title: Purification, characterization and immobilization of proteinase inhibitors from Stichodactyla helianthus.
abstract: Isolation of proteinase inhibitors from the sea anemone Stichodactyla helianthus was achieved by trichloroacetic acid treatment of the aqueous extract followed by affinity chromatography on trypsin-Sepharose and ion-exchange chromatography on CM-cellulose. The average molecular mass of the major inhibitor (ShPI-I) obtained by fast atom bombardment mass spectrometry (FAB-MS) was 6110.6 Da. The amino acid sequence was determined by FAB-MS combined with manual Edman degradation, digestions with endopeptidases and exopeptidases and automatic sequencing. The sequence of ShPI-I (55 amino acids) was compared with those reported in the SwissProt database for several proteinase inhibitors and significant similarity to inhibitors belonging to the Kunitz family was observed. ShPI-I exhibits a broad specificity for serine, cysteine and aspartic proteinases. The dissociation constants of the complexes formed with different enzymes were determined. The affinity-purified fraction (PI) was immobilized on Sepharose and used in the purification of different classes of proteinases.
authors.affiliation: Facultad de Biologia, Universidad de la Habana, Cuba.
authors.forename: J
authors.initials: J
authors.lastname: Delfín
authors.forename: I
authors.initials: I
authors.lastname: Martínez
authors.forename: W
authors.initials: W
authors.lastname: Antuch
authors.forename: V
authors.initials: V
authors.lastname: Morera
authors.forename: Y
authors.initials: Y
authors.lastname: González
authors.forename: R
authors.initials: R
authors.lastname: Rodríguez
authors.forename: M
authors.initials: M
authors.lastname: Márquez
authors.forename: A
authors.initials: A
authors.lastname: Saroyán
authors.forename: N
authors.initials: N
authors.lastname: Larionova
authors.forename: J
authors.initials: J
authors.lastname: Díaz
authors.forename: G
authors.initials: G
authors.lastname: Padrón
authors.forename: M
authors.initials: M
authors.lastname: Chávez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002847:Chromatography, Agarose; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D012615:Sea Anemones / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D011480:Protease Inhibitors; C080476:ShPI proteinase inhibitor; D014360:Trypsin Inhibitor, Kunitz Soybean