Human serum alpha 1-antichymotrypsin is an inhibitor of pancreatic elastases

Incubation of human serum alpha 1-antichymotrypsin with human pancreatic elastase 2 or porcine pancreatic elastase results in the complete inhibition of each enzyme as determined by spectrophotometric assays. alpha 1-Antichymotrypsin reacts much more rapidly with the human than with the porcine enzyme. The inhibitor: enzyme molar ratio, required to obtain full inhibition of enzymatic activity, is equal to 1.25/1 when alpha 1-antichymotrypsin reacts with human pancreatic elastase 2 while it is markedly higher with porcine pancreatic elastase (5.5/1). Patterns obtained by SDS/polyacrylamide gel electrophoresis of the reaction products show the formation with both enzymes of an equimolar complex (Mr near 77 000) and the release of a fragment migrating as a peptide of Mr near 5000. Moreover a free proteolytically modified form of alpha 1-antichymotrypsin, electrophoretically identical with that obtained in the reaction with cathepsin G or bovine chymotrypsin, is produced in the reaction with each elastase but in a much greater amount when alpha 1-antichymotrypsin reacts with porcine elastase than with human elastase. As a consequence of our findings, the specificity of alpha 1-antichymotrypsin, so far limited to the inhibition of chymotrypsin-like enzymes from pancreas and leukocyte origin, has to be extended to the two pancreatic elastases investigated in this work. A contribution of alpha 1-antichymotrypsin to the regulatory balance between plasma inhibitors and human pancreatic elastase 2 in pancreatic diseases is suggested.     

Investigation of the active center of rat pancreatic elastase

We have isolated rat pancreatic elastase I (EC 3.4.21.36) using a fast two-step procedure and we have investigated its active center with p-nitroanilide substrates and trifluoroacetylated inhibitors. These ligands were also used to probe porcine pancreatic elastase I whose amino acid sequence is 84% homologous to rat pancreatic elastase I as reported by MacDonald, et al. (Biochemistry 21, (1982) 1453-1463). Both proteinases exhibited non-Michaelian kinetics for substrates composed of three or four residues: substrate inhibition was observed for most enzyme substrate pairs, but with Ala3-p-nitroanilide, rat elastase showed substrate inhibition, whereas porcine elastase exhibited substrate activation. With most of the longer substrates, Michaelian kinetics were observed. The kcat/Km ratio was used to compare the catalytic efficiency of the two elastases on the different substrates. For both elastases, occupancy of subsite S4 was a prerequisite for efficient catalysis, occupancy of subsite S5 further increased the catalytic efficiency, P2 proline favored catalysis and P1 valine had an unfavorable effect. Rat elastase has probably one more subsite (S6) than its porcine counterpart. The rate-limiting step for the hydrolysis of N-succinyl-Ala3-p-nitroanilide by rat elastase was essentially acylation, whereas both acylation and deacylation rate constants participated in the turnover of this substrate by porcine elastase. For both enzymes, trifluoroacetylated peptides were much better inhibitors than acetylated peptides and trifluoroacetyldipeptide anilides were more potent than trifluoroacetyltripeptide anilides. A number of quantitative differences were found, however, and with one exception, trifluoroacetylated inhibitors were less efficient with rat elastase than with the porcine enzyme.     

Effect of oxidation of methionine in a peptide substrate for human elastases: a model for inactivation of alpha 1-protease inhibitor.

Human α₁-protease inhibitor which is an important plasma protein, contains a methionine residue at its reactive site. A model synthetic peptide substrate, succinyl-L-alanyl-L-alanyl-L-prolyl-L-methionine p-nitroanilide, has been employed to study the effect of oxidation of methionine on the rate of hydrolysis of this substrate by human elastases. The methionine sulfoxide derivative obtained by mild oxidation of this substrate is hydrolyzed by pancreatic elastase 2 and leukocyte elastase at rates that are 5% and 0.3% of the rates measured for hydrolysis of the parent compound by the respective enzymes. These results suggest that oxidation of the active site methionine residue of human α₁-protease inhibitor may decrease the rate of reaction of pancreatic or leukocyte elastase with this inhibitor.     

