Purification, characterization and substrate specificity of a basic proteinase in the venom of habu (Trimeresurus flavoviridis)

A basic proteinase was purified and characterized from the venom of Habu (Trimeresurus flavoviridis). Its molecular weight, isoelectric point and optimum pH were approx. 24,000, 9.2 and 9, respectively. Susceptibility to several reagents was examined. The proteinase had endopeptidase activity cleaving the Gly-Leu bond in synthetic peptides but no exopeptidase activity. It did not hydrolyze a peptide, Z-Gly-Pro-Leu-Gly-Pro, which had been a good substrate for the major proteinase in the venom. The proteinase cleaved oxidized insulin B chain at five positions: His10-Leu11, Ala14-Leu15, Tyr16-Leu17, Gly23-Phe24 and Phe24-Phe25. From the disappearance of intermediate peptides and the peptides accumulated, the order and the intensity of cleavage of these positions were determined, and the substrate specificity was compared with those hitherto described for hemorrhagic and nonhemorrhagic venom proteinases.     

Contribution of peptide bonds to inhibitor-protease binding: crystal structures of the turkey ovomucoid third domain backbone variants OMTKY3-Pro18I and OMTKY3-psi[COO]-Leu18I in complex with Streptomyces griseus proteinase B (SGPB) and the structure of the free inhibitor, OMTKY-3-psi[CH2NH2+]-Asp19I

X-ray crystallography has been used to determine the 3D structures of two complexes between Streptomyces griseus proteinase B (SGPB), a bacterial serine proteinase, and backbone variants of turkey ovomucoid third domain (OMTKY3). The natural P1 residue (Leu18I) has been substituted by a proline residue (OMTKY3-Pro18I) and in the second variant, the peptide bond between Thr17I and Leu18I was replaced by an ester bond (OMTKY3-psi[COO]-Leu18I). Both variants lack the P1 NH group that donates a bifurcated hydrogen bond to the carbonyl O of Ser214 and O(gamma) of the catalytic Ser195, one of the common interactions between serine proteinases and their canonical inhibitors. The SGPB:OMTKY3-Pro18I complex has many structural differences in the vicinity of the S1 pocket when compared with the previously determined structure of SGPB:OMTKY3-Leu18I. The result is a huge difference in the DeltaG degrees of binding (8.3 kcal/mol), only part of which can be attributed to the missing hydrogen bond. In contrast, very little structural difference exists between the complexes of SGPB:OMTKY3-psi[COO]-Leu18I and SGPB:OMTKY3-Leu18I, aside from an ester O replacing the P1 NH group. Therefore, the difference in DeltaG degrees, 1.5 kcal/mol as calculated from the measured equilibrium association constants, can be attributed to the contribution of the P1 NH hydrogen bond toward binding. A crystal structure of OMTKY3 having a reduced peptide bond between P1 Leu18I and P'1 Asp19I, (OMTKY3-psi[CH2NH2+]-Asp19I) has also been determined by X-ray crystallography. This variant has very weak association equilibrium constants with SGPB and with chymotrypsin. The structure of the free inhibitor suggests that the reduced peptide bond has not introduced any major structural changes in the inhibitor. Therefore, its poor ability to inhibit serine proteinases is likely due to the disruptions of the canonical interactions at the oxyanion hole.     

Proteinase inhibitors in rat serum. Purification and partial characterization of three functionally distinct trypsin inhibitors.

Three different serine proteinase inhibitors were isolated from rat serum and purified to apparent homogeneity. One of the inhibitors appears to be homologous to alpha 1-proteinase inhibitor isolated from man and other species, but the other two, designated rat proteinase inhibitor I and rat proteinase inhibitor II, seem to have no human counterpart. alpha 1-Proteinase inhibitor (Mr 55000) inhibits trypsin, chymotrypsin and elastase, the three serine proteinases tested. Rat proteinase inhibitor I (Mr 66000) is active towards trypsin and chymotrypsin, but is inactive towards elastase. Rat proteinase inhibitor II (Mr 65000) is an effective inhibitor of trypsin only. Their contributions to the trypsin-inhibitory capacity of rat serum are about 68, 14 and 18% for alpha 1-proteinase inhibitor, rat proteinase inhibitor I and rat proteinase inhibitor II respectively.     

