Chymotrypsin materials

Markowitz et al. developed a different approach, again in an attempt to overcome some of the inherent difficulties that arise when imprinted bulk materials are used as catalysts [82], Here, the authors used a template-directed method to imprint an a-chymotrypsin TSA at the surface of silica nanoparticles, prepared with a number of organically modified silanes as functional monomers. Silica particle formation was performed in a microemulsion, where a mixture of a non-ionic surfactant and... 

Further study of this third material by Holman and Cook (10) showed that its biological activity was destroyed by pronase, but not by chymotrypsin. Moreover, this peptide was found in extracts of hindguts, terminal ganglia, proctodeal nerves, and heads of L. maderae. 

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 - ). 

Materials. Trypsin, Lys C, chymotrypsin and adrenocorticotropic hormone fragment (18-39) were purchased from Sigma (St. Louis, Mo). Tris(hydroxymethyl)aminomethane (Tris), sucrose, acetonitrile, HPLC grade water and acetic acid were purchased from Fisher Scientific (Pittsburgh, PA). Matrix (a-cyano 4-hydroxy-cinnamic acid) was purchased from Hewlett Packard (Palo Alto, CA). The low-molecular weight calibration standard was purchased from Bio-Rad (Richmond, CA). 

Numerous studies done on conjugated proteins were oriented toward the differentiation of the material without performing special separations. For example, direct pyrolysis of several enzymes showed a significant difference in the chromatographic profile of the pyrolysate. The enzymes analyzed by this procedure included a-chymotrypsin, creatine kinase, lactate dehydrogenase, catalase, acetylcholinesterase, and urease [19,20]. 

The optimum pH for an enzymatic reaction may shift by as much as 3 pH units upon immobilization.24 This shift is a result of both the charge of the support material and the chemical modification of the enzyme. Figure 4.4 illustrates the dramatic shifts that occur in the pH optimum of chymotrypsin, a proteolytic enzyme, following covalent immobilizations onto a polyornithine (positive) carrier and an ethylene-maleic anhydride copolymer (negative). 

Because the local pH at the surface of the support is lower than the bulk, or measured pH, the apparent pH optimum shifts to higher pH values with this and other negatively charged support materials. Figure 4.4 shows that chymotrypsin immobilized onto an ethylene-maleic anhydride support exhibits a pH optimum of 9.4, almost one full pH unit higher than that observed for the native enzyme. 

Potent natriuretic activity was evident in a simple phosphate buffered saline extract of whole atria. 4 Subsequently, the material was found to be acid stable.76 7,88,100,101 while the extract is both acid and heat76.101 stable, trypsin,75,89,93 as well as other proteolytic enzymes76 such as chymotrypsin, ami nopeptidase A and carboxypeptidases B and C destroy natriuretic activity. However, treatment with carboxy-peptidase A only blunted activity,76 and concanavalin A had no effect.75 These observations are consistent with ANF being a small peptide. 

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