Irreversible inhibition of serine proteases by peptide derivatives of (alpha-aminoalkyl)phosphonate diphenyl esters

Peptidyl derivatives of diphenyl (alpha-aminoalkyl)phosphonates have been synthesized and are effective and specific inhibitors of serine proteases at low concentrations. Z-PheP(OPh)2 irreversibly reacts with chymotrypsin (kobsd/[I] = 1200 M-1 s-1) and does not react with two elastases. The best inhibitor for most chymotrypsin-like enzymes including bovine chymotrypsin, cathepsin G, and rat mast cell protease II is the tripeptide Suc-Val-Pro-PheP(OPh)2 which corresponds to the sequence of an excellent p-nitroanilide substrate for several chymases. The valine derivative Z-ValP(OPh)2 is specific for elastases and reacts with human leukocyte elastase (HLE, 280 M-1 s-1) but not with chymotrypsin. The tripeptide Boc-Val-Pro-ValP(OPh)2, which has a sequence found in a good trifluoromethyl ketone inhibitor of HLE, is the best inhibitor for HLE (kobsd/[I] = 27,000 M-1 s-1) and porcine pancreatic elastase (PPE, kobsd/[I] = 11,000 M-1 s-1). The rates of inactivation of chymotrypsin by MeO-Suc-Ala-Ala-Pro-PheP(OPh)2 and PPE and HLE by MeO-Suc-Ala-Ala-Pro-ValP(OPh)2 were decreased 2-5-fold in the presence of the corresponding substrate, which demonstrates active site involvement. Only one of two diastereomers of Suc-Val-Pro-PheP(OPh)2 reacts with chymotrypsin (146,000 M-1 s-1), and the enzyme-inhibitor complex had one broad signal at 25.98 ppm in the 31P NMR spectrum corresponding to the Ser-195 phosphonate ester. Phosphonylated serine proteases are extremely stable since the half-time for reactivation was greater than 48 h for the inhibited elastases and 7.5-26 h for chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trifluoroacetyl tripeptides as potent inhibitors of human leukocyte elastase

Trifluoroacetylated peptides are much more potent inhibitors of human leukocyte elastase than the corresponding unblocked, acylated or benzyloxycarbonylated peptides. The most active compound was trifluoroacetyl-Val-Tyr-Val (Ki = 1.3 micron). A number of free and NH2-terminal-substituted peptides exhibited similar affinities for porcine pancreatic and human leukocyte elastase, indicating that these two enzymes must have similar specificity sites.     

The interaction of trifluoroacetyl peptide chloromethyl ketones with porcine pancreatic elastase. Direct evidence for nonproductive enzyme.inhibitor complexes.

19F NMR investigations of the interactions between elastase and the reversible inhibitors CF3CO-Ala3, CF3CO-Lys-Ala2 and CF3CO-Ala4 show that these peptides have a single mode of binding to the enzyme. Furthermore the results indicate that the CF3CO-group experiences the same environment in all of the reversible complexes formed with these inhibitors. This agrees with the higher affinity of these peptides for the enzyme as compared to the corresponding acetylated inhibitors and confirms our earlier hypothesis of the existence of a specific binding site for the CF3CO-group on the enzyme. The interactions between elastase and the irreversible inhibitors CF3CO-Alan chloromethyl ketone (n = 2, 3, 4, 5) and CF3CO-Lys-Ala4 chloromethyl ketone have been investigated by enzymatic measurements and 19F NMR spectroscopy. The kinetic constants k2 and KI describing the irreversible inhibition are significantly lower for all the CF3CO-peptide chloromethyl ketones with exception of CF3CO-Ala2 chloromethyl ketone, than for the corresponding acetylated ones. Moreover, 19F NMR spectroscopy enabled us to demonstrate, for the tri- and tetrapeptide derivatives, the parallel formation of reversible nonproductive enzyme.inhibitor complexes. The spectroscopic properties of these complexes are completely different from those of the irreversible complexes but are similar to those observed with the reversible complexes described above. In the case of the pentapeptide chloromethyl ketones, fast hydrolysis of the peptide or fast inactivation of the enzyme does not allow observations to be made but does not exclude the existence of reversible nonproductive complexes. In fact, their existence is strongly suggested by the enzyme reaction rate measurements. The similarity of the properties of all the reversible complexes, their striking differences with those of all the irreversible complexes, as well as their mutual exclusivity, permit the conclusion that the CF3CO-group does not bind at one of the classical S subsites of elastase.     

