Proteolytic enzymes assay

Proteolytic Enzymes, Assay of (Davis and Smith) Purification of Biologically Active Compounds by Affinity Chromatography, The Purines, New Methods for Purfication and Separation of 2 215... 

Proteolytic Enzyme Assays Using Conjugated Polyelectrolytes... 

Fig. 2.3. (a) Fluorescence spectroscopic changes observed in a proteolytic enzyme assay using PPE-SO3 (3) and K-pNA. Initial addition of KpNA quenches fluorescence, and then addition of peptidase gives rise to fluorescence recovery. Solid line Initial fluorescence, [PPE-SO3] = 1.0 pM, phosphate buffer solution, pH 7.1 Dotted line fluorescence after addition of 167 nM K-pNA. Fluorescence intensity as a function of time (5-200 min) after addition of porcine intestinal peptidase (3.3 pg mL ). (b) Mechanism of the turn-on CPE-based sensor... 

Proteolytic enzymes in the respiratory mucosa play important role(s) in the regulation of lung inflammation and remodelling [123, 124], Pulmonary proteolytic enzymes, however, also comprise one of the barriers which pulmonary-administered protein/peptide drugs have to overcome in order to achieve adequate bioavailability [125]. Intriguingly, the pulmonary enzymatic barrier is an aspect that has been little investigated and is poorly understood. Inconsistencies in the data available to date are most likely a result of the use of different techniques (e.g., PCR, immunotechniques and enzyme activity assays), different species and different cell (pheno)types, for example primary cells vs. cell lines. 

The hbraries of enzyme substrates were obtained by spht-pool synthesis to yield one-bead-one-compound hbraries. The substrate assay was performed with a range of proteolytic enzymes such as subtilisin Carlsberg [26], cruzipain [27], protein disulfide isomerase [28-29], matrix metalloprotease MM P-9 [30], papain [31],... 

Reyes (1971) described a very sensitive assay for measuring the milkclotting activity of low concentrations of residual proteolytic enzymes in curd and whey. The substrate, buffered at pH 5.7, consisted of 1 g MSNF dissolved in a mixture of 70 ml 6.6 x 10 2 M cacodylic acid, 30 ml 6.6 x 10 2 M triethanolamine, and 1 ml 3 M CaCl2. 

Lawrence and Sanderson proposed another micro-method for measuring chymosin and other proteolytic enzymes. Measurement of concentration was based on the rate of radial diffusion of the enzyme through a thin layer of caseinate-agar gel. The limit of diffusion was marked by a zone of precipitated casein (Emstrom and Wong 1974). Holmes et al (1977) developed a microdiffusion assay for residual proteolytic enzymes in curd and whey that is more sensitive than the method of Lawrence and Sanderson or the clotting-time assay of Reyes (1971). 

As an example, this protocol describes how to perform the reaction using a casein substrate and a commercial proteolytic enzyme cocktail. The activity of the commercial four-enzyme cocktail is 35 U/ml, but should be tested prior to the experiment in order to determine the appropriate amount of enzyme needed for the reaction. The proteolytic activity assay (Garcia-Carreno et al., 1994) is described below. 

Knight, C.G. 1995. Fluori metric assays of proteolytic enzymes. Methods Enzymol. 248 18-34. 

Sarath, G., Zeece, M.G., and Penheiter, A.R. 2001. Protease assay methods. In Proteolytic Enzymes A Practical Approach (R. Benyon and J.S. Bond, eds.) pp. 45-76. Oxford University Press, Oxford. 

CG Knight. Fluorometric assays of proteolytic enzymes. Meth Enzymol 248 18-35, 1995. 

Ichishima, E. (1970). Purification and mode of assay for acid proteinase of Aspergillus saitoi. In Perlmann, G. E., and Lorand, L. (Eds.), Methods in Enzymology Proteolytic Enzymes. Vol. XIX, (pp.397-406). New York Academic Press. 

Grant, S.K., Sklar, J.G., and Cummings, R.T. 2002. Development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates. J. Biomol. Screen. 7, 531-540. 

Lipase (Microbial) Activity for Medium- and Long-Chain Fatty Acids, (S3)105 Lysozyme Activity, (S3)106 Maltogenic Amylase Activity, 804 Milk-Clotting Activity, 805 Pancreatin Activity, 805 Pepsin Activity, 807 Phospholipase A2 Activity, 808 Phytase Activity, 808 Plant Proteolytic Activity, 810 Proteolytic Activity, Bacterial (PC), 811 Proteolytic Activity, Fungal (HUT), 812 Proteolytic Activity, Fungal (SAP), 813 Pullulanase Activity, 814 Trypsin Activity, 814 Enzyme Assays, 786 Enzyme-Hydrolyzed (Source) Protein,... 

Papain is a proteolytic enzyme from plants. In the HPLC assay developed to measure its activity, the water-soluble N-benzyl-L-arginine ethyl ester (B AEE) is used as the substrate. 

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