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Identification of a highly reactive sulphydryl group in human placental glutathione transferase by a site‐directed fluorescent reagent
Author(s) -
Lo Bello Mario,
Petruzzelli Raffaele,
De Stefano Ester,
Tenedini Cristiana,
Barra Donatella,
Federici Giorgio
Publication year - 1990
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(90)81421-j
Subject(s) - maleimide , chemistry , trypsin , thiol , glutathione , cysteine , enzyme , fluorescence , peptide , reagent , biochemistry , glutathione s transferase , affinity label , organic chemistry , physics , quantum mechanics
A fluorescent maleimide derivative, N ‐(4‐anilino‐1‐naphthyl) maleimide (ANM), a specific probe for thiol groups, reacted with human placental glutathione transferase (GST, EC 2.5.1.18), causing a complete inactivation of the enzyme in a few minutes. The modified enzyme was denatured, alkylated and digested with (L‐1‐tosylamide‐2‐phenylethyl chloromethyl ketone)‐trypsin. The tryptic digest was analysed by HPLC and a fluorescent peptide was obtained. The sequence of this peptide allowed us, by a comparison with a well known primary structure, to assign the position 47 to the most reactive cysteine of GST enzyme.