Trypsin characteristics

FIGURE 2.16 pH versus enzymatic activity. The activity of enzymes is very sensitive to pH. The pH optimum of an enzyme is one of its most important characteristics. Pepsin is a protein-digesting enzyme active in the gastric fluid. Trypsin is also a proteolytic enzyme, but it acts in the more alkaline milieu of the small intestine. Lysozyme digests the cell walls of bacteria it is found in tears. 

In addition to blood, certain types of specimens are submitted to the Pediatric laboratory which would not be commonly seen elsewhere. An example of this is sweat for analysis of chloride. The process of obtaining the sweat by iontophoresis usually falls to the personnel of the Laboratory of Neonatology (17). Stool for analysis of lipids and trypsin is more commonly submitted to the Laboratory of Neonatology than to the laboratory which services the adult population. The reason for this is that one is screening for certain intestinal diseases characteristic of infants and newborns which are rare in adults. Such conditions would be celiac disease, cystic fibrosis and others. 

Bromomethyl-3,4-dibromo-3,4-dihydrocoumarin 1 (Fig. 11.4) and its chloro-methylated analogue 2b rapidly and progressively inactivate a-chymotrypsin and also the activities of a series of trypsin-like proteases. A benzyl substituent characteristic of good substrates of a-chymotrypsin was introduced at the 3-position to make inhibition more selective. This substituted dihydrocoumarin 3 irreversibly inhibited a-chymotrypsin and other proteases. These functionalized six-membered aromatic lactones, and their five- and seven-membered counterparts, 3//-benzofuran-2-ones 2a26 and 4,5-dihydro-3//-benzo[b]oxepin-2-ones 2c,27 were the first efficient suicide inhibitors of serine proteases. Their postulated mechanism of action is shown in Scheme 11.2. 

Kai and Imakubo(76) found that the temperature at which emission from the exposed tryptophan is no longer observed appears to be characteristic of the protein, having values of 180 K for trypsin, 200 K for aldolase, and 230 K for alkaline phosphatase. 

Fig. 3. Amino acid sequence of several somatic human histone HI proteins to illustrate the microheterogeneity of linker histones. The sequences for human histone HI variants (H1.1-H1.4) were obtained from Ref [373] and Hl-5 was from Ref [412]. The nomenclature followed for the designation of these histone variants was Doenecke (e.g., see Ref. [412]). The nomenclature of Parseghian et at. [373] is shown in parentheses. The regions corresponding to the trypsin-resistant (winged helix motif [96]) which is characteristic of the protein members of the histone HI family are indicated by a boxed inset.

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). 

Cleavage of the polypeptide chain prior to partial reduction is an efficient way of controlling the reaction. For example, co-agatoxin IVB was completely reduced with TCEP, but upon treatment with trypsin, partially reduced species were detected under the same conditions 48 In contrast to these all or none characteristics in reduction, the four disulfide bonds of echistatin (1) can be reduced in a stepwise manner, which even allowed the elucidation of its reductive unfolding pathway 52 ... 

Beuchat (60) investigated the performance of enzyme-hydrolyzed defatted peanut flour in a cookie formula. Flour slurries were treated with pepsin at pH 2.0, bromelain at pH 4.5, and trypsin at pH 7.6. After readjustment to pH 6.9, materials were freeze-dried, pulverized (60-mesh), and then substituted for wheat flour at 5, 15, and 25%. Adjustment of peanut flour to pH 2.0, as well as treatment with pepsin at this pH, greatly improved the handling characteristics of dough in which these flours were incorporated. Use of peanut flours treated at pH 4.5, with or without bromelain, and at pH 7.6, with or without trypsin, improved handling properties of cookie dough. These doughs did not tend to crumble... 

Heinegard, D., Hascall, V. G Aggregation of cartilage proteoglycans. 3. Characteristics of the proteins isolated from trypsin digests of aggregates. J. Biol. Chem. 249, 4250 (1974)... 

Tryptic peptide mapping. The technique of generating a chromatographic profile characteristic of the fragments resulting from trypsin enzyme cleavage of the protein. 

As mentioned above, the rectal route is very attractive for systemic delivery of peptide and protein drugs, but rectal administration of peptides often results in very low bioavailability due to not only poor membrane penetration characteristics (transport barrier) but also due to hydrolysis of peptides by digestive enzymes of the GI tract (enzymatic barrier). Of these two barriers, the latter is of greater importance for certain unstable small peptides, as these peptides, unless they have been degraded by various proteases, can be transported across the intestinal membrane. Therefore, the use of protease inhibitors is one of the most promising approaches to overcome the delivery problems of these peptides and proteins. Many compounds have been used as protease inhibitors for improving the stability of various peptides and proteins. These include aprotinin, trypsin inhibitors, bacitracin, puromycin, bestatin, and bile salts such as NaCC and are frequently used with absorption enhancers for improvement in rectal absorption. 

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