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Detection of trypsin- and chymotrypsin-like proteases using p-nitroanilide substrates after sodium dodecyl sulphate polyacrylamide gel electrophoresis.
J Chromatogr. 1989 May 26; 470(2):401-6.JC

Abstract

Specific chromogenic p-nitroanilide substrates have proved useful for localizing proteolytic enzymes, such as trypsin, chymotrypsin and elastase after separation by agarose gel electrophoresis and when immobilized on nitrocellulose. This procedure was further developed for use with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). After SDS-PAGE, proteins were transferred electrophoretically to a nitrocellulose membrane. The membrane was incubated for 10-60 min with Bz-Ile-Glu-Gly-Arg-p-nitroanilide as a substrate for detection of trypsin-like proteases and with MeO-Suc-Arg-Pro-Tyr-p-nitroanilide for detection of chymotrypsin. The yellow p-nitroanilide released at the site of proteolytic activity was converted into a visible and stable red azo dye. By this method was identified and determined the molecular weight of a trypsin-like protease that occurs at high concentrations in mucinous ovarian tumour cyst fluid together with its specific inhibitor peptide, tumour-associated trypsin inhibitor (TATI). The method was also used to visualize trypsin and chymotrypsin in human pancreatic juice. Using the trypsin substrate, three proteolytic bands, corresponding to Mr of 22,000, 24,000 and 26,000 daltons, were visualized in pancreatic juice, while the proteolytic zones in cyst fluid had Mr of 25,000 and 28,000 daltons. With the chymotrypsin substrate, a band of 29,000 daltons was visualized in pancreatic juice, whereas no activity was detected in cyst fluid. By incubation of the blotted cyst fluid proteins with 125I-labelled TATI, a pattern of bands at 25,000 and 28,000 daltons was detected identical to that obtained with the chromogenic substrate.

Authors+Show Affiliations

Koivunen E
Department I of Obstetrics and Gynecology, Helsinki University Central Hospital, Finland.

MeSH

AnilidesAnimalsBlotting, WesternCattleChymotrypsinElectrophoresis, Polyacrylamide GelHumansPancreatic JuiceProtease InhibitorsSubstrate SpecificityTrypsinTrypsin Inhibitor, Kazal Pancreatic

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

2768384

Citation

Koivunen, E. "Detection of Trypsin- and Chymotrypsin-like Proteases Using P-nitroanilide Substrates After Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis." Journal of Chromatography, vol. 470, no. 2, 1989, pp. 401-6.
Koivunen E. Detection of trypsin- and chymotrypsin-like proteases using p-nitroanilide substrates after sodium dodecyl sulphate polyacrylamide gel electrophoresis. J Chromatogr. 1989;470(2):401-6.
Koivunen, E. (1989). Detection of trypsin- and chymotrypsin-like proteases using p-nitroanilide substrates after sodium dodecyl sulphate polyacrylamide gel electrophoresis. Journal of Chromatography, 470(2), 401-6.
Koivunen E. Detection of Trypsin- and Chymotrypsin-like Proteases Using P-nitroanilide Substrates After Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis. J Chromatogr. 1989 May 26;470(2):401-6. PubMed PMID: 2768384.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Detection of trypsin- and chymotrypsin-like proteases using p-nitroanilide substrates after sodium dodecyl sulphate polyacrylamide gel electrophoresis. A1 - Koivunen,E, PY - 1989/5/26/pubmed PY - 1989/5/26/medline PY - 1989/5/26/entrez SP - 401 EP - 6 JF - Journal of chromatography JO - J Chromatogr VL - 470 IS - 2 N2 - Specific chromogenic p-nitroanilide substrates have proved useful for localizing proteolytic enzymes, such as trypsin, chymotrypsin and elastase after separation by agarose gel electrophoresis and when immobilized on nitrocellulose. This procedure was further developed for use with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). After SDS-PAGE, proteins were transferred electrophoretically to a nitrocellulose membrane. The membrane was incubated for 10-60 min with Bz-Ile-Glu-Gly-Arg-p-nitroanilide as a substrate for detection of trypsin-like proteases and with MeO-Suc-Arg-Pro-Tyr-p-nitroanilide for detection of chymotrypsin. The yellow p-nitroanilide released at the site of proteolytic activity was converted into a visible and stable red azo dye. By this method was identified and determined the molecular weight of a trypsin-like protease that occurs at high concentrations in mucinous ovarian tumour cyst fluid together with its specific inhibitor peptide, tumour-associated trypsin inhibitor (TATI). The method was also used to visualize trypsin and chymotrypsin in human pancreatic juice. Using the trypsin substrate, three proteolytic bands, corresponding to Mr of 22,000, 24,000 and 26,000 daltons, were visualized in pancreatic juice, while the proteolytic zones in cyst fluid had Mr of 25,000 and 28,000 daltons. With the chymotrypsin substrate, a band of 29,000 daltons was visualized in pancreatic juice, whereas no activity was detected in cyst fluid. By incubation of the blotted cyst fluid proteins with 125I-labelled TATI, a pattern of bands at 25,000 and 28,000 daltons was detected identical to that obtained with the chromogenic substrate. UR - https://www.unboundmedicine.com/medline/citation/2768384/Detection_of_trypsin__and_chymotrypsin_like_proteases_using_p_nitroanilide_substrates_after_sodium_dodecyl_sulphate_polyacrylamide_gel_electrophoresis_ DB - PRIME DP - Unbound Medicine ER -