Assay of alpha-cysteine proteinase inhibitor in serum or plasma

A new proteinase inhibitor has recently been found in human serum or plasma which specifically inhibits cysteine proteinases such as ficin, papain, bromelain and cathepsin B. However, serum contains alpha 2-macroglobulin which also inhibits these cysteine proteinases and, consequently, interferes with the assay of the new alpha-cysteine proteinase inhibitor. Therefore, assay of the inhibitor in serum has not been established previously. In the present method, the alpha 2-macroglobulin is inactivated by preincubating the serum in methylamine solution at 55 degrees C, while the alpha-cysteine proteinase inhibitor retains its activity. The inhibitory power against cysteine proteinases is found to be due mainly to this protein in human serum. This inhibitor is also found in mammals such as cows, pigs and rats. Vitamin E deficient rats show a very high inhibitor level. Therefore, the present method will enable us to investigate the relation between diseases and the activity of the alpha-cysteine proteinase inhibitor. Also, this method is simple and inexpensive. The necessary amount of serum is only 10 microliter.     

Proteinase binding and inhibition by the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3

The inhibitory capacity of the alpha-macroglobulins resides in their ability to entrap proteinase molecules and thereby hinder the access of high molecular weight substrates to the proteinase active site. This ability is thought to require at least two alpha-macroglobulin subunits, yet the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3 (alpha 1I3) also inhibits proteinases. We have compared the inhibitory activity of alpha 1I3 with the tetrameric human homolog alpha 2-macroglobulin (alpha 2M), the best known alpha-macroglobulin, in order to determine whether these inhibitors share a common mechanism. alpha 1I3, like human alpha 2M, prevented a wide variety of proteinases from hydrolyzing a high molecular weight substrate but allowed hydrolysis of small substrates. In contrast to human alpha 2M, however, the binding and inhibition of proteinases was dependent on the ability of alpha 1I3 to form covalent cross-links to proteinase lysine residues. Low concentrations of proteinase caused a small amount of dimerization of alpha 1I3, but no difference in inhibition or receptor binding was detected between purified dimers or monomers. Kininogen domains of 22 and 64 kDa were allowed to react with alpha 1I3- or alpha 2M-bound papain to probe the accessibility of the active site of this proteinase. alpha 2M-bound papain was completely protected from reaction with these domains, whereas alpha 1I3-bound papain reacted with them but with affinities several times weaker than uncomplexed papain. Cathepsin G and papain antisera reacted very poorly with the enzymes when they were bound by alpha 1I3, but the protection provided by human alpha 2M was slightly better than the protection offered by the monomeric rat alpha 1I3. Our data indicate that the inhibitory unit of alpha 1I3 is a monomer and that this protein, like the multimeric alpha-macroglobulins, inhibits proteinases by steric hindrance. However, binding of proteinases by alpha 1I3 is dependent on covalent crosslinks, and bound proteinases are more accessible, and therefore less well inhibited, than when bound by the tetrameric homolog alpha 2M. Oligomerization of alpha-macroglobulin subunits during the evolution of this protein family has seemingly resulted in a more efficient inhibitor, and we speculate that alpha 1I3 is analogous to an evolutionary precursor of the tetrameric members of the family exemplified by human alpha 2M.     

Characterization of neutral proteinases from human spleen]

Several proteinases hydrolyzing histone and caseine in neutral media were obtained by Sephadex G-100 fractionation of water and salt (1 M KCl) extracts of human spleen. The level of the activity of proteinases in the extracts was very low as a result of the presence of an inhibitor. Neutral proteinases were found in two protein fractions. The "high-molecular-weight-" proteinases were inhibited by DFP and therefore they were attributed to a group of serine proteinases. The "low-molecular-weight" fraction contained neutral SH-dependent proteinase(s) and DFP-inhibited enzymes. In this fraction, the kininogenase activity and the hydrolysis of Boc-1-ananine p-nitrophenyl ester, N-benzoyl-L-tryosine ethyl ester and N-benzoyl-DL-arginine p-nitroanilide were observed.     

Purification of an acid proteinase from Aspergillus saitoi and determination of peptide bond specificity.

The specificity and mode of action of an acid proteinase (EC 3.4.23.6) from Aspergillus saitoi were investigated with oxidized B-chain of insulin, angiotensin II and bradykinin. Further purification of acid proteinase was performed with N,O-dibenzyloxycarbonyl-tyrosine hexamethylene-diamino-Sepharose 4B affinity chromatography and isoelectric focusing. The purified enzyme was free of any other proteolytic activity demonstrated in Asp. saitoi. Acid proteinase from Asp. saitoi hydrolyzed primarily two peptide bonds in the oxidized B-chain of insulin, the Leu(15)-Tyr(16) bond and the Phe(24)-Phe(25) bond. Additional cleavages of the bonds His(10)-Leu(11), Ala(14)-Leu(15) and Tyr(16)-Leu(17) were also noted. Primary splitting sites at Leu(15)-Tyr(16) and Phe(24-)-Phe(25) with acid proteinase from Asp. saitoi were identical with those reported in the work of cathepsin D (EC 3.4.23.5) from human erythrocyte. Hydrolysis of angiotensin II was observed at the Tyr(4)-Ile(5) bond. In conclusion, peptide bonds which have a hydrophobic amino acid such as phenylalanine, tyrosine, leucine and isoleucine in the P'1 position (as defined by Berger and Schechter, [29]) are preferentially cleaved by the trypsinogenactivating acid proteinase from Asp. saitoi.     

Inter-alpha-trypsin inhibitor. Inhibition spectrum of native and derived forms

The conversion of inter-alpha-trypsin inhibitor (I alpha I) into active, acid-stable derivatives by proteolytic degradation has been tested with 10 different proteinases. Of these, only plasma kallikrein, cathepsin G, neutrophil elastase, and the Staphylococcus aureus V-8 proteinase were found to be effective, each releasing more than 50% of this activity. However, a strong correlation between inhibitor degradation and significant release of acid-stable activity could only be found with the V-8 enzyme. Inhibition kinetics for the interaction of native I alpha I, the inhibitory fragment released by digestion with S. aureus V-8 proteinase, or the related urinary trypsin inhibitor, with seven different proteinases indicated that all had essentially identical Ki values with an individual enzyme and, where measurements were possible, nearly identical second order association rate constants. Significantly, none of the five human proteinases tested, including trypsin, chymotrypsin, plasmin, neutrophil elastase, and cathepsin G, would appear to have low enough Ki values to be physiologically relevant. Thus, the role of native I alpha I or its degradation products in controlling a specific proteolytic activity is still unknown.     

The role of inter-alpha-trypsin inhibitor and other proteinase inhibitors in the plasma clearance of neutrophil elastase and plasmin

The plasma clearance of neutrophil elastase, plasmin, and their complexes with human inter-alpha-trypsin inhibitor (I alpha I) was examined in mice, and the distribution of the proteinases among the plasma proteinase inhibitors was quantified in mixtures of purified inhibitors, in human or murine plasma, and in murine plasma following injection of purified proteins. The results demonstrate that I alpha I acts as a shuttle by transferring proteinases to other plasma proteinase inhibitors for clearance, and that I alpha I modulates the distribution of proteinase among inhibitors. The clearance of I alpha I-elastase involved transfer of proteinase to alpha 2-macroglobulin and alpha 1-proteinase inhibitor. The partition of elastase between these inhibitors was altered by I alpha I to favor formation of alpha 2-macroglobulin-elastase complexes. The clearance of I alpha I-plasmin involved transfer of plasmin to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Results of distribution studies suggest that plasmin binds to endothelium in vivo and reacts with I alpha I before transfer to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Evidence for this sequence of events includes observations that plasmin in complex with I alpha I cleared faster than free plasmin, that plasma obtained after injection of plasmin contained a complex identified as I alpha I-plasmin, and that a murine I alpha I-plasmin complex remained intact following injection into mice. Plasmin initially in complex with I alpha I more readily associated with alpha 2-plasmin inhibitor than did free plasmin.     

Novel roles of protease inhibitors in infection and inflammation

The local balance between proteinase inhibitors and proteinases determines local proteolytic activity. Various studies have demonstrated the importance of serine proteinase inhibitors in regulating the activity of serine proteinases that are released by leucocytes during inflammation. Recently it has been shown that these inhibitors may also display functions that are distinct from those associated with the inhibition of leucocyte-derived proteinases. In this review the results of selected studies focusing on three inhibitors of neutrophil elastase, i.e. alpha(1)-proteinase inhibitor, secretory leucocyte proteinase inhibitor and elafin, are presented, with the aim of illustrating their possible involvement in the regulation of inflammation, host defence against infection, tissue repair and extracellular matrix synthesis.     

