Trypsin, 3X, Bovine Pancreas, TPCK Treated (EC 3.4.21.4)

Trypsin is a pancreatic serine protease with substrate specificity based upon positively charged lysine and arginine side chains (Brown and Wold, 1973a). It has been isolated from a number of sources including dogfish (Titani, et al., 1975), moose (Stevenson and Voordouw, 1975), whale (Bricteux-Gregoire, et al., 1975), elephant seal (Bricteux-Gegoire, et al., 1974), Streptomyces griseus (Jurasek and Smillie, 1974, Olafson and Smillie, 1975), canine (Ohlsson and Tegner, 1973), African lungfish (Reeck and Neurath, 1972), swine (Hermodson, et al., 1973, Walker and Keil, 1973), rat (Vandermeers, et al., 1973), human ( Mallory and Travis, 1973, Figarell, et al., 1975). Trypsinogen and trypsin have been reviewed by Walsh (1970). The enzyme exhibits esterase and amidase activities. Activity (Dry Weight): >180 units per mg Dry Weight, Activity (Protein): >180 units per mg protein, Unit Definition: 1 unit hydrolyzes 1 umole of p-toluene-sulfonyl-L-arginine methyl ester (TAME) per minute at 25ºC, pH 8.2, in the presence of 0.01M calcium ion. (1 TAME unit = 19.2 USP/NF units = 57.5 BAEE units). Chymotrypsin: <0.02%, Protein: >80%, Quality Control: SDS-PAGE, Characteristics of Trypsin from Bovine Pancreas: MW:, Trypsinogen: 24,000D (Walsh and Neurath, 1964)., Trypsin: 23,800D (Cunningham, 1954). Optimum pH: ~8.0. , Extinction coefficient: = 14.3. Isoelectric point: Trypsinogen: pH 9.3 (Walsh and Neurath, 1964)., Trypsin: pH 10.5 (Cunningham, 1954). Composition: Trypsinogen has been shown to have certain intrinsic activity. See Knights and Light (1974) for further references. It is usually considered as the inactive precursor of trypsin which may be activated by removal of a terminal hexapeptide to yield single-chain [[beta]]-trypsin. Subsequent limited autolysis produces other active forms having two or more peptide chains bound by disulfide bonds. The predominant forms are [[alpha]]-trypsin, having two peptide chains and [[beta]]-, a single chain. Different activity and thermal stability are shown by [[alpha]]-and [[beta]]-trypsin. , Benzamidine prevents conversion of [[beta]] to [[alpha]] (Beardslee and Zahnley, 1973, Schroeder and Shaw, 1968). Trypsin can, by chromatography, be separated into the [[alpha]] and [[beta]] forms. (Jameson and Elmore, 1974). See also Keil-Dlouhá, et al. (1971a and b) and Smith and Shaw (1969). It has been pointed out by Schroeder (1972) that the several active species of trypsin have different catalytic constants and molecular properties and are present in all regularly purified preparations. The structure of trypsin and a molecular model have been reported by Stroud, Kay and Dickerson (1971). Active site studies have been reported by Berliner and Wong (1974 and 1973), Wong and Shaw (1974), Liem and Scheraga (1974), Nishino, et al. (1974), Chambers, et al. (1974), Royer and Uy (1973), Ghiron, et al. (1973), Robinson, et al. (1973a and b), Kasai and Ishii (1973), Brown and Wold (1973a and b), and Keil-Dlouhá (1972). , Inhibitors: Trypsin is inhibited by organophosphorus compounds such as diisopropyl fluorophosphate and natural "trypsin inhibitor" from pancreas. Soybean, lima bean, and egg white are sources of inhibitors. Chambers, et al. (1974) found silver ion to be a potent inhibitor. Inhibition by benzamidine is reversible (Mares-Guia and Shaw, 1965). Other reports on inhibitors include Gaudin and Stevens (1974), Krebs and Jacquot-Armand (1973), Moroi and Yamasaki (1974), Morgan, et al. (1974) and Geratz, et al. (1975). Activators: The rate of trypsinogen conversion is enhanced by using lanthanide in place of calcium ions (Gomez, et al. 1974). , Specificity: The protease activity of trypsin is highly specific toward positively charged side chains with lysine and arginine. See also Keil-Dlouhá, et al. (1971a). Stabilizers: Calcium ion retards trypsin autolysis and promotes activation of trypsinogen. Sipos and Merkel (1970) have reported on the calcium-trypsin complex. See also Griffiths and Brechner (1973). , Stability: Trypsin is stable as a dry powder at 4°C for years. Storage and Stability: Lyophilized powder may be stored at -20°C. Stable for 6 months after receipt at -20°C. Reconstitute with 0.001N HCl. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Reconstituted product is stable for 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.