Enzyme Assays Chymotrypsin Activity

The derivatization of an enzyme by DFP is accomplished simply by incubating DFP with the protein. Aliquots of the incubation mixture can then be assayed for activity in the presence of DFP or after its removal by either gel filtration or diafysis. The second-order rate constant at neutral pH for the inactivation of chymotrypsin by DFP is roughly 3 x 10 sec (Main 1964). This is the most rapid rate of... 

A second elastolytic enzyme (Af, 21,900) has been isolated from porcine pancreas. It shows higher activity than chymotrypsin in the hydrolysis of acetyltyrosine ester, which is used routinely to assay chymotrypsin. Another E,-like enzyme, a-lytic proteinase (Af, 19,900, 198 amino acids) has been isolated from the soil bacterium Myxobacter 495. This enzyme is remarkably similar to pancreatic E. both in structure (41 % homology, sequence in the active center Gly-Asp-Ser-Gly, 3 homologous disulfide bridges) and substrate specificity. Another E. (Af, 22,300) has been isolated from Pseudomonas aeruginosa. 

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 of an enzyme solution, historically tied to a rate assay, depends on the correlation between the rate and a concentration (determined by some means, usually spectroscopic) of the purest possible sample of that enzyme. Since with even the most thoroughly characterised enzymes, such as a-chymotrypsin, a substantial proportion of the matter is inactive enzyme and water (up to about 30% in some cases), the concentration of active enzyme from rate assays is liable to a wide margin of error. 

Two libraries of a-ketoamide derivatives were generated on pins to map the S and S subsites of human heart chymase [234]. The libraries were screened directly on the pins by a method that measured enzyme absorption onto the pins and by an enzyme depletion assay. From the first library which was based on the sequence Z-Ile-Aa2-Aai-Phe-CO-Gly-GIy it was found that Pro-Gly provided the P3-P2 combination that gave rise to potent inhibition and maximum selectivity over chymotrypsin (library 17 Kj 1.0 nM). From the second library of peptidyl-3-fluorobenzylpyruvamides, (F)-Phe-CO-Glu-Asp-Arg-OMe was identified as an inhibitor of the chymase (library 18 K 1 /iM) but it was less active than the corresponding ketoamide. However it exhibited 100-fold selectivity over chymotrypsin [234]. 

The first enzyme to catalyze prolyl isomerization was identified by Fischer and colleagues (1984). This discovery was possible because they developed an ingenious assay for prolyl isomerases based on the conformational specificity of chymotrypsin. This protease cleaves a chro-mogenic reporter group from a tetrapeptide (such as succinyl-Ala-Ala-Pro-Phe-4-nitroanilide) only when the Ala—Pro bond of this peptide is in the trans conformation. In aqueous solution the assay peptide exists as a 90 10 mixture of molecules with the Ala—Pro bond in trans and cis, respectively. Therefore, in the presence of a high concentration of chymotrypsin, 90% of the peptide molecules are cleaved within the dead time of manual mixing. Hydrolysis of the remaining 10% is slow because it is limited in rate by the cis —trans isomerization of the Ala—Pro bond. Acceleration of this reaction serves as a sensitive probe for prolyl isomerase activities. 

Figure 6. Effect of chemical modification of turkey ovomucoid on its inhibition of bovine a-chymotrypsin (by delay time assays). Abbreviations used Chy, chy-motrypsin TO, turkey ovomucoid AcTO, acetylated turkey ovomucoid AmTO, amidinated turkey ovomucoid SucTO, succinylated turkey ovomucoid 1TO, iodinated turkey ovomucoid I AcTO, iodinated and acetylated turkey ovomucoid. To a mixture of enzyme-buffer and substrate (benzoyl tyrosine ethyl ester, plus m-nitrophenol as indicator) was added the inhibitor solution within 18-25 sec and the enzyme activities recorded on a chart at 395 mfi. The weight ratio for chymotrypsin and the turkey ovomucoids was 22 15. The percent change in transmission is proportional to the amount of enzyme activity. At the time of the additions of the inhibitor, the enzyme was hydrolyzing the substrate (28).

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]... 

Free and immobilized chymotrypsin were assayed using 2mM of N-acetyl-L-tyrosine ethyl ester (ATEE) in 0.05M PBS at pH 8.0. The absorbance of the solution or the supernatant at 256 nm was plotted against the enzyme weight in the reaction mixture. The initial slope of the curve was used to evaluate the activity. 

---