Inhibition by elastase inhibitors of the formyl Met Leu Phe-induced chemotaxis of rat polymorphonuclear leukocytes

Rat leukocyte elastase has been purified successively by AH-Sepharose Kappa-elastin affinity chromatography and by ion exchange chromatography on a carboxymethyl Sephadex resin. It has great similarities with human leukocyte elastase in its molecular weight, substrate specificity and inhibitory profile. The effect of rat leukocyte elastase inhibitors in influencing the chemotactic response of rat PMN to fMetLeuPhe has been compared to that of other proteinase inhibitors. The results indicated that oleoyl (Ala)2ProValCH2Cl, a specific inhibitor of human and rat leukocyte elastases and Eglin C, which also inhibits human and rat cathepsin G, are among the powerful inhibitors of rat PMN chemotaxis induced by the formyl oligopeptide. This suggests that these neutral proteinases, in addition to their known participation in connective tissue catabolism, could play a role in PMN locomotion and chemotaxis.     

Identification of a novel class of elastase isozyme, human pancreatic elastase III, by cDNA and genomic gene cloning

By using porcine elastase I cDNA as a probe, we have isolated two different but closely related cDNAs encoding elastase-like proteases from a human pancreatic cDNA library. The amino acid sequences deduced from the cloned cDNA sequences showed 56-61% identity with those of both pancreatic elastases I and II, similar to the homology between elastases I and II. The active form of the elastase-like proteases appeared to be composed of 242 amino acids and preceded by a signal peptide and propeptide of 28 amino acids. Dot blot analysis of various tissue mRNAs demonstrated that the genes for the cloned cDNAs are expressed at a high level only in the pancreas. In addition, sequence analysis of the cloned genomic genes corresponding to one of the cDNAs showed that they are members of the elastase gene family. These results indicate that the two enzymes encoded by the cDNAs should be classified into a third class of elastase isozyme. Therefore, we designated them as human pancreatic elastases IIIA and IIIB. They strongly resembled cholesterol-binding pancreatic protease, suggesting that they may possess not only a digestive function but also function(s) related to cholesterol metabolism or transport in the intestine.     

Sbustrate specificity of the elastase and the chymotrypsin-like enzyme of the human granulocyte.

Human granulocyte elastase (EC 3.4.21.11) differs from hog pancreatic elastase in its specificity for synthetic substrates. Although hydrolyzing peptide bonds adjacent to the carboxyl group of alanine, the granulocyte enzyme prefers valine at the cleaved bond, in contrast to the pancreatic enzyme which prefers alanine. Peptide bonds involving the carboxyl group of isoleucine can be hydrolyzed by the granulocyte enzyme but are not hydrolyzed to any significant extent extent by pancreatic elastase. This difference in specificty could explain the lower sensitivity of the granulocyte enzyme to inhibitors containing alanine analogs, such as the peptide chloromethyl ketones and elastatinal. The human granulocyte chymotrypsin-like enzyme differs from pancreatic chymotrypsin by being able to cleave substrates containing leucine in addition to those containing the aromatic amino acids.     

Killing of schistosomes by elastase and hydrogen peroxide: implications for leukocyte-mediated schistosome killing

Activated leukocytes participate in immunity to infection by the parasitic blood fluke Schistosoma mansoni. They attach to the surface of schistosomes and secrete schistosomicidal substances. Cationic proteins, hydrolytic enzymes, and oxidants, produced by the leukocytes, have been implicated in the damage to the schistosomes. To examine the possible involvement of elastase in the killing of schistosomes by leukocytes, young and adult stages of S. mansoni were treated in vitro with pancreatic elastase (PE) and neutrophil elastase (NE). Schistosomula, lung-stage schistosomula (LSS), and adult worms (AW) have been found to be sensitive to both PE and NE. Male AW were more sensitive to PE than female AW. The enzymatic activity of elastase is essential for its toxic effect because heat-inactivation and specific elastase inhibitors prevented elastase-mediated schistosome killing. Thus, alpha1-antitrypsin and the chloromethyl ketone (CMK)-derived tetrapeptides Ala-Ala-Pro-Val-CMK and Ala-Ala-Pro-Ala-CMK but not Ala-Ala-Pro-Phe-CMK and Ala-Ala-Pro-Leu-CMK blocked PE caseinolytic and schistosomulicidal activities. As shown previously, schistosomes are also efficiently killed by hydrogen peroxide. LSS appear to be more resistant than AW and early-stage schistosomula to the lytic effects of hydrogen peroxide. Cotreatment experiments with both elastase and hydrogen peroxide indicated that they exert an additive toxic effect and that hydrogen peroxide sensitizes schistosomula to the toxic effect of elastase but not vice versa. These results demonstrate, for the first time, that elastases may be toxic molecules used by neutrophils, eosinophils, and macrophages to kill various developmental stages of S. mansoni.     

Elastolysis of normal and partially cross-linked elastin

Rate of elastolysis by pancreatic and leukocyte elastases of normal and copper deficient porcine aortic elastins were measured using a conductimetric method. Kinetics obey to Michaelis-Menten model for both substrates and enzymes. KM and Vmax values derived from Lineweaver-Burk plots indicate that, if a near uniformity exists in KM, differences were observed in catalytic rates, kcat increasing approximately 40% for copper deficient elastin elastolysis by leukocyte elastase. This higher susceptibility to proteolysis may have implications for understanding turnover of elastin in tissues.     

Elastases from human and canine granulocytes, I. Some proteolytic and esterolytic properties.

The lysosome-like granules of human and canine granulocytes contain an enzyme with elastinolytic activity. The enzymatic behaviour of these elastases was further characterized using the protein substrates elastin-orcein and azocasein and the synthetic substrates tert.-butyloxycarbonyl-alanine p-nitrophenylester (Boc-Ala-ONp) and 3-carboxypropionyl-L-alanyl-L-alanyl-L-alanine p-nitroanilide (Suc-Ala3-NHNp) in photometric assays. The affinities of the granulocyte elastases and of porcine pancreatic elastase to these substrates are very similar, e.g. human granulocyte elastase: KM (Boc-Ala-ONp) = 0.35mM, KM (Suc-Ala3-NHNp) = 1.25mM, porcine pancreatic elastase: KM (Boc-Ala-ONp) = 0.3mM, KM (Suc-Ala3-NHNp) - 1.15mM. The most convenient substrate for the assay of human and dog granulocyte elastases and for kinetic measurements with these enzymes is Suc-Ala3-NHNp. Using this substrate, the dissociation constant of the complex of human granulocyte elastase with human alpha1-antitrypsin could be determined (Ki = 3.5 x 10(-10)M).     

Human pancreatic enzymes: purification and characterization of a nonelastolytic enzyme, protease E. resembling elastase.

?An enzyme with proteolytic activity has been isolated from activated extracts of human pancreatic tissue. The purification procedure included salt fractionation followed by ion-exchange chromatography on SE-TSephadex C-25 and on DEAE-Sephadex A-50. The homogeneity of this enzyme, designated protease te, was demonstrated by disc electrophoresis and by sedimentation equilibrium centrifugation stidues. The homogeneous enzyme shows the ability to hydrolyze many of the conventional synthetic substrates used for the identification of elastase activity; however, it demonstrates no significant elastolytic activity. A comparison of human protease E with porcine elastase reveals a high degree of similarity between the two proteases with respect to inhibition by active-site directed peptide chloromethyl ketones, stability, decreased susceptibility to naturally occurring proteinase inhibitors, and specificity for synthetic substrates as well as several other physical properties. The major difference between human protease E and porcine elastase, other than the lack of elastolytic activity by human protease E, seems to be in the ionic character and the amino acid composition of these two proteins. Porcine elastase is a cationic enzyme, while human protease E appears to be anionic in nature. These dissimilarities concerning elastolytic activity and ionic character appear to be directly related.     

Partial purification and characterization of mouse peritoneal exudative macrophage elastase

Mouse peritoneal exudate macrophage elastase can be significantly purified with 60% recovery of the starting activity by affinity chromatography against SDS-treated alpha-elastin covalently linked to agarose beads. The enzyme has an apparent Mr of 26 500 based on SDS-acrylamide gel electrophoresis. Molecular sieving chromatography on Sephadex gel gives a Mr for macrophage elastase of 21 000--28 000. The enzyme is not inhibited by chloromethyl ketone inactivators specific for pancreatic and leukocyte elastase nor by phenylmethylsulfonyl fluoride. Macrophage elastase also does not bind to tritiated diisopropylphosphorofluoridate. The enzyme is inhibited by EDTA and thus appears to be a metallo-protease. Macrophage elastase is resistant to human alpha 1-proteinase inhibitor and to human and mouse alpha 2-macroglobulin. In view of its lack of susceptibility to these endogenous serum proteinase inhibitors, macrophage elastase may play an important role in physiological and pathological remodeling of connective tissues.     

