Affinity partitioning combines the partitioning behavior of biological macromolecules in aqueous two-phase systems with the principle of biorecognition. Among the numerous substances that have been evaluated as ligands, the reactive dyes constitute a group of low cost textile dyes which have proved to act as biomimetic ligands for many enzymes. The ability of reactive yellow 2 (RY2) to interact with trypsin (TRP) and chymotrypsin (ChTRP) and its behavior in aqueous two-phase systems formed by polyethylene glycol (PEG) and sodium citrate (NaCit)- were investigated. Different variables such as PEG molecular weight, tie line length and dye concentration were analyzed. RY2 showed to bind specifically to both TRP and ChTRP with affinity constants near to 10(3)M(-1). Its partition equilibrium is practically displaced to the top phase in systems formed by PEG of different molecular weight. Addition of this dye to PEG 8000/NaCit systems until a final concentration of 0.196%(w/w) induced an increase in TRP and ChTRP partition coefficients of at least 2 times over that in the absence of the ligand. These findings demonstrate that RY2 fulfils all the requirements to be considered as an affinity ligand in aqueous two-phase partitioning of TRP and ChTRP.

A trypsin-like, membrane-bound protease from Bacteroides gingivalis was solubilized by Triton X-100 and partially purified by a combination of DEAE-Sepharose and aminophenylmercuric Sepharose chromatography, by taking advantage of the thiol group on the enzyme. The purified enzyme hydrolysed the synthetic substrates benzoyl-L-arginine-p-nitroanilide (L-BAPA), benzoyl-D,L-arginine-beta-naphthylamide (BANA) and tosyl-L-arginine methyl ester, as well as bovine serum albumin and ovalbumin, but not tosyl-L-lysine methyl ester. The enzyme activity was enhanced by SH-reagents and was inhibited to different degrees by SH-inhibitors, chelators and microbial low-molecular-weight inhibitors such as leupeptin, antipain and chymostatin. These microbial inhibitors could be of practical use as ligands for affinity chromatography for further purification. The possible involvement of the protease in periodontal diseases is also discussed.

Faecally enriched extracts of Dermatophagoides pteronyssinus were shown to contain a trypsin-like enzyme which was allergenic. Chromatofocusing studies revealed the presence of nine major isoforms in D. pteronyssinus, with pI in the range 4 to greater than 8, but only two (range 4-5) in D. farinae. Trypsin isolated from D. pteronyssinus by benzamidine-Sepharose 6B affinity chromatography and gelfiltration was found to be a 31-kDa protein which was enzymatically similar to both invertebrate and vertebrate trypsins. The N-terminal sequence obtained (IVGGEXALAGEXPYQISL) was identical to that reported for the mite allergen Der p III and showed homology with crayfish trypsin and Der f III from D. farinae. Mite trypsin underwent autolysis and the N-terminal sequences of two fragments were found to be ALAGEXPYQI and NNQVXGI respectively. Both showed homology with crayfish trypsin, and the former sequence was identical to residues 7-18 of the native enzyme and Der p III. All isoforms of mite trypsin were showed to be allergenic by radioallergosorbent assay and further studies indicated that the trypsin degradation products were also allergenic. The enzyme was compared with other mite allergens and the rank order of allergenic potency was shown to be: whole mite extract greater than Der p I greater than trypsin. However, all sera from a panel of mite allergic individuals showed IgE reactivity to trypsin, comparable to that seen using whole mite extract and Der p I. These data indicate that mite trypsin is a major allergen corresponding to the previously described allergen, Der p III.

We designed degenerated oligodeoxyribonucleotide primers derived from amino acid (aa) sequences of the highly conserved active sites of mammalian serine proteases (SPs). These primers were used to selectively amplify, in polymerase chain reactions (PCRs), cDNA fragments coding for a SP. We used poly(A)+RNA from human brain to obtain cDNA fragments and amplified one cDNA encoding a novel SP. The full-length nucleotide (nt) sequence was identified by PCR and screening a genomic library in order to obtain the 5'-region. The deduced as sequence shows a high degree of homology to trypsinogens, except for the first exon. In addition to this brain-specific trypsinogen, there exists a variant of the cDNA in pancreas, differing only in the nt sequence of the first exon. An active form of the trypsin was synthesized in vitro and purified by affinity chromatography using soybean trypsin inhibitor (STI) agarose to demonstrate the trypsin-specific interaction with a naturally occurring inhibitor of trypsins.

1,1'-Carbonyldiimidazole, a carbonylating reagent, has been shown to be suitable for the activation of cross-linked agaroses for affinity chromatography. The activated matrix (an imidazolyl carbamate) is relatively stable to hydrolysis but smoothly reacts with N-nucleophiles such as those present in either affinity chromatography ligands or leashes, e.g. ethylenediamine or 6-aminohexanoic acid. If butylamine was attached via the 1,1'-carbonyldiimidazole method, the resulting product was devoid of charged groups and thus had the same titration curve as agarose. The suitability of this new matrix for affinity chromatography was demonstrated by the successful purification of trypsin by several different systems.

We have isolated and identified a new zymogen in human pancreatic tissue and fluid. It is secreted as a minor component of pancreatic juice and resembles the two known trypsinogen variants in many properties. Its electrophoretic mobility and isoelectric pH lie between those of the cationic and anionic trypsinogen variants, and we propose the name "mesotrypsinogen" for the new enzyme precursor. It is activated by enteropeptidase or trypsin, and the free enzyme possesses a substrate specificity similar to that of the trypsins. Its pH optimum is at 8.2, and it appears to require Ca2+ for full enzymatic activity. The molecular weight of the new enzyme is approximately 25,000, similar to that of the known trypsin variants. Its stability resembles that of anionic trypsin extending over a pH range of 4-8.5. Activity is lost gradually at pH 2. The enzyme is inactivated rapidly by diisopropylfluorophosphate, but in contrast to the trypsins, it reacts only slowly with tosyllysine chloromethylketone. Immunologically, it is different from the cationic trypsin variant with which it does not cross-react. The most remarkable property of mesotrypsin is its almost total resistance to biological trypsin inhibitors, such as pancreatic trypsin inhibitor, soybean, lima bean, ovomucoid inhibitor, alpha 1-antitrypsin, etc. It is capable of activating trypsinogen in the presence of excess pancreatic trypsin inhibitor and thus inducing activation of other pancreatic zymogens, but it also possesses the ability to degrade trypsinogen rapidly to inert products. The physiological or pathophysiological role of this unique enzyme remains to be explored.

The structural determinants of the primary substrate specificity of rat anionic trypsin were examined by using oligonucleotide-directed mutagenesis coupled to a genetic selection. A library was created that encoded trypsins substituted at amino acid positions 189 and 190 at the base of the substrate binding pocket. A genetic selection, with a dynamic range of 5 orders of proteolytic activity, was used to search 90,000 transformants of the library. Rapid screening for arginyl amidolysis and esterolysis confirmed the activity of the purified isolates. Trypsin and 15 mutant trypsins with partially preserved function were identified and characterized kinetically on arginyl and lysyl peptide substrates. Alternative arrangements of amino acids in the substrate binding pocket sustained efficient catalysis. A negative charge at amino acid position 189 or 190 was shown to be essential for high-level catalysis. With the favored aspartic acid residue at position 189, several amino acids could replace serine at position 190. Modulation of the specificity for arginine and lysine substrates was shown to depend on the amino acid at position 190. The regulatory effect of the amino acid side chain at position 190 on the substrate specificity is also reflected in substrate binding pockets of naturally occurring trypsin homologs.

The ability of plasma proteinase inhibitors to inactivate human chymase, a chymotrypsin-like proteinase stored within mast cell secretory granules, was investigated. Incubation with plasma resulted in over 80% inhibition of chymase hydrolytic activity for small substrates, suggesting that inhibitors other than alpha 2-macroglobulin were primarily responsible for chymase inactivation. Depletion of specific inhibitors from plasma by immunoadsorption using antisera against individual inhibitors established that alpha 1-antichymotrypsin (alpha 1-AC) and alpha 1-proteinase inhibitor (alpha 1-PI) were responsible for the inactivation. Characterization of the reaction between chymase and each inhibitor demonstrated in both cases the presence of two concurrent reactions proceeding at fixed relative rates. One reaction, which led to inhibitor inactivation, was about 3.5 and 4.0-fold faster than the other, which led to chymase inactivation. This was demonstrated in linear titrations of proteinase activity which exhibited endpoint stoichiometries of 4.5 (alpha 1-AC) and 5.0 (alpha 1-PI) instead of unity, and SDS gels of reaction products which exhibited a banding pattern indicative of both an SDS-stable proteinase-inhibitor complex and two lower Mr inhibitor degradation products which appear to have formed by hydrolysis within the reactive loop of each inhibitor. At inhibitor concentrations approaching those in plasma where inhibitor to chymase concentration ratios were in far excess of 4.5 and 5.0, the rate of chymase inactivation by both serpin inhibitors appeared to follow pseudo-first order kinetics. The "apparent" second order rate constants of inactivation determined from these data were about 3000-fold lower than the rate constants reported for human neutrophil cathepsin G and elastase with alpha 1-AC and alpha 1-PI, respectively. This suggests that chymase would be inhibited about 650-fold more slowly than these proteinases when released into plasma. These studies demonstrate that although chymase is inactivated by serpin inhibitors of plasma, both inhibitors are better substrates for the proteinase than they are inhibitors. This finding along with the slow rates of inactivation indicates that regulation of human chymase activity may not be a primary function of plasma.