Abstract
Papain was labelled by attachment of the fluorescent groups 2-(4'-acetamidoanilino)naphthalene-6-sulphonic acid (AANS) or N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid (AEDANS) to the active-site cysteine residue, with the aim of using the labelled papains as probes in competitive titrations of unlabelled cysteine proteinases with their inhibitors. The interaction between the labelled papains and cystatins was accompanied by an increase in fluorescence emission of up to 38-fold for AANS-papain and approximately 3.5-fold for AEDANS-papain. Fluorescence titrations gave dissociation equilibrium constants of 3.1 and 0.6 microM for the binding of chicken cystatin and recombinant human cystatin C respectively to AANS-papain and of 11.9 microM for the binding of chicken cystatin to AEDANS-papain. The kinetics of interaction of chicken cystatin with AANS-papain showed an unusual biphasic dependence of the observed pseudo-first-order rate constant on inhibitor concentration, consistent with the reaction occurring via both pathways of a general two-step binding mechanism. AANS-papain was selected as the most suitable probe for competitive titrations of unlabelled active or inactivated cysteine proteinases with inhibitors. This technique, which provides stoichiometries and dissociation constants for the interaction between unlabelled enzyme and inhibitor, allows monitoring of the interactions by a large fluorescent signal in a wavelength region where the interacting proteins do not contribute to the observed fluorescence. Such competitive titrations of active papain or actinidin with chicken cystatin or recombinant human cystