Trypsin Activity

The first intermediate—7r-chymotrypsin—displays chymo-trypsin activity. Suggest proteolytic enzymes that might carry out these cleavage reactions effectively. 

Due to their response mechanism the polyion-selective electrodes are not sensitive to the small fragments of polyionic macromolecules. Thus, if an enzyme cleaves the polyionic molecule these sensors can be used for detection of enzyme activity. Polycation protamine is rich in arginine residues that make it a suitable substrate for protease-sensitive electrochemical assays. Real-time detection of trypsine activity was demonstrated with the protamine-selective electrode as a detector [38],... 

The methods used were acrosin proteolytic activity (APA) assay (with gelatin) and acrosin activity assay with N-a-benzoyl-DL-arginine-p-nitroanihde (BAPNA)-Triton X 100, and BAPNA assay for trypsin activity [9-11]. The antioxidant activity was tested spectrometrical with ABTS+ [12]. Cytotoxicity of extracts was determined by MTT Cell Proliferation Assay [13]. 

The experiment with 0.5 g/mL of erode extracts of different parts of Allium plants (bulb, green leaf, white stalk) showed the inhibition of acrosin and trypsin activities, mostiy the red onion and garlic extracts. These results demonstrated that spermicidal effect of Allium extracts is determined also by their capacity of protease inhibition. The red onion extract shows the higher antioxidant capacity and a very low cytotoxicity. 

The aim of this work was to evaluate the inhibition effect of ome Allium aqueous extracts on acrosin and trypsin activities, a new aspect in the wide spectrum of Allium compound activities. The antioxidant capacity, probably responsible for antithrombosis and antitumoral effects, was determined for different parts of Allium plants (bulb, green leaf, white stalk). The toxicity of crude extracts was also determined. 

Fig. 41.2 Inhibition activities of plant extracts on trypsin activity (mean value SD)...

Trypsin inhibition activity was determined by measuring the decrease of trypsin activity in the presence of Allium extracts. The results obtained with different types of extracts (bulb, stalk, green leaf-0.5 g/iuL) are presented in Fig. 41.2. All determinations were carried out in triplicate. The higher inhibition activity was obtained with red onion bulb extract (18.55 0.14 Ul/min or 27.94 0.21 U/g fresh plants). Onion bulb extract shows also a high trypsin inhibition (18.48 0.035 UFmin, 27.83 0.053U/g), and all extracts of garlic (bulb, leaf and stalk) show activities between 11.05 1.615 - 13.185 0.205 Ul/min (16.64 2.42 - 19.86 0.31 U/g). 

Collagenase assays were carried out in 50 mM Tricine, 0.2 M NaCl, 10 mM CaCl2 (pH 7.5) containing 0.05% Brij-35 and 50 //M ZnS04 at 30 C on p-chloromercuribenzoate-activated HNC and trypsin-activated HFC using rat type I collagen as substrate. a-Ms denote a-... 

Figure 5. Effect of stirrer rate on trypsin activity in the hydrolysis of BAEE at under the mass transfer limited conditions at 308 K.

F5. Frossard, J. L., Trypsin activation peptide (TAP) in acute pancreatitis From pathophysiology to clinical usefulness. JOP. J. Pancreas (Online) 2, 69-77 (2001). 

Briand, L., Chobert, J.-M., Tauzin, J., Declerck, N., Leonil, J., Molle, D., Tran, V., and Haertle, T. 1997. Regulation of trypsin activity by Cu2+ chelation of the substrate binding site. Protein Engng 10, 551—560. 

A substantial inhibition (>30%) of trypsin activity was measured for the whole library Ax. A first iteration of 6 sublibraries, where 3 out of 18 monomers (Cys was removed to reduce artifacts) were removed from the set,... 

While the presence of families of positives made the determination of the most active compounds more difficult, their activities contributed to the 34% inhibition of trypsin activity seen for the whole library. In other words, a moderately active single family of inhibitors could easily be lost due to the low activity of the whole library, thus this approach is only suitable for libraries where either very few highly potent or many weakly potent compounds are present, and for biological assays able to reliably detect activities in the medium micromolar range. The redundancy of the library (65,321 different individuals compared to the theoretical 130,321 permutations, producing multiple copies of some individuals) was due to the scaffold symmetry and could have caused anomalies in the assay results due to the different concentrations of the individuals. 

For enzyme inhibition assays, urine is the preferred specimen [4]. Interestingly, Bik can be measured by the inhibition of trypsin in urine but not in plasma. Urinary Bik analysis may also be performed by antibody staining, latex agglutination, and radioimmunoassay (RIA) [4]. Despite the analytical approach used, all Bik forms are measured together. The enzyme inhibition method involves adding known amounts of trypsin to the specimen and monitoring trypsin inhibition. Trypsin activity is assessed by detection of by-products from a cleavable substrate. Dipstick methods are available for the rapid detection of trypsin inhibitors in urine [15, 17 19]. 

The reaction process of trypsin-catalyzed hydrolysis of the inverse substrates is illustrated in Fig. 4. Here the process is compared to that of normal-type substrates. After specific binding and efficient acylation, the site-specific amidinophenyl moiety is cleaved (leaving group) to give the acyl enzyme in a very specific manner. As a result, inverse substrates are expected to be applicable as a general method for specific introduction of any acyl group of non-specific structure into the trypsin active site. 

For the first time, inverse substrates provide a general method for the specific introduction of an acyl group into the trypsin active site without recourse to cation-containing acyl compounds. The preparation of various new acyl enzymes is expected to lead to the discovery of novel features of the enzymatic reaction mechanism. In addition, any desired reporter groups might be specifically introduced into the trypsin... 

The microenvironment of the trypsin active site was estimated by spectrometric analysis of these acyl trypsin preparations. 

Induction of mRNAs for several other specific rat hepatic proteins by GH has also been demonstrated [81-83]. The effect could be demonstrated in vivo and in vitro and involved a relatively rapid induction with a 5-fold increase in mRNA levels within 4 h of the administration of GH, although synergism with cortisol (possibly and/or thyroxine) was necessary for a maximal response [83]. cDNAs corresponding to two of the induced proteins have been cloned [82,83] and found to have sequences homologous to those of a known family of serine protease inhibitors. One of these proteins was shown to be secreted as a heavily glycosylated serum protein, and to have potent anti-trypsin activity [83]. Regulation of the production of this protein by GH was shown to occur mainly at the transcriptional level [83]. 

Figure 26-2. The pancreatic enzyme cascade. Pancreatic proteases enter the intestinal lumen as inactive zymogens. Within the duodenum, a specific enzyme of the duodenal mucosa, enterokinase, activates trypsinogen by releasing the trypsinogen activation peptide (TAP). Subsequently, active trypsin activates the other zymogens and acts autocat-alytically.

Involvement of sperm proteolytic enzymes in fertilization processes has a long history. Perhaps the first definitive observation that sperm proteases affected egg envelopes was that of Yamane in 1935. He demonstrated that an extract of rabbit sperm dispersed the cumulus cells and solubilized the zona pellucida (ZP) or egg envelope of the rabbit egg (1.). The presence of proteases in the sperm extract was presumed by analogy with the dispersing action of the trypsin activity in pancreatin. In 1939, Tyler obtained an extract from sperm of the giant keyhole limpet Meeathura crenulata which dissolved the egg envelope without affecting the egg itself. 

The third criterion of the inhibition of fertilization by active site inhibitors was observed both In vitro (63) and in vivo (64) fertilization. However, this inhibition was never total, and once the sperm had tightly bound the ZP, trypsin active site inhibitors did not prevent further penetration or hydrolysis of the ZP (65). Therefore, while acrosin does not strictly follow all of the criteria for a ZP lysin, it does cause limited hydrolysis of the ZP and it is in the correct place to act in penetration of the ZP. 

Application and Principle This procedure is used to determine the trypsin activity of trypsin preparations derived from purified extracts of porcine or bovine pancreas. 

Lipase (Microbial) Activity for Medium- and Long-Chain Fatty Acids, (S3)105 Lysozyme Activity, (S3)106 Maltogenic Amylase Activity, 804 Milk-Clotting Activity, 805 Pancreatin Activity, 805 Pepsin Activity, 807 Phospholipase A2 Activity, 808 Phytase Activity, 808 Plant Proteolytic Activity, 810 Proteolytic Activity, Bacterial (PC), 811 Proteolytic Activity, Fungal (HUT), 812 Proteolytic Activity, Fungal (SAP), 813 Pullulanase Activity, 814 Trypsin Activity, 814 Enzyme Assays, 786 Enzyme-Hydrolyzed (Source) Protein,... 

Ten female Ae. aegypti midguts were removed and analyzed for trypsin activity 24 h later. Results taken from (15). 

---