Amino Acid Sequence Of Trypsin Research Articles

Trypsin from the Pyloric Ceca of Pectoral Rattail (Coryphaenoides pectoralis): Purification and Characterization

Trypsin from the pyloric ceca of pectoral rattail (Coryphaenoides pectoralis) was purified and characterized. Purification was carried out by ammonium sulfate precipitation, followed by column chromatographies on Sephacryl S-200, DEAE-cellulose and Sephadex G-50. The enzyme was purified 89-fold with a yield of 2.2%. Purified trypsin had an apparent molecular weight of 24 kDa when analyzed using SDS-PAGE and size exclusion chromatography. Optimal profiles of pH and temperature of the enzyme were 8.5 and 45 degrees C, respectively, using N(alpha)-p-tosyl-l-arginine methyl ester hydrochloride as a substrate. It was stable in a wide pH range of 6-11 but unstable at a temperature greater than 40 degrees C. Trypsin was stabilized by calcium ion. The activity of purified trypsin was effectively inhibited by soybean trypsin inhibitor and TLCK and was partially inhibited by EDTA. Activity continuously decreased with increasing NaCl concentration (0-30%). The kinetic trypsin constants K(m) and K(cat) were 0.15 mM and 210 s(-1), respectively, while the catalytic efficiency (K(cat)/K(m)) was 1400 s(-1) mM(-1). The N-terminal amino acid sequence of trypsin was determined to be 12 residues (IVGGYECQEHSQ), and the sequence showed high homology to other fish trypsins.

Purification and characterisation of trypsins from the pyloric caeca of mandarin fish (Siniperca chuatsi)

Two trypsins of anionic form (trypsin A) and cationic form (trypsin B) from the pyloric caeca of mandarin fish (Siniperca chuatsi) were highly purified by a series of chromatographies, including DEAE-Sephacel, Sephacryl S-200 HR, Q-Sepharose or SP-Sepharose. Purified trypsins revealed a single band on native-PAGE. The molecular weights of trypsin A and B were 21kDa and 21.5kDa, respectively, as estimated by SDS–PAGE, both under reducing and non-reducing conditions. Zymography analysis showed that both trypsins were active in degrading casein. Trypsin A and B exhibited maximal activity at 35°C and 40°C, respectively, and shared the same optimal pH of 8.5, using Boc-Phe-Ser-Arg-MCA as substrate. The two trypsins were stable up to 45°C and in the pH range from 4.5 to 11.0. Trypsin inhibitors are effective on these two enzymes and their susceptibilities were similar. Both trypsins were activated by metal ions such as Ca2+ and Mg2+ and inactivated by Fe2+, Zn2+, Mn2+, Cu2+, Al3+, Ba2+ and Co2+ to different degrees. Apparent Km values of trypsin A and B were 2.18μM and 1.88μM, and Kcat values were 81.6S−1 and 111.3S−1 for Boc-Phe-Ser-Arg-MCA, respectively. Immunoblotting analysis using anti-common carp trypsin A positively cross-reacted with the two enzymes, suggesting their similarity. The N-terminal amino acid sequence of trypsin B was determined as IVGGYECEAH, which is highly homologous with trypsins from other species of fish.

Trypsin from the pyloric caeca of bluefish ( Pomatomus saltatrix)

Trypsin was purified from the pyloric caeca of bluefish ( Pomatomus saltatrix) by ammonium sulfate precipitation, acetone precipitation and soybean trypsin inhibitor-Sepharose 4B affinity chromatography. Bluefish trypsin migrated as a single band using both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE and had a molecular mass of 28 kDa. The optima pH and temperature for the hydrolysis of benzoyl- dl-arginine- p-nitroanilide (BAPNA) were 9.5 and 55 °C, respectively. The enzyme was stable over a broad pH range (7 to 12), but was unstable at acidic pH, and at temperatures greater than 40 °C. The enzyme was inhibited by specific trypsin inhibitors: soybean trypsin inhibitor (SBTI), N- p-tosyl- l-lysine chloromethyl ketone (TLCK) and the serine protease inhibitor phenylmethyl sulfonylfluoride (PMSF). CaCl 2 partially protected trypsin against activity loss at 40 °C, but NaCl (0 to 30%) decreased the activity in a concentration dependent manner. The N-terminal amino acid sequence of trypsin was determined as IVGGYECKPKSAPVQVSLNL and was highly homologous to other known vertebrate trypsins.

