Chymotrypsin assay

This same approach was used for assaying other protease activities such as chy-motrypsin and renin [49], Chymotrypsin and renin cleave only at specific sites of... 

Cycloamyloses have been studied extensively as models of a-chymotrypsin and other serine proteases. Chymotypsin works via a double displacement pathway in which the hydroxyl group of serine-195 acts as a nucleophile. This is shown in Scheme I. In nature, the substrate is an amide. Synthetic esters are also used in model studies and routine assays. Nitrophenyl and... 

The pH-stat method can be modified and applied to a particular enzyme or substrate to assay any enzyme/substrate combination. The substrate can come from any source of protein such as poultry, milk, soybean, or fish processing byproducts. The amount of protein in the reaction should not exceed 8% (as calculated in Basic Protocol 1). The enzyme can be any alkaline endopeptidase such as Alcalase, trypsin, or chymotrypsin, and should be used in the proportions indicated in Basic Protocol 1. The selection of the appropriate enzyme depends on its efficiency and cost. 

In this assay, enzymes with chymotrypsin-like specificity readily bind the side chain of Phe at the primary substrate binding site (SI) and subsequently hydrolyze the adjacent amide bond linking the Phe residue to the p-NA moiety. On cleavage, the release of p-NA is measured by the increase in absorbance at 410 nm (e4l0 = 8480 M 1 cm-1) with time, using a recording spectrophotometer. 

The optimal concentration of the enzyme test solution depends on the proteinase being assayed, and must be determined experimentally. For chymotrypsin, 2.5 to 25 pg/ml (0.1 to I pM) is appropriate. Concentrations of 5 to 50 pg/ml are appropriate for most proteinoses assayed with this procedure. 

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. 

In Table II are shown the results from kinetic studies with commercially available gastric and pancreatic enzymes. Trypsin was strongly inhibited, at least at a low concentration of casein as substrate. The hydrolysis of benzoyl arginine ethyl ester (BAEE) by trypsin was non-competitively inhibited, giving a 30% reduction of Vmax at 0.5 mg/ml of the LMW fraction. Carboxypepti-dase A, and to a lesser extent carboxypeptidase B, were non-competitively inhibited as well. Pepsin and chymotrypsin were not affected by the conditions used in these assays. 

Trypsin and factor Xa (fXa) are two members of the chymotrypsin family that have 38% sequence identity on the amino acid level and have distinguishable substrate specificities. Recently, the N-terminal 13-barrel of fXa and the C-terminal /3-barrel of trypsin were fused at a rationally designed site in the linker region between the two domains in order to create a hybrid fXa-trypsin protease (Hopfner et al., 1998). The fXa-trypsin hybrid was highly active and more active than either parent on three of the ten substrates assayed, as determined by k /Km. For most substrates, the activity of fXa-trypsin was an admixture of the two parents, probably because trypsin had higher activity than fXa for all the substrates tested. 

Calculations One USP Chymotrypsin Unit is defined as the activity causing a change in absorbance at the rate 0.0075/ min under the conditions of the assay. Determine the average absorbance change per min using only those values within the 3-min portion of the curve where the rate of change is constant. Plot a curve of absorbance against time. 

The search for an enzymatic activity that would catalyze prolyl peptide bond isomerization began soon after the proposal of the proline hypothesis. The success came in 1984, when Fischer and co-workers discovered a peptidylprolyl m—tram-isomerase activity in porcine kidney and other tissues by an assay that is based on the conformational specificity of chymotrypsin. This protease cleaves the 4-nitroanilide moiety from the peptide glutaryl-Ala-Ala-Pro-Phe-4-nitroanilide only when the Ala-Pro peptide bond is in the trans conformation. In aqueous solution 90% of the molecules are trans in the assay peptide and only 10% are cis. Therefore, in the presence of a high concentration of chymotrypsin, 90% of the hydrolysis reaction occurs within the dead time of manual mixing. Hydrolysis of the remaining 10% is slow, limited in rate by the cis — ... 

Serum albumin labeled with an iodine radionuclide was firstly used as a substrate for determining protease activity by Absolon This method was later on modified several times and applied for assaying various proteolytic activities in different materials. Mego et al. injected denaturated I-human %rum albumin into the tail vein of rats and measured the rate of intralysosomal proteolysis on isolated lysosomes containing endocytosed substrate. This method was also used for the determining the intralysosomal pH on the basis of differences found in the rate of I-albumin breakdown in intact and lysed lysosomes C-bovine serum albumin, I-casein or I-albumin have been alternatively used as substrate for measuring the activity of trypsin, chymotrypsin and papain - ). 

We may also mention that peptide-bond absorption at somewhat longer wavelengths has been employed by Mitz and Schleuter (1957), and by Schmitt and Seibert (1961) to estimate the rates of digestion by peptidases acting on simple peptides. Schwert and Takenaka (1955) describe difference-spectral methods for assaying chymotrypsin and trypsin with acetyl-L-tyrosine ethyl ester and benzoyl-L-arginine ethyl ester as the respective substrates. 

The report by Haas and Guardia (18) pertains to their efforts to apply immunological methods for the assay of pesticide residues, and expresses their aim to test the suitability of methods for field analytical purposes. Haas and Guardia also used DDT and malathion to represent two of the most important classes of insecticides, chlorinated hydrocarbons and organophosphorus compounds. They first attempted to prepare insecticide-protein antigens in which enzymes were used as the protein carrier. Antiserum of rabbits injected with DDA-carbonic anhydrase or malathion-chymotrypsin failed to show the presence of the respective antibodies. 

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... 

For assaying the pH-dependent activity of subtilisin crystals as mentioned above, low buffer capacities were used [6,102]. Thus, the formation of a dynamic pH-gradient during hydrolysis may explain the observed pH-shift in activity. Also carrier-fixed a-chymotrypsin reveals exactly the same phenomena [86, 97], which is discussed in more detail elsewhere [103]. 

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