Characterization of specificity of subtilisin Carlsberg towards peptide T by high-performance liquid chromatography and electrospray mass spectrometry

Peptide T has a sequence (Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr) belonging to HIV envelope that is involved in the interaction with CD4 receptor of T lympho cytes. Research of protease activities towards this peptide is very relevan t for AIDS therapy. Characterization of specificity of subtilisin Carlsberg towards this very hydrophilic peptide is proposed by using high-performanc e liquid chromatography and mass spectrometry. Peptide T was totally hydrol ysed by the protease after 24 h. Separation of hydrophilic fragments was pe rfected with an hydrophilic stationary phase and a reversed acetonitrile gr adient. Peptide masses were determined by ion spray mass spectrometry. Four primary and one secondary hydrolysis products were found, corresponding to cleavage at the carboxylic side of threonine. Specifities of subtilisin Ca rlsberg towards the Segments 19 to 26 of bovine pancreatic ribonuclease A, an homologous fragment of peptide T, and peptide T were compared. (C) 2000 Elsevier Science Inc. All rights reserved.