Enzyme Assays Trypsin Activity

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],... 

Evidence demonstrating that the degradation observed is catalyzed by enzyme(s) was obtained by typical denaturing treatments. Late extracellular protein fractions from both strains present the same characteristics resistance to heat up to 100°C, partial resistance to acidity as low as pH 1.0 (samples being returned to pH 7.8 prior to assaying for activity), but are completely inactivated by proteolysis with a mixture of trypsin and chy-motrypsin (Table II). 

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]. 

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,... 

Treatment of -ABSC-HEMA with glutaraldehyde produced enzyme supports capable of binding up to 55 wt % trypsin. Incorporation of hydrophobic styrene units Into the support reduced the capacity to 2-. 4 wt X but enhanced the specific activity of the trypsin. The esterase activity of bound trypsin, assayed with TAME, was found to range from 11% to 45% of that exhibited by the free trypsin. Active-site titration of a PHEMA-trypsln conjugate with p-nltrophenyl-p -guanadlnobenzoate HCl Indicated the active species to be 31% of the total amount of protein bound. 

The crude fish enzyme extracts were prepared as per the method of Baranowski et al. (1984), and stored in ice for use in the pressure treatments and subsequent enzyme assays. The spectrophotometric methods of Hummel (1959) and Erlanger et al (1961) were used to assay for chymotrypsin-like and trypsin-like enzyme activities using BTEE and BAPNA as substrates, respectively. Cathepsin C activity was assayed using gly-phe-NA as substrate (Lee et al, 1971), and collagenase activity in the fish extracts were assayed as per the method of Wunsch and Heidrich (1963). Protein content of the crude enzyme extracts from fish was determined by the method of Hartree (1972). 

Trypsin inhibitors in animal diets are known to cause pancreatic hypertrophy and hyperplasia. We therefore monitored pancreas weight and enzymic contents in all of the above experiments. The pancreata were removed from the chicks at the termination of the experiments, weighed, and assayed for activities of trypsin, chymotrypsin and carboxypeptidases A and B, after activating their zymogens with either trypsin or enterokinase. 

The assay is suitable for assaying enzyme in serum and plasma, and also recombinant human prorenin expressed in Chinese hamster ovary cells. Prorenin was activated by trypsin digestion. 

AAT can be quantified by all immunochemical methods, with immunoturbidimetry and immunonephelometry the most commonly used methods. Because it constitutes about 90% of the serum inhibition of trypsin or elastase activity against small substrates, such as benzoyl-DL-arginine p-nitroanilide, it can also be semiquantified by the inhibitory capacity of serum for these enzymes however, this assay is not specific for AAT. 

Table III also shows that trypsln-cvb-hydrogel derivatives retain in the range of 11-45% the original enzyme activity, which is about average for the covalently bound trypsin on polymers containing hydrogel. For comparison, Mosbach (2.) immobilized trypsin on a crosslinked copolymer of acrylamide and hydroxyethyl-methacrylate via CNBr coupling, and analyzed it photometrically with a- -benzoyl-Dl-arginine-p-nltroanlllde (BAPNA), reporting a 35% retention of activity. o Driscoll and co-workers (1 ) however, reported an equivalent of 1.3% efficiency when trypsin was physically entrapped in a crosslinked PHEMA gel and assayed with TAME.

Assay of the isozymers from L rubellus is based on the enzymic reaction with various substrates. F-T and F-III are thought to represent a chymotrypsin-like and a trypsin-like protease, respectively [24], F-II appears to act as a trypsin-like protease or an elastase. Interestingly, F-II1-1 and -2, also with strong caseinolylic activity, have much higher fibrinolytic activity than plasmin as described by Mihara et al. [1,3]... 

Background Trypsinogen is synthesized exclusively by the acinar cells of the pancreas, and measurement of this zymogen by a trypsin-like immunoreactivity (TLl) assay provides an indirect index of pancreatic damage. TLl detects both trypsinogen and trypsin forms and hence the use of the term TLl to describe the total concentration of these two immunoreactive species. It should be noted that the active enzyme (trypsin) is only present in the serum when there is pancreatic inflammation. As a consequence, TLl concentration has been shown to be useful for the diagnosis of AP, although it has a low sensitivity (Steiner, 2003). 

---