The activating system of chitin synthetase from Saccharomyces cerevisiae. Purification and properties of the activating factor.

The yeast proteinase that causes activation of the chitin synthetase zymogen has been purified by a procedure that includes affinity chromatography on an agarose column to which the proteinaceous inhibitor of the enzyme had been covalently attached. The purified enzyme yielded a single band upon disc gel electrophoresis at pH 4.5 in the presence of urea. At the same pH, but without urea, a faint band was detected in coincidence with enzymatic activity, whereas at pH 9.5, either in the absence or in the presence of sodium dodecyl sulfate, no protein zone could be seen. From sedimentation and gel filtration data, a molecular weight of 44,000 was estimated. The proteinase was active within a wide range of pH values, with an optimum between pH 6.5 AND 7. Titraton of the activity with the protein inhibitor from yeast required 1 mol of inhibitor/mol of enzyme. A similar result was obtained with phenylmethylsulfonyl fluoride, an indication that 1 serine residue is required for enzymatic activity. The enzyme exhibited hydrolytic activity with several proteins and esterolytic activity with many synthetic substrates, including benzoylarginine ethyl ester and acetyltyrosine ethyl ester.A comparison of the properties of the enzyme with those of known yeast proteinases led to the conclusion that the chitin synthestase activating factor is identical with the enzyme previously designated as proteinase B (EC 3.4.22.9). This is the first time that a homogeneous preparation of proteinase B has been obtained and characterized.     

Inactivation of human plasma alpha 1-antichymotrypsin by microbial proteinases

Incubation of human plasma alpha 1-antichymotrypsin with proteinases from various microbial sources resulted in the enzymatic inactivation of the inhibitor as determined by loss of inhibitory activity against alpha-chymotrypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the reaction products indicated that intact alpha 1-antichymotrypsin (Mr 67000) had been converted to an inactive form (63000) by limited proteolysis. No stable proteinase/inhibitor complexes were detected, and no random proteolysis of the inactivated inhibitor occurred even after prolonged incubation with the proteinases. Metallo- and serine proteinases from several microbial sources all readily inactivated alpha 1-antichymotrypsin. Since alpha 1-antichymotrypsin is also an early stage acute phase reactant, its inactivation may be important in disrupting bodily defense mechanisms.     

Mechanism of action of inter-alpha-trypsin inhibitor

Inter-alpha-trypsin inhibitor (I alpha I) is a unique proteinase inhibitor that can be proteolyzed by the same enzymes that are inhibited, to generate smaller inhibitors. This study examines the reactions of I alpha I with trypsin, chymotrypsin, plasmin, and leukocyte elastase. Complexes of I alpha I and proteinase were demonstrated by gel filtration chromatography. Complete digestion of I alpha I by each proteinase was not accompanied by a comparable loss of inhibition of that enzyme or a different enzyme. Following proteolysis, inhibitory activity was identified in I alpha I fragments of molecular weight 50,000-100,000 and less than 40,000. Addition of a second proteinase inhibitor prevented proteolysis. Both I alpha I and its complex with proteinase were susceptible to degradation. Kinetic parameters for both the inhibition and proteolysis reactions of I alpha I with four proteinases were measured under physiological conditions. On the basis of these results, a model for the mechanism of action of I alpha I is proposed: Proteinase can react with either of two independent sites on I alpha I to form an inhibitory complex or a complex that leads to proteolysis. Both reactions occur simultaneously, but the inhibitory capacity of I alpha I is not significantly affected by proteolysis since the product of proteolysis is also an inhibitor. For a given proteinase, the inhibition equilibrium constant and the Michaelis constant for proteolysis describe the relative stability of the inhibition and proteolysis complexes; the second-order rate constants for inhibition and proteolysis indicate the likelihood of either reaction. The incidence of inhibition or proteolysis reactions involving I alpha I in vivo cannot be assessed without knowledge of the exact concentrations of inhibitor and proteinases; however, analysis of inhibition rate constants suggests that I alpha I might be involved in plasmin inhibition.     

Preparation and properties of polyethylene glycol-trypsin adducts.

The covalent attachment of polyethylene glycol of 5000 daltons to non-essential groups on trypsin produces an adduct that no longer precipitates with anti-trypsin antibody. In comparison with trypsin, polyethylene glycol-trypsin preparations show equal or greater activity against N-alpha-benzoyl-L-arginine ethyl ester, about one-fourth activity against angiotensin II, and little activity against bovine liver catalase. The polyethylene glycol-trypsin adduct dissolves soft blood clots at one-fourth the rate of trypsin. Soybean trypsin inhibitor produces two-thirds inhibition of the adduct under conditions that cause complete inhibition of trypsin.     

Antibody raised against extracellular proteinases ofSporothrix schenckii inS. schenkii inoculated hairless mice

Sporothrix schenckii produces two extracellular proteinases, namely proteinase I and II. Proteinase I is a serine proteinase, inhibited by chymostatin, while proteinase II is an aspartic proteinase, inhibited by pepstatin. Studies on substrate specificity and the effect of proteinase inhibitors on cell growth suggest an important role for these proteinases in terms of fungal invasion and growth. There has, however, been no evidence presented demonstrating thatS. schenckii produces 2 extracellular proteinases in vivo. In order to substantiate the in vivo production of proteinases and to attempt a preliminary serodiagnosis of sporotrichosis, serum antibodies against 2 proteinases were assayed usingS. schenckii inoculated hairless mice. Subsequent to an intracutaneous injection ofS. schenckii to the mouse skin, nodules spontaneously formed and disappeared for a period of 4 weeks. Histopathological examination results were in accordance with the microscopic observations. Micro-organisms disappeared during the fourth week. Serum antibody titers against purified proteinases I and II were measured weekly, using enzyme-linked immunosorbent assay (EIA). As a result, the time course of the antibody titers to both proteinases I and II were parallel to that of macroscopic and microscopic observations in an experimental mouse sporotrichosis model. These results suggest thatS. schenckii produces both proteinases I and II in vivo. Moreover, the detection of antibodies against these proteinases can contribute to a serodiagnosis of sporotrichosis.     

Chymotrypsin inhibitory activity of normal C1-inhibitor and a P1 Arg to His mutant: evidence for the presence of overlapping reactive centers.

C1-inhibitor is a serine proteinase inhibitor that is active against C1s, C1r, kallikrein, and factor XII. Recently, it has been shown that it also has inhibitory activity against chymotrypsin. We have investigated this activity of normal human C1-inhibitor, normal rabbit C1-inhibitor, and P1 Arg to His mutant human C1-inhibitors and find that all are able to inhibit chymotrypsin and form stable sodium dodecyl sulfate-resistant complexes. The Kass values show that the P1 His mutant is a slightly better inhibitor of chymotrypsin than normal human C1-inhibitor (3.4 x 10(4) compared with 7.3 x 10(3)). The carboxy-terminal peptide of normal human C1-inhibitor, derived from the dissociated protease-inhibitor complex, shows cleavage between the P2 and P1 residues. Therefore, as with alpha 2-antiplasmin, C1-inhibitor possesses two overlapping P1 residues, one for chymotrypsin and the other for Arg-specific proteinases. In contrast, with the P1 His mutant, the peptide generated from the dissociation of its complex with chymotrypsin demonstrated cleavage between the P1 and P'1 residues. Therefore, unlike alpha 2-antiplasmin, chymotrypsin utilizes the P2 residue as its reactive site in normal C1-inhibitor but utilizes the P1 residue as its reactive site in the P1 His mutant protein. This suggests that the reactive center loop allows a degree of induced fit and therefore must be relatively flexible.     

In vivo catabolism of alpha 1-antichymotrypsin is mediated by the Serpin receptor which binds alpha 1-proteinase inhibitor, antithrombin III and heparin cofactor II

The in vivo catabolism of 125I-labeled alpha 1-antichymotrypsin was studied in our previously described mouse model. Native alpha 1-antichymotrypsin cleared with an apparent t1/2 of 85 min, but alpha 1-antichymotrypsin in complex with chymotrypsin or cathepsin G cleared with a t1/2 of 12 min. Clearance of the complex was blocked by a large molar excess of unlabeled complexes of proteinases with either alpha 1-antichymotrypsin or alpha 1-proteinase inhibitor. These studies indicate that the clearance of alpha 1-antichymotrypsin-proteinase complexes utilizes the same pathway as complexes with the homologous inhibitor alpha 1-proteinase inhibitor. Previous studies have demonstrated that this pathway is also responsible for the catabolism of two other serine proteinase inhibitors, antithrombin III and heparin cofactor II. This pathway is thus responsible for removing several proteinases involved in coagulation and inflammation from the circulation, thereby decreasing the likelihood of adventitious proteolysis.     

Rapamycin induces binding activity to the terminal oligopyrimidine tract of ribosomal protein mRNA in rats

Cathepsin G, elastase, and proteinase 3 are serine proteinases released by activated neutrophils. Cathepsin G can cleave angiotensinogen to release angiotensin II, but this activity has not been previously reported for elastase or proteinase 3. In this study we show that elastase and proteinase 3 can release angiotensin I from angiotensinogen and release angiotensin II from angiotensin I and angiotensinogen. The relative order of potency in releasing angiotensin II by the three proteinases at equivalent concentrations is cathepsin G > elastase > proteinase 3. When all three proteinases are used together, the release of angiotensin II is greater than the sum of the release when each proteinase is used individually. Cathepsin G and elastase can also degrade angiotensin II, reactions which might be important in regulating the activity of angiotensin II. The release and degradation of angiotensin II by the neutrophil proteinases are reactions which could play a role in the local inflammatory response and wound healing.     

Proteolytic inactivation of alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin by oxidatively activated human neutrophil metalloproteinases.

Human neutrophils use the H2O2-myeloperoxidase-chloride system to generate chlorinated oxidants capable of activating metalloproteinase zymogens that hydrolyze not only native and denatured collagens, but also the serine proteinase inhibitor (serpin) alpha 1-proteinase inhibitor (alpha 1 PI). To identify the metalloenzyme that hydrolyzes and inactivates alpha 1 PI, neutrophil releasates were chromatographed over gelatin-Sepharose and divided into fractions containing either progelatinase or procollagenase. The gelatinase-containing fraction cleaved alpha 1 PI in a manner inhibitable by native type V, but not type I, collagen. Conversely, while the collagenase-containing fraction also cleaved alpha 1 PI, this activity was inhibited by type I, but not type V, collagen. Because type I and V collagens are competitive substrates for collagenase and gelatinase, respectively, each of the metalloproteinase zymogens were purified to apparent homogeneity and examined for alpha 1 PI-hydrolytic activities. Both purified gelatinase and collagenase inactivated alpha 1PI by hydrolyzing the serpin within its active-site loop at the Phe352-Leu353 and Pro357-Met358 bonds, albeit with distinct kinetic properties. Furthermore, purified collagenase, but not gelatinase, cleaved a second serpin, alpha 1-antichymotrypsin, by hydrolyzing the Ala362-Leu363 bond within its active-site loop. These data demonstrate that human neutrophils use chlorinated oxidants to activate collagenolytic metalloproteinases whose substrate specificities can be extended to members of the serpin superfamily.     

Inhibition of human granulocyte proteinases by a heat and acid-stable proteinase inhibitor from rabbit serum]

Several human granulocyte proteinases sensitive to the thermo- and acid-resistant proteinase inhibitor from rabbit serum (TASPI) were revealed, using TASPI-Sepharose 4B. It was found that TASPI inhibits the following human granulocyte proteinases: granules-localized kininogenase and chymotrypsin-like kininase (serine proteinases), elastase-like proteinase and benzoyl arginine ethyl ester esterase, as well as chymotrypsin-like kininase from the post-granule supernatant. These enzymes were compared to known granulocyte proteinases. Some carboxylic kininogenase sensitive to TASPI was identified in the granulocyte membrane debris fraction. The capability to inhibit neutral kininogenase suggests that TASPI is a first natural proteinase inhibitor, which can differentiate granulocyte and blood plasma kininogenases. Using trypsin-Sepharose 4B in the granulocyte post-granule supernatant, the acid-resistant trypsin and chymotrypsin inhibitor was identified. The data obtained are indicative of an antiinflammatory function of TASPI in mammals.