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PubMed1984

Activation, desensitization, and recycling of frog erythrocyte beta-adrenergic receptors. Differential perturbation by in situ trypsinization.

We have utilized limited in situ trypsinization of the adenylate cyclase-coupled beta-adrenergic receptor of frog erythrocytes to probe the processes of receptor activation, desensitization, and recycling. Treatment of intact erythrocytes with trypsin (1 mg/ml) for 1 h at 20 degrees C converts all the receptor peptides (identified by photoaffinity labeling with p-azido-125I-benzylcarazolol) from a Mr approximately 58,000 to a Mr approximately 40,000 species. Nonetheless, the trypsinized beta-adrenergic receptors bind agonists and antagonists with unaltered affinity and with no change in the number of binding sites. Moreover, the ability of the proteolyzed receptors to interact with the nucleotide regulatory protein to form a high affinity guanine nucleotide-sensitive state and to activate adenylate cyclase were also unaltered. However, upon exposure of intact cells to the agonist isoproterenol, trypsinized beta-adrenergic receptors were more rapidly and more completely cleared from the plasma membranes ("down-regulated") than untrypsinized receptors. Whereas down-regulated receptors from nontrypsinized cells appear to recycle to the cell surface after removal of the agonist, internalized trypsinized beta-adrenergic receptors do not recycle to the plasma membrane and appear to be degraded within the cell. Moreover, when internalized receptors, recovered in a light vesicle fraction, were fused with a heterologous adenylate cyclase system, untreated but not trypsinized receptors reconstituted catecholamine stimulation of the enzyme. These data suggest that the beta-adrenergic receptor contains a trypsin-sensitive site which is exposed on the outer surface of the plasma membrane. Proteolysis at this site releases a fragment which though not critically involved in either ligand binding or "effector coupling" might be important for anchoring the receptors in the plasma membrane. These data also suggest that in situ proteolysis of the receptors might serve as a physiological trigger for their internalization and degradation.

Adenylyl Cyclases↗
PubMed1976

Effect of endotoxin and trypsin on the blood pressor response to catecholamines in normo- and hypothermic rabbits.

The action of endotoxin, trypsin or hypothermia on the vascular reactivity to catecholamines was investigated in rabbits, and with trypsin in normothermic dogs as well. In rabbits, LD10 of Escherichia coli 0111 endotoxin increased the blood pressor effect of i.v. adrenaline or noradrenaline with maxima at 60-90 min. LD50 endotoxin elicited a vascular hyporeactivity to catecholamines within an hour. 22 hours later, however, a hyperreactivity to catecholamines developed. At this time, repeated administration of LD50 endotoxin did not reduce the increased catecholamine responsiveness. Trypsin (i.v. 1.5 mg/kg) also potentiated the pressor effect of catecholamines in rabbits and dogs with maxima at 5-10 min. Higher trypsin doses induced a hyporeactivity. LD50 endotoxin in a single or a repeated dose after 22 hours did not decrease the blood pressure of cooled rabbits and failed to alter the vascular reactivity to adrenaline or noradrenaline. The blood pressure effect of trypsin differed in character in normo- and hypothermic rabbits, depending on the depth of cooling. Low body temperature eliminated the potentiating effect of trypsin to the blood pressor action of catecholamines. In normothermic rabbits pretreated with amino-pyrine and phenylbutazone, the blood pressure and the catecholamine potentiation effects of endotoxin or trypsin were inhibited or considerably reduced. The results support the significance of altered vascular reactivity to catecholamines under different pathologic conditions where endotoxin and/or proteases may occur.

Aminopyrine↗
PubMed1996

Attempts to convert chymotrypsin to trypsin.