Purification and Characterization of Trypsin from the Spleen of Tongol Tuna (Thunnus tonggol)

Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin appearing as a single band on native PAGE showed the maximal activity at pH 8.5 and 65 degrees C. It was stable in a wide pH range of 6-11 but unstable at the temperatures greater than 50 degrees C. The enzyme required calcium ion for thermal stability. The activity was strongly inhibited by 1.0 g/L soybean trypsin inhibitor and 5 mM TLCK and partially inhibited by 2 mM ethylenediaminetetraacetic acid. Activity was lowered with an increasing NaCl concentration (0-30%). The enzyme had a Km for Nalpha-p-tosyl-L-arginine methyl ester hydrochloride of 0.25 mM and a Kcat of 200 s-1. The N-terminal amino acid sequence of trypsin was determined as IVGGYECQAHSQPHQVSLNA and was very homologous to other trypsins.

N-terminal amino acid sequence of trypsin from the pyloric ceca of starfish Asterias amurensis

N-terminal amino acid sequence of trypsin from the pyloric ceca of starfish Asterias amurensis

Purification and partial characterization of the pancreatic proteolytic enzymes trypsin, chymotrypsin and elastase from the chicken

The purpose of this work was to isolate, purify and partially sequence trypsin, chymotrypsin and elastase from the chicken pancreas. The extraction of the pancreatic zymogens with 0.5 M CaCl2 at pH 7.5 for 9 h appeared to be most effective in obtaining maximum recovery of the three enzymes. The sequential Cucurbita maxima trypsin inhibitor I/bovine pancreas trypsin inhibitor/soybean trypsin inhibitor affinity chromatography gave the best result for the isolation of trypsin, chymotrypsin and elastase, respectively, from the same extract. For each proteinase, multiple form of enzymatic activity could be observed after gel electrophoresis and each form was further purified on an ion-exchange column. The N-terminal amino acid sequence of trypsin and chymotrypsin showed homologies with the bovine enzymes whereas elastase showed homologies with the porcine enzyme. The molecular mass of trypsin, chymotrypsin and elastase were estimated to be 23 500, 25 700 and 25 000, respectively, which are values close to those in mammalian species. Although some kinetic constants (Km and kcat/Km) appeared different from those observed in other species, the pH dependent enzymatic activities were similar to those reported in other animal species.

Amino acid sequence of crayfish (Astacus fluviatilis) trypsin If.

The complete amino acid sequence of trypsin from the crayfish Astacus fluviatilis has been determined. The protein was fragmented with cyanogen bromide after S-carboxymethylation of the reduced disulfide bonds and by trypsin after S-carboxymethylation as well as after succinylation of lysine residues and aminoethylation of the reduced disulfide bonds. Peptides were purified by gel filtration and by reversed-phase high-performance liquid chromatography. Stepwise degradation was performed in a spinning cup sequencer. The enzyme contains 237 amino acid residues and has a molecular weight of 25 030. In contrast to bovine trypsin, it contains three rather than six disulfide bonds which are paired in the same fashion as those in trypsin from Streptomyces griseus. The constituents of the active site of bovine trypsin are present in corresponding positions in the crayfish enzyme. Crayfish trypsin shows 43.6% sequence identity with the bovine enzyme as compared to 40.0% identity with the S. griseus enzyme. The present analysis affords the first detailed view into the evolution of trypsins at the invertebrate level.