradation using a special peptide program. The phenylthiohydantoins were all identified by chemical ionization mass spectrometry, except four cysteic acid residues that were identified on an amino acid analyzer after acid hydrolysis. Quantitative evaluation of the phenylthiohydantoins by chemical ionization mass spectrometry using total molecular-ion beam integration greatly facilitated sequencing. The mass spectrum of the dipeptide less than Glu-Gly revealed that the N-terminus was blocked by pyroglutamic acid. The complete amino acid sequence of isoinhibitor K was determined. An almost 50% homology between the sequences of the snail inhibitor and the bovine trypsin-kallikrein inhibitor (Kunitz) became obvious. A comparison of all homologous sequences of this particular class of proteins known to date is presented.

Trypsin-kallikrein isoinhibitor K (type Kunitz) from snails (Helix pomatia). Purification and characterization.

A basic proteinase inhibitor, isoinhibitor K, was purified by SE-Sephadex C-25 column chromatography from the mixture of acid-stable and heat-stable isoinhibitors of the snail (Helix pomatia). Isoinhibitor K is homogeneous in polyacrylamide gel, cellulose acetate and polyacrylamide-dodecylsulfate electrophoresis. From the electrophoretic mobility in dodecylsulfate-polyacrylamide gel and apparent molecular weight of 6500 +/- 200 was estimated. From the amino acid composition the inhibitor consists of 58 amino acid residues. It contains three disulfide bridges, a C-terminal valine and a lysine residue at the reactive site. Isoinhibitor K inhibits the enzymes: bovine trypsin and chymotrypsin, porcine plasmin and pancreatic kallikrein, the trypsin-like component of Streptomyces griseus proteinase-pronase E, and fungi proteinase K from Tritirachium album Limber, which is only inhibited very slightly in contrast to the effect of the mixture of isoinhibitors. The inhibitory effect of isoinhibitor K against these enzymes is compared to that of the mixture or of other isoinhibitors. The following enzymes are not inhibited by isoinhibitor K: Aspergillus proteinase P and alkaline bacillus proteinase 2231 (Röhm), which both are inhibited by the mixture of isoinhibitors. Porcine elastase, bacterial proteinase N (M) (Röhm), and a trypsin-like proteinase from wheat are not inhibited, porcine acrosin and porcine serum kallikrein only to a very minor extent by the mixture of isoinhibitors. Reactive-site peptide-bond cleavage during inhibition could not be detected. Thus, the inhibitory behaviour is just as broad in specificity and as unusual as that of the trypsin-kallikrein inhibitor (Kunitz) from bovine organs. The N-terminus is blocked by pyroglutamic acid. Isoinhibitor K is the main component of the isoinhibitors secreted into the mucus and amounts to 35-40% of the mixture.

Kinetics of binding of bovine trypsin-killikrein inhibitor (K unitz) in which the reactive-site peptide bond Lys-15--Ala-16 is cleaved, to alpha-chymotrypsin and beta-trypsin.

Equilibrium measurements of the binding of reactive-site-cleaved (modified) bovine trypsin-kallikrein inhibitor (Kunitz) to alpha-chymotrypsin and beta-trypsin show a stoichiometric 1:1 association with high binding constants. At least in the case of chymotrypsin much evidence is presented that the reaction with modified inhibitor leads to the same complex as the reaction with virgin inhibitor does. The association rate constant of modified inhibitor with chymotrypsin at pH 7, 22.5 degrees C is 15.8 M-1 S-1. This is about 2 x 10(4) times slower than the binding of virgin inhibitor to that enzyme. In the analogous reaction of modified inhibitor with beta-trypsin, however, the association rate constant (1.2 x 10(4) M-1 s-1 at pH 6.9, 22.5 degrees C) is of about the same order of magnitude as it is in the reaction of virgin inhibitor and trypsin. These and analogous phenomena observed in the reactions of virgin and modified soybean trypsin inhibitor (Kunitz) with alpha-chymotrypsin and beta-trypsin suggest that the specificity of both inhibitors to trypsin is strongly reflected in the association rate constants of the modified forms. The dissociation rate constants of the complexes of trypsin-kallikrein inhibitor with chymotrypsin or with trypsin towards the modified inhibitor are estimated to be unmeasurably slow (half-life times of 45 or 1.5 x 10(4) years, respectively).

The effect of cleaving the reactive-site peptide bond Lys-15--Ala-16 on the conformation of bovine trypsin-kallikrein inhibitor (K unitz) as revealed by solvent-perturbation spectra, circular dichroism and fluorescence.

Spectroscopic measurements of virgin bovine trypsin-kallikrein inhibitor and its modified species (in which the reactive-site peptide bond Lys-15--Ala-16 is split) indicate a conformational difference between both proteins. The inhibitor contains four tyrosines but no tryptophan. In the modified inhibitor a tyrosyl blue shift is seen in the difference absorption spectrum of modified against virgin inhibitor. The solvent perturbation spectra show an increase of the fraction of exposed tyrosyls from 0.45 in the virgin inhibitor to 0.59 in the modified form. Comparison of the circular dichroism spectra of the modified and virgin inhibitors reveals a decrease of the mean residue ellipticity in the tyrosine and peptide bond region of the modified inhibitor. In the fluorescence spectra a 50% increase in the quantum yield of the tyrosine fluorescence is observed in the modified inhibitor. All these spectroscopic data support the idea, which is also evidenced by the X-ray crystallographic model, that in the modified inhibitor up to five residues from Ala-16 to Arg-20 gain rotational freedom.

Inactivation of the trypsin-kallikrein inhibitor (Kunitz) by cleavage of the dipeptide Ala 16--Arg 17 in the reactive site.

Inactivation of the trypsin-kallikrein inhibitor (Kunitz) by cleavage of the dipeptide Ala 16--Arg 17 in the reactive site.