Trypsin and chymotrypsin have specificity pockets of essentially the same geometry, yet trypsin is specific for basic while chymotrypsin for bulky hydrophobic residues at the P1 site of the substrate. A model by Steitz, Henderson and Blow suggested the presence of a negative charge at site 189 as the major specificity determinant: Asp189 results in tryptic, while the lack of it chymotryptic specificity. However, recent mutagenesis studies have shown that a successful conversion of the specificity of trypsin to that of chymotrypsin requires the substitution of amino acids at sites 138, 172 and at thirteen other positions in two surface loops, that do not directly contact the substrate. For further testing the significance of these sites in substrate discrimination in trypsin and chymotrypsin, we tried to change the chymotrypsin specificity to trypsin-like specificity by introducing reverse substitutions in rat chymotrypsin. We report here that the specificity conversion is poor: the Ser189Asp mutation reduced the activity but the specificity remained chymotrypsin-like; on further substitutions the activity decreased further on both tryptic and chymotryptic substrates and the specificity was lost or became slightly trypsin-like. Our results indicate that in addition to structural elements already studied, further (chymotrypsin) specific sites have to be mutated to accomplish a chymotrypsin --> trypsin specificity conversion.

Amino Acid Sequence↗
PubMed1996

Attempts to convert chymotrypsin to trypsin.

Trypsin and chymotrypsin have specificity pockets of essentially the same geometry, yet trypsin is specific for basic while chymotrypsin for bulky hydrophobic residues at the P1 site of the substrate. A model by Steitz, Henderson and Blow suggested the presence of a negative charge at site 189 as the major specificity determinant: Asp189 results in tryptic, while the lack of it chymotryptic specificity. However, recent mutagenesis studies have shown that a successful conversion of the specificity of trypsin to that of chymotrypsin requires the substitution of amino acids at sites 138, 172 and at thirteen other positions in two surface loops, that do not directly contact the substrate. For further testing the significance of these sites in substrate discrimination in trypsin and chymotrypsin, we tried to change the chymotrypsin specificity to trypsin-like specificity by introducing reverse substitutions in rat chymotrypsin. We report here that the specificity conversion is poor: the Ser189Asp mutation reduced the activity but the specificity remained chymotrypsin-like; on further substitutions the activity decreased further on both tryptic and chymotryptic substrates and the specificity was lost or became slightly trypsin-like. Our results indicate that in addition to structural elements already studied, further (chymotrypsin) specific sites have to be mutated to accomplish a chymotrypsin-->trypsin specificity conversion.

Amino Acid Sequence↗
PubMed1976

Preparation and characterization of a dextran-trypsin conjugate.

Bovine pancreatic trypsin was coupled to dextran after activation of the polysaccharide by cyanogen bromide. The soluble dextran-trypsin conjugated was purified by molecular sieve chromatography. After coupling, 53% of the esterase activity of trypsin remained, but the conjugate had only 7% of the caseinolytic activity of the native enzyme. The modified trypsin showed greater resistance than the native enzyme to inactivation by heat treatment, autodigestion, or denaturing agents, and was also more resistant to inhibition by trypsin inhibitors, particularly ovomucoid. Treatment with dextranase partly removed the improved stability properties and resistance to inhibition of the trypsin-dextran conjugate. The conjugated enzyme preparation consists of a heterogenous mixture of macromolecular aggregates, each containing many trypsin and many dextran molecules linked together. Intramolecular cross-linking of enzyme molecules by polysaccharide chains is considered to be responsible for stabilization of the tertiary structure of the enzyme molecules in the conjugate.

Animals↗
PubMed1988

Zymogen activation: effect of peptides sequentially related to the bovine beta-trypsin N-terminus on Kazal inhibitor and benzamidine binding to bovine trypsinogen.

The activating effect of peptides sequentially related to the Ile 16-Val17-Gly18 N-terminus of bovine beta-trypsin (namely Ile-Val-Gly, Ile-Val, Ile-Leu, Ile-Ala, Val-Val, Leu-Val, and Val-Leu) on the thermodynamic parameters for the binding of the porcine pancreatic secretory trypsin inhibitor (Kazal inhibitor) and benzamidine to bovine trypsinogen was investigated at pH 5.5 (Bis tris-HCl buffer, I = 0.1 M) and T = 21 +/- 0.5 degrees C. Thermodynamic parameters for Kazal inhibitor and benzamidine association to the binary peptide/zymogen adducts are more favorable than those observed for ligand binding to the proenzyme alone, although never as much as those reported for the formation of bovine beta-trypsin/Kazal inhibitor and bovine beta-trypsin/benzamidine adducts. Analogously, the affinity of activating peptides for the binary proenzyme/Kazal inhibitor and binary proenzyme/benzamidine complexes is higher than that observed for peptide binding to free bovine trypsinogen. Differences in affinity for ligand binding to free bovine trypsinogen, to its binary adducts and to bovine beta-trypsin suggest the presence of different activation levels of the proenzyme, none of which structurally coincide with that achieved in bovine beta-trypsin. The existence of different discrete states suggests that the zymogen-to-active enzyme transition should not be considered as a two-state process but as a multistep event.

Amidines↗
PubMed1978

The elimination in dogs of trypsin-alpha-macroglobulin complexes inactivated by the Kazal or the Kunitz inhibitor.

The elimination of trypsin-alpha-macroglobulin complexes and similar complexes with the trypsin inactivated by low-molecular weight inhibitor was studied in anesthetized dogs. The complex was inactivated either by the Kazal (pancreatic secretory trypsin inhibitor, PSTI) or the Kunitz inhibitor (Trasylol BE). The inhibitors were labelled with 125I and in the case of the trypsin-alpha-macroglobulin complex the trypsin was labelled with 125I. All of the inactivated complexes exhibited a half-life of about 5 min in the dog. The elimination in plasma was exponential until 80 - 85% of the initial dose was cleared in 30 min and nearly negligible thereafter as seen by radioactivity measurements. Simultaneously increasing amounts of dialyzable radioactive substances with a lower molecular weight than the inhibitors were recovered in the urine. No significant differences in the elimination of trypsin-alpha-macroglobulin complexes were detected in plasma or in the urine before and after inactivation with the Kazal inhibitor (PSTI) or the Kunitz inhibitor (Trasylol BE).

Animals↗
PubMed1980

A calorimetric comparison of trypsin and its anhydro modification in complex formation with Kunitz soybean inhibitor.

The heat of complex formation between native trypsin or its anhydro modification (Ako, H., Foster, R. J., and Ryan, C. J. (1972) Biochem. Biophys. Res Commun. 47, 1402--1407) with native or cleaved Kunitz soybean inhibitor (Finkenstadt, W. R., and Laskowski, M., Jr. (1965) J. Biol. Chem. 240, 962--963) has been observed from pH 3.5 to 7.5. Steep dependence of reaction heat upon pH between pH 3.5 and 4.25 is observed with native inhibitor in reaction with both trypsin and anhydrotrypsin. The character of this heat-pH relationship is consistent with a cooperative process involving three to four ionizable groups. Above pH 4.25, trypsin and anhydrotrypsin in reaction with native inhibitor have quite different pH dependencies. Native trypsin shows an apparent pK near pH 5, whereas anhydrotrypsin + inhibitor reaction heat remains nearly constant from pH 4.5 to 6, and shows an apparent pK near pH 7.0. Above pH 4, the reaction heat-pH relations for native or cleaved inhibitor in reaction with trypsin are only slightly different (Barnhill, M. T., Jr., and Trowbridge, C. G. (1975) J. Biol. Chem. 250, 5501--5507). On the other hand, substitution of cleaved for native inhibitor in reaction with anhydrotrypsin causes a greatly reduced reaction heat, with no clearly developed pH dependence from pH 5.5 to 7.5. The reaction heat-pH relationships are analyzed thermodynamically in terms of the temperature coefficient of a pH-dependent equilibrium constant. It is clear that conversion of the active site serine side chain of trypsin to dehydroalanine changes the pH dependence of its reaction heat with soybean inhibitor, and that the complex formed is sensitive to the state of the scissile inhibitor bond. These differences provide a comparison of a protein-protein association with and without covalent bond formation between reactants.

Animals↗
PubMed1990

Trypsin treatment of bovine embryos after in vitro exposure to infectious bovine rhinotracheitis virus or bovine herpesvirus-4.

The objectives of this study were to evaluate the efficacy of trypsin treatment for the removal/inactivation of infectious bovine rhinotracheitis virus (IBRV) adhering to zona pellucida-intact (ZP-I) bovine embryos and to determine if bovine herpesvirus-4 (BHV-4) adheres to ZP-I bovine embryos. When adherence of BHV-4 was demonstrated, an additional objective was to determine whether trypsin treatment removes or inactivates this virus. A total of 139 ZP-I embryos was collected from superovulated donor cows at 7 d after estrus. Embryos were exposed to 10(6) to 10(7) plaque-forming units (pfu) of either IBRV or BHV-4 for 1 to 2 h. Subsequently, approximately equal numbers of embryos exposed to each virus were either washed 12 times and the washes and embryos examined for the presence of infectious virus, or they were treated with trypsin and the embryos examined for the presence of infectious virus. Although the fourth wash was the last positive wash, an average of 18 pfu of virus was detected from each of six groups (a total of 24 embryos) after exposure to IBRV and washing. Infectious bovine rhinotracheitis virus was not isolated from any of nine trypsin-treated groups (a total of 43 embryos). The seventh wash was the last positive wash for any group after exposure to BHV-4, yet an average of 2 pfu of virus was detected from each of six groups (a total of 29 embryos) after washing. No BHV-4 was isolated from any of eight trypsin-treated groups (a total of 43 embryos). The study confirmed previous reports that IBRV adheres to the bovine ZP after in vitro exposure and that trypsin treatment is effective in keeping ZP-I embryos free of this virus. Adherence of BHV-4 to ZP-I bovine embryos was demonstrated for the first time. Trypsin treatment was also effective in removing this herpesvirus.

Journal Article↗
PubMed2005

Characterization of cDNAs encoding three trypsin-like proteinases and mRNA quantitative analysis in Bt-resistant and -susceptible strains of Ostrinia nubilalis.

Our previous studies suggested that Bacillus thuringiensis (Bt) resistance in a Dipel-resistant strain of Ostrinia nubilalis was primarily due to reduced trypsin-like proteinase activity. In this study, we demonstrated a 254-fold resistance to Cry1Ab protoxin but only 12-fold to trypsin-activated Cry1Ab toxin in the Dipel-resistant strain. Significantly higher resistance to Cry1Ab protoxin than to trypsin-activated Cry1Ab toxin further supports the hypothesis that reduced trypsin-like proteinase activity leading to reduced activation of the Bt protoxin is a major resistance mechanism in the Dipel-resistant strain. To understand the molecular basis of reduced proteinase activity, three cDNAs, OnT2, OnT23, and OnT25, encoding full-length trypsin-like proteinases, were sequenced in Bt-resistant and -susceptible O. nubilalis larvae. Although a number of nucleotide differences were found in sequences from the Bt-resistant and -susceptible strains, the differences were not consistent with reduced trypsin-like activity in the Bt-resistant strain. However, the mRNA levels of OnT23 in the resistant strain were 2.7- and 3.8-fold lower than those of the susceptible strain as determined by northern blotting and real-time quantitative PCR, respectively. Thus, reduced trypsin-like activity may be attributed to reduced expression of OnT23 in Bt-resistant O. nubilalis. Our study provides new insights into Bt resistance management strategies, as resistance mediated by reduced Bt protoxin activation would be ineffective if resistant insects ingest a fully activated form of Cry1Ab toxin, either in spray formulations or transgenic Bt crops.

Amino Acid Sequence↗
PubMed2000

Characterization of SNP1, a cell wall-degrading trypsin, produced during infection by Stagonospora nodorum.

Stagonospora (= Septoria) nodorum when grown in liquid culture with wheat cell walls as the sole carbon and nitrogen source secretes numerous extracellular depolymerases, including a rapidly produced, alkaline, trypsin-like protease (SNP1). The enzyme was purified 417-fold by cation exchange chromatography and has a molecular mass of 25 kDa on sodium dodecyl sulfate gels, pI 8.7, and pH optimum of 8.5. It cleaved peptide bonds on the carboxyl side of lysine or arginine, was strongly inhibited by the trypsin inhibitors aprotinin and leupeptin and weakly by phenylmethylsulfonyl fluoride, and its activity was stimulated by calcium. SNP1 has the characteristic, conserved, fungal, trypsin N terminus. Polymerase chain reaction (PCR) primers based on this sequence and the conserved trypsin active site were used to amplify a DNA fragment that facilitated isolation of the corresponding genomic clone from a lambda library of S. nodorum. The full-length sequence confirmed its identity as a trypsin-like protease containing the N-terminal sequence of the previously purified enzyme. Infected leaf tissue contained a protease, not present in controls, that coeluted with the fungal trypsin from cation exchange, and had properties (pI and inhibitor characteristics) similar to those of the fungal trypsin. SNP1 expression in planta was detected by Northern (RNA) blotting, reverse transcription PCR, and green fluorescent protein confocal microscopy. SNP1 released hydroxyproline from wheat cell walls. The release of hydroxyproline, together with its early expression in planta, suggests that SNP1 participates in the degradation of host cell walls during infection.

Amino Acid Sequence↗
PubMed2002

Remarkable phylum selectivity of a Schistocerca gregaria trypsin inhibitor: the possible role of enzyme-inhibitor flexibility.

A 35-mer polypeptide isolated from the hemolymph of desert locust Schistocerca gregaria (SG) proved to be a canonical inhibitor of bovine trypsin (K(i) = 0.2 microM). Despite having a trypsin-specific arginine at the primary specificity P(1) site, it inhibits bovine chymotrypsin almost as well (K(i) = 2 microM). Furthermore, while the latter reactivity improves 10(4)-fold by the single replacement of P(1) Arg by Leu, changing P(1)' from Lys to Met only moderately improves trypsin affinity (K(i) = 30 nM). The apparent low compatibility to trypsin, however, is not observed vs two arthropodal trypsins: SG peptides with P(1) Arg inhibit crayfish and shrimp trypsins with K(i) values in the picomolar range. This unprecedented high discrimination between orthologous enzymes is postulated to derive from flexibility differences in the protein-protein interaction. The more than four orders of magnitude phylum selectivity makes these peptides prospective candidates for agricultural use.

Amino Acid Sequence↗
PubMed2001

X-ray crystallographic analyses of complexes between bovine beta-trypsin and Schiff base copper(II) or iron(III) chelates.

To establish the structural basis underlying the activity of a novel series of metal-chelate trypsin inhibitors, the structures of p-amidinosalicylidene-l-alaninato(aqua)copper(II) (1a), m-amidinosalicylidene-l-alaninato(aqua)copper(II) (1b), bis(p-amidinosalicylidene-l-alaninato)iron(III) (2a), and bis(m-amidinosalicylidene-l-alaninato)iron(III) (2b) bound to bovine beta-trypsin were studied by X-ray crystallography. The amidinium group of the inhibitor donates hydrogen bonds to Asp189, Gly219 and Ser190, as seen before in trypsin-benzamidine complexes. The copper(II) ion of 1a is situated away from trypsin's catalytic triad residues, and is octahedrally coordinated by a Schiff base and three water molecules. In contrast, the copper(II) ion of 1b is situated close to the catalytic triad and adopts a square pyramidal coordination geometry. The iron(III) ion of 2a is octahedrally coordinated by two Schiff base ligands and, like the copper(II) ion of 1a, is situated away from the catalytic triad. The p-amidinophenyl ring of a second Schiff base ligand of 2a is directed toward a hydrophobic groove formed by Trp215 and Leu99. Finally, the iron(III) ion of 2b appears to be replaced by magnesium(II), which is octahedrally coordinated by a Schiff base, Gln192 and two water molecules. One of the Schiff base ligands seen in the trypsin-2a complex or in the unbound form of 2b is replaced by water molecules and Gln192. His57 and Ser195 form water-mediated interactions with the magnesium(II) ion of 2b, and Ser195 also forms a hydrogen bond with the phenolic oxygen atom of the Schiff base ligand. These structures reveal a novel mode of interaction between metal-chelate inhibitors and serine proteases, thus providing a structural basis for the development of more potent inhibitors against a variety of trypsin-like enzymes.

Animals↗
PubMed1986

Antigenicity of native and modified Kunitz soybean trypsin inhibitors.

Food provides a continuous antigenic stimulus to the immune system and the antigenicity of processed food proteins should be considered in toxicological evaluations. The antigenicity of the Kunitz trypsin inhibitor was studied using antibodies prepared by inoculating rabbits with native, heat-denatured, and N-acetylcysteine-treated Kunitz soybean trypsin inhibitors. Immunochemical studies using a competitive solid-phase enzyme immunoassay and two groups of sera revealed two patterns of antigenicity. Antibodies elicited with the denatured inhibitor were specific for the denatured conformation of the protein. In contrast, native inhibitor elicited antibodies that selectively recognized determinants in both native and heat-treated protein, but that did not bind trypsin inhibitors treated with N-acetylcysteine. These results imply that: the disulfide bonds must be intact to maintain the native antigenic conformation and the cysteine treatment may suppress allergic manifestations of soybean trypsin inhibitors and possibly other food proteins. These studies were extended by analyzing a panel of monoclonal antibodies prepared against native Kunitz trypsin inhibitor. The inhibitor has at least two distinct antigenic sites (epitopes), one of which is retained under denaturing conditions. The measurement of native Kunitz trypsin inhibitor in food samples by immunoassay appears practical. The relevance of these findings to food processing, food safety, and health is also discussed.

Animals↗
PubMed1977

Neutrophilic leukocytes in immunologic reactions in vitro: IV. The effect of trypsin.

Prior trypsinization of rabbit PMN prevented the normal selective release of lysosomal constituents induced by contact with zymosan-C3 and abolished the adherence of these cells to sheep RBC sensitized with IgM antibody and complement (PMN rosettes). The effect of trypsin could be completely reversed by exposure of the cells to soybean trypsin inhibitor after trypsinization. Trypsin did not inhibit the lysosomal release provoked by contact with immune complexes of interfere with rosette formation between PMN and sheep RBC sensitized with IgG antibody. The action of trypsin on the PMN C3b receptor may not be enzymatic.

Animals↗
PubMed1993

Inhibition of Glossina morsitans midgut trypsin activity by D-glucosamine.

The effect of the amino sugar D-glucosamine on trypsin in crude midgut homogenates of Glossina morsitans morsitans was studied in vitro. The results showed that the midgut trypsin was specifically and competitively inhibited by D-Glucosamine. Glucose, fructose, mannose, inositol, galactose, galactosamine, N-acetyl-D-glucosamine, and methyl-alpha-D-glucosamine were ineffective as inhibitors, even at concentrations exceeding 600 mM. D-glucosamine also had a similar inhibitory effect on bovine pancreatic trypsin. In both cases, the inhibition was incomplete as shown by nonlinear Dixon plots. The Michaelis and inhibition constants estimated for the midgut trypsin were 41 +/- 2 microM and 68 +/- 3 mM, respectively. These results suggest that the susceptibility of tsetse flies to trypanosome infection, which is associated with high midgut glucosamine levels, could be due to inhibition of trypsin or trypsin-like enzymes by this sugar.

Animals↗
PubMed1998

Peritoneal lavage with aprotinin in patients with severe acute pancreatitis. Effects on plasma and peritoneal levels of trypsin and leukocyte proteases and their major inhibitors.

CONCLUSION: Although high-dose aprotinin given intraperitoneally to patients with severe acute pancreatitis seems to inhibit activated trypsin in the peritoneal cavity, the treatment has little effect on the balance between proteases and antiproteases. Plasma levels of leukocyte proteases were high in all the patients, indicating leukocyte activation to be an important feature of the pathophysiology of severe acute pancreatitis. A surprise finding was that the patients had higher peritoneal levels of pancreatic secretory trypsin inhibitor (PSTI) after the lavage procedure. BACKGROUND: Although most studies have shown protease inhibitor therapy to have little or no effect on acute pancreatitis, in an earlier study we found that very high doses of the protease inhibitor aprotinin given intraperitoneally to patients with severe acute pancreatitis seemed to reduce the need of surgical treatment for pancreatic necrosis. In the present study we have further analyzed plasma and peritoneal samples from the same patients to ascertain whether the aprotinin treatment affects the balance between proteases and endogenous antiproteases. METHODS: In a prospective double-blind randomized multicenter trial, 48 patients with severe acute pancreatitis were treated with intraperitoneal lavage. One group (aprotinin group, n = 22) was also treated with high doses (20 million KIU given over 30 h) of aprotinin intraperitoneally. The remaining 26 patients made up the control group. The protease-antiprotease balance was studied by measuring immunoreactive anionic trypsin (irAT), cationic trypsin (irCT), complexes between cationic trypsin and alpha 1-protease inhibitor (irCT-alpha 1 PI), leukocyte elastase and neutrophil proteinase 4 (NP4), as well as the endogenous protease inhibitors, pancreatic secretory trypsin inhibitor (PSTI), alpha 2-macroglobulin (alpha 2M), alpha 1-protease inhibitor (alpha 1 PI), antichymotrypsin (ACHY), and secretory leukocyte protease inhibitor (SLPI). Intraperitoneal levels were studied before and after the lavage procedure, and plasma levels were followed for 21 d. RESULTS: The control group had lower plasma levels of SLPI and analysis of peritoneal fluid showed the reduction of irCT-alpha 1 PI to be more pronounced in the aprotinin group. None of the other variables measured differed significantly between the two groups. All patients had very high levels of leukocyte elastase and NP4 both in peritoneal exudate and in plasma. Peritoneal levels of PSTI were higher after the lavage procedure in contrast to the other measured variables that all showed lower peritoneal levels after the lavage.

Acute Disease↗
PubMed1988

Isolation of soybean trypsin inhibitors by affinity chromatography on anhydrotrypsin-Sepharose 4B.

By repeated treatments of trypsin with phenylmethylsulfonyl fluoride (PMSF), followed by base elimination of PMS from the PMS-trypsin, a catalytically inactive anhydrotrypsin preparation of low (less than 1%) active trypsin content was obtained. Inactive material was removed by affinity chromatography on trypsin inhibitor-Sepharose 4B and the purified anhydrotrypsin with full binding capacity for trypsin inhibitors was coupled to cyanogen bromide-activated Sepharose 4B. When used below its maximum capacity for trypsin inhibitors the anhydrotrypsin-Sepharose-4B affinity column absorbed both classes of inhibitors present in soybean. When overloaded, the Kunitz type was bound preferentially. Based on this observation, conditions for the partial separation of the two types of inhibitors were worked out.

Chromatography, Affinity↗