Fourteen 3-carboxypropionyl-tripeptide-p-nitroanilides of the general formula 3-carboxypropionyl-alanyl-alanyl-Y-p-nitroanilide (Y = glycine, norvaline, S-methylcysteine, valine, norleucine, S-ethylcysteine, methionine, leucine, isoleucine, phenylalanine, tyrosine, S-benzylcysteine, C"-phenylglycine, and proline) were synthesized and their cleavage by elastase, trypsin, and chymotrypsin (Km, kcat and k,at/Km) was determined.The significance of amino acid residues in the position of Y was evaluated firstly with respect to their structure (topographically), and secondly with respect to their free energy (thermodynamically). The alanine residue substrate was cleaved best by elastase, the phenylalanine substrate by chymotrypsin. Trypsin cleaved two substrates only, that is those containing a phenylalanine and a tyrosine residue.The optimum length of the elastolytic substrates was studied in a series of N-3-carboxypropionyl-(Ala),-p-nitroanilides (n = 1, 2, 3, 4, 5), N-3-carboxypropionyl-(Gly),-p-nitroanilides (n = 1, 2, 3), and in p-nitroanilides of fatty acids with two to seven carbon atoms. Elastase cleaved tri, tetra, and pentapeptides of alanine.p-Nitroanilides of the glycine series, as well as p-nitroanilides of fatty acids were not cleaved. 3-Carboxypropionyl-tetra-alanine-p-nitroanilide was the most suitable substrate so far found for elastase cleavage; it is not cleaved by trypsin nor chymotrypsin. The optimum distance between Y and the terminal anionic carboxyl residue was found to be 1.8 nm in elastolytic substrates.Pancreatic elastase has lately been the subject of interest concerning the mechanism of its effect, and also concerning its presumed participation in the pathogenesis of some diseases, such as pancreatitis [l -31, lung emphysema [4,5], and atherosclerosis [6].A series of various peptides has already been prepared to elucidate and determine the optimum amino acid sequences in synthetic substrates [7 -101. The bond between C-terminal alanine and the corresponding amide or ester bond is the most easily split. Succinyl-alanyl-alanyl-alanine-p-nitroanilide, described in the preceding communication, has been shown to be the most specific substrate [ll]. (The positions of amine acid residues, discussed below, are defined in this peptide as P4-P3-PI-P1-Pi .) The principal reason for the synthesis of the anionic substrates has been perceived as the proof of electrostatic interactions between the substrate and elastase (in the sense of Gertler's conception of the natural substrate bonding) [12]. Electrostatic induction brings about a change in the elastase conformation according to Koshland's theory of 'induced fit' [13]. Since this change is caused also by natural ionogenic substances (salts of cholic acids) [14] and thus probably occurs under physiological conditions, it has been considered adequate to modify our type of ionogenic peptide and to prove 'optimalization' of the amino acid residue in the position PI [15]. The use of p-nitroaniline as chromogenic residue permits the cleavage between the pos...

Out of a series of alkylamides of N-acylated tripeptides, Glt-(Ala)2-Pro-NH-Et and Glt-(Ala)3-NH-Pr were found to be potent inhibitors of porcine and human pancreatic elastase, and because they are free of toxic groups they might be considered for in vivo application.

Two newly synthetized chromogenic substrates N a -tosyl-Z,-arginine-p-nitroanilide (ΤΑΡΑ) and N a -benzyloxycarbonyl-£-arginine-pnitroanilide (ΖΑΡΑ) were used for the determination of trypsin activity in duodenal contents before and after stimulation with cholecystokinin-rpancreozymin and secretin. In addition, the trypsin activity of the same duodenal samples was determined with the aid of N^-benzoyl-jD, Ζ,-arginine-p-nitroanilide (ΒΑΡΑ) and N a -tosyl-Z,-arginirie-methyl ester (TAME). Duodenal contents from a total of 27 control subjects and 8 patients with pancreatic exocrine insufficiency were studied. The values for trypsin concentration and output displayed a high degree of correlation with all investigated substrates. It was possible to establish the recalculation coefficients for both concentration (ΖΑΡΑ = 5.63 ΤΑΡΑ, ΒΑΡΑ = 1.20 ΤΑΡΑ, TAME = 1.20 ΤΑΡΑ) and output (ΖΑΡΑ = 5.51 ΤΑΡΑ, ΒΑΡΑ = 0.58 ΤΑΡΑ, TAME = 0.57 ΤΑΡΑ) of duodenal trypsin. The use of ΤΑΡΑ and ΖΑΡΑ for the determination of trypsin in biological material is suggested.