Trypsin and

To the remainder of the gelatin solution, add 0 5 to o 8 g. of finely powdered commercial trypsin and incubate at 40 . Carry out the formaldehyde titration on 25 ml. samples at intervals as above. 

Protein inhibitors are often active against a variety of en2ymes, although each molecule may possess a separate and very distinct binding site for each en2yme. For example, many trypsin and chymotrypsin inhibitors are identical compounds (12). 

Lupine seed, though used primarily in animal feeds (see Feeds AND FEED ADDITIVES), does have potential for use in human appHcations as a replacement for soy flour, and is reported to contain both trypsin inhibitors and hemagglutenins (17). The former are heat labile at 90°C for 8 minutes the latter seem much more stable to normal cooking temperatures. Various tropical root crops, including yam, cassava, and taro, are also known to contain both trypsin and chymotrypsin inhibitors, and certain varieties of sweet potatoes may also be impHcated (18). 

Many enzymes have been the subject of protein engineering studies, including several that are important in medicine and industry, eg, lysozyme, trypsin, and cytochrome P450. SubtiHsin, a bacterial serine protease used in detergents, foods, and the manufacture of leather goods, has been particularly well studied (68). This emphasis is in part owing to the wealth of stmctural and mechanistic information that is available for this enzyme. 

Mucolytics reduce the viscosity of tenacious and purulent mucus, thus faciUtating removal. The distinction between mucolytics and other classes of expectorants is frequently blurred. Steam, sometimes in conjunction with surfactants or volatile oils, has long been used to decrease viscosity by physical hydration. However, agents that chemically depolymerize certain components of mucus are available. Trypsin and other proteolytic enzymes have shown good clinical activity because of their abiUty to cleave glycoproteins. Pancreatic domase, which depolymerizes DNA found in purulent mucus, also has shown clinical utihty. 

Phenylmethanesulfonyl fluoride (PMSF) [329-98-6] M 174.2, m 90-91 , 92-93 . Purified by recrystn from ""CgHe, pet ether or CHCl3-pet ether. [Davies and Dick J Chem Soc 483 1932 cf Tullock and Coffman J Org Chem 23 2016 I 960.] It is a general protease inhibitor (specific for trypsin and chymotrypsin) and is a good substitute for diisopropylphosphoro floridate [Fahrney and Gould 7 Am Chem Soc 85 997 1963]. 

This is nicely illustrated by members of the chymotrypsin superfamily the enzymes chymotrypsin, trypsin, and elastase have very similar three-dimensional structures but different specificity. They preferentially cleave adjacent to bulky aromatic side chains, positively charged side chains, and small uncharged side chains, respectively. Three residues, numbers 189, 216, and 226, are responsible for these preferences (Figure 11.11). Residues 216... 

Figure 11.11 Schematic diagrams of the specificity pockets of chymotrypsin, trypsin and elastase, illustrating the preference for a side chain adjacent to the scisslle bond In polypeptide substrates. Chymotrypsin prefers aromatic side chains and trypsin prefers positively charged side chains that can interact with Asp 189 at the bottom of the specificity pocket. The pocket is blocked in elastase, which therefore prefers small uncharged side chains.

Ruhlman, A., et al. Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. /. Mol. Biol. 77 417-436, 1973. 

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

Look up the structure of human insulin (Section 26.7), and indicate w here in each chain the molecule is cleaved by trypsin and chymotrypsin. 

Figure 49-4. Diagram of a myosin moiecuie showing the two intertwined a-heiices (fibrous portion), the giobuiar region or head (G),the iight chains (L), and the effects of proteoiytic cieavage by trypsin and papain. The giobuiar region (myosin head) contains an actin-binding site and an L chain-binding site and aiso attaches to the remainder of the myosin moiecuie.

Moreover, other proteolytic products from a chemokine have been proposed to modulate HIV infection. Both CCL14 processing by trypsin and CCL5 processing... 

Secondary cell cultures, which can be prepared by taking cells from some types of primary culture, usually those derived from embryonic tissue, dispersing them by treatment with trypsin and inoculating some into a fresh batch of medium. A limited number of subcultures can be performed with these sorts of cells, up to a maximum of about 50 before the cells degenerate. 

Some of the best investigated anti-nutrients are the enzyme inhibitors present in legumes and other plants. The Bowman-Birk and the Kunitz inhibitors of trypsin and other proteases are among the best characterized. In contrast to the non-specific and widespread influences of tannins and lectins (Carmona, 1996), the Bowman-Birk, Kunitz and other such inhibitors target specific enzymes. Corresponding with this, proteases and other digestive enzymes vary in sensitivity to the different inhibitors. 

Figure 6 High-speed tryptic fingerprint. Horse cytochrome c was digested with trypsin and the peptide chromatographed in acetonitrile water 0.1% TFA at various temperatures and flow rates on a 15 x 0.2-cm PS-DVB column packed with 3-p, 300-A particles, (a) 26°C and 0.5 ml/min. (b) 42°C and 0.7 ml/min. (c) 70°C and 1.1 ml/min. Detection at 220 nm. Note that the resolution rises with the speed of separation.89 (From Swadesh, ]., BioTechniques, 9, 626, 1990. With permission.)...

Titani, K., Sasagawa, T., Resing, K., and Walsh, K. A., A simple and rapid purification of commercial trypsin and chymostrypsin by reverse-phase high-performance liquid chromatography, Anal. Biochem., 123, 408, 1982. 

Figure 2.4. Peptide fingerprinting by MALDI-TOF mass Spectrometry. Proteins are extracted and separated on by 2D gel electrophoresis. A spot of interest is excised from the gel, digested with trypsin, and ionized by MALDI. The precise mass of proteolytic fragments is determined by time-of- flight mass spectrometry. The identity of the protein is determined by comparing the peptide masses with a list of peptide masses generated by a simulated digestion of all of the open reading frames of the organism.

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