NMR and enzymatic investigation of the interaction between elastase and sodium trifluoroacetate.

At pH 5.5, sodium trifluoroacetate is a potent competitive inhibitor of porcine elastase (Ki = 2.6 mM) and human leukocyte elastase (Ki = 9.3 mM). For both enzymes the Ki increases strongly with pH. Sodium fluoride is inactive on pancreatic elastase and sodium acetate is a weak inhibitor of this enzyme. Trifluoroethanol inhibits both enzymes but is less active than trifluoroacetate in acidic pH conditions. Bovine trypsin and alpha-chymotrypsin are resistant to the action of sodium trifluoroacetate and trifluoroethanol. The interaction between sodium trifluoroacetate and pancreatic elastase is also demonstrated by 19F NMR spectroscopy. Trifluoroacetyltrialanine is able to displace trifluoroacetate from its complex with pancreatic elastase. In addition, a method using turkey ovomucoid for the active site titration of leukocyte and pancreatic elastase is described.     

Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme

Elastase-like enzymes are involved in important diseases such as acute pancreatitis, chronic inflammatory lung diseases, and cancer. Structural insights into their interaction with specific inhibitors will contribute to the development of novel anti-elastase compounds that resist rapid oxidation and proteolysis. Proteinaceous Kunitz-type inhibitors homologous to the bovine pancreatic trypsin inhibitor (BPTI) provide a suitable scaffold, but the structural aspects of their interaction with elastase-like enzymes have not been elucidated. Here, we increased the selectivity of ShPI-1, a versatile serine protease inhibitor from the sea anemone Stichodactyla helianthus with high biomedical and biotechnological potential, toward elastase-like enzymes by substitution of the P1 residue (Lys¹³) with leucine. The variant (rShPI-1/K13L) exhibits a novel anti-porcine pancreatic elastase (PPE) activity together with a significantly improved inhibition of human neuthrophil elastase and chymotrypsin. The crystal structure of the PPE·rShPI-1/K13L complex determined at 2.0 Å resolution provided the first details of the canonical interaction between a BPTI-Kunitz-type domain and elastase-like enzymes. In addition to the essential impact of the variant P1 residue for complex stability, the interface is improved by increased contributions of the primary and secondary binding loop as compared with similar trypsin and chymotrypsin complexes. A comparison of the interaction network with elastase complexes of canonical inhibitors from the chelonian in family supports a key role of the P3 site in ShPI-1 in directing its selectivity against pancreatic and neutrophil elastases. Our results provide the structural basis for site-specific mutagenesis to further improve the binding affinity and/or direct the selectivity of BPTI-Kunitz-type inhibitors toward elastase-like enzymes.     

Elafin: an elastase-specific inhibitor of human skin. Purification, characterization, and complete amino acid sequence

A potent inhibitor of human leukocyte elastase (EC 3.4.21.37) and porcine pancreatic elastase (EC 3.4.21.36) was purified to homogeneity from human horny layers. It inhibits human leukocyte elastase and porcine pancreatic elastase in a 1:1 molar ratio and shows equilibrium dissociation constants of 6 x 10(-10) M and 1 x 10(-9) M, respectively. Inhibition of plasmin, trypsin, alpha-chymotrypsin, and cathepsin G was not observed. This inhibitor proved to be an acid stable basic peptide with an isoelectric point of 9.7. The complete amino acid sequence appears to be unique with 38% homology to the C-terminal half of antileukoprotease. The sequence shows that the inhibitor is composed of 57 amino acids and predicts a Mr of 7017. The high affinity as well as the apparent specificity for elastases suggests a functional role in preventing elastase-mediated tissue proteolysis. It is suggested that the term "elafin" be used to designate this inhibitor.     

Binding of amino acid side chains to preformed cavities: interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P1 residues.

In the association of serine proteinases with their cognate substrates and inhibitors an important interaction is the fitting of the P1 side chain of the substrate or inhibitor into a preformed cavity of the enzyme called the S1 pocket. In turkey ovomucoid third domain, which is a canonical protein proteinase inhibitor, the P1 residue is Leu18. Here we report the values of equilibrium constants, Ka, for turkey ovomucoid third domain and 13 additional Leu18X variants with six serine proteinases: bovine alpha chymotrypsin A, porcine pancreatic elastase, subtilisin Carlsberg, Streptomyces griseus proteinases A and B, and human leukocyte elastase. Eight of the Xs are coded amino acids: Ala, Ser, Val, Met, Gln, Glu, Lys, and Phe, and five are noncoded: Abu, Ape, Ahx, Ahp, and Hse. They were chosen to simplify the interamino acid comparisons. In the homologous series of straight-chain side chains Ala, Abu, Ape, Ahx, Ahp, free energy of binding decreases monotonically with the side-chain length for chymotrypsin with large binding pocket, but even for this enzyme shows curvature. For the two S. griseus enzymes a minimum appears to be reached at Ahp. A minimum is clearly evident for the two elastases, where increasing the side-chain length from Ahx to Ahp greatly weakens binding, but much more so for the apparently more rigid pancreatic enzyme than for the more flexible leukocyte enzyme. beta-Branching (Ape/Val) is very deleterious for five of the six enzymes; it is only slightly deleterious for the more flexible human leukocyte elastase. The effect of gamma-branching (Ahx/Leu), of introduction of heteroatoms (Abu/Ser), (Ape/Hse), and (Ahx/Met), and of introduction of charge (Gln/Glu) and (Ahp/Lys) are tabulated and discussed. An important component of the free energy of interaction is the distortion of the binding pocket by bulky or branched side chains. Most of the variants studied were obtained by enzymatic semisynthesis. X18 variants of the 6-18 peptide GlyNH2 were synthesized and combined with natural reduced peptide 19-56. Disulfide bridges were formed. The GlyNH2 was removed and the reactive-site peptide bond X18-Glu19 was synthesized by complex formation with proteinase K. The resultant complexes were dissociated by sudden pH drop. This kinetically controlled dissociation afforded virgin, reactive-site-intact inhibitor variants.     

Inhibition of leucocyte elastase by heparin and its derivatives.

Leucocyte proteinases, e.g. leucocyte elastase and cathepsin G, are inhibited by heparin. The activities of pig pancreatic and Pseudomonas aeruginosa elastases are unaffected by this polysaccharide. Heparin derivatives of known Mr and degree of sulphation were isolated. The inhibition of leucocyte elastase by these oligosaccharides can be classified as tight-binding hyperbolic non-competitive. Ki values ranged from 40 nM to 100 microM and were found to be inversely correlated with the chain length of the oligosaccharides. Desulphated compounds lacked inhibitory potential towards leucocyte elastase. Over-O-sulphated di- and tetra-saccharides are more potent inhibitors than their over-N-sulphated counterparts. It is proposed that the therapeutic use of heparin and its derivatives could be extended to disease states such as emphysema and rheumatoid arthritis, where the role of leucocyte elastase has been clearly established.     

Anionic inhibitors of pancreatic and leukocyte elastase. Alkylamides of 3-carboxypropionyl- and 4-carboxybutyrylalanine peptides

Low-molecular inhibitors of pancreatic and leukocyte elastase were synthesized of the general formula X-[Ala]n-A [X = Suc and Glt, A = ethylamide, n = 1, 2, 3 and 4; for n = 2 X = H and A = (2-phenylethyl)amide] and of the formula X-[Ala]3-A (X = H, Suc and Glt, A = methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, (2-phenylethyl)- and diethylamide; for A = ethylamide, X = maleyl or acetyl). Inhibition constants Ki of these pancreatic and leukocyte elastase inhibitors were determined using the chromogenic substrates Suc-[Ala]4-Nan or Glt-[Ala]4-Nan and Glt-[Ala]3-Val-Nan, respectively. The series of anionic inhibitors containing three alanine residues has the best inhibitory properties. Of the acyl groups, 4-carboxybutyryl appears most advantageous, propylamide is the most suitable of alkylamides for pancreatic elastase, and isobutylamide for leukocyte elastase. Peptides containing a free amino group show an inhibition for pancreatic elastase lower by one order than that of the corresponding acylated derivatives. Glt-[Ala]3-NH-Pr is the best inhibitor of pancreatic elastase with Ki = 0.003mM and Suc-[Ala]3-NH-iBu is the best inhibitor of leukocyte elastase with Ki = 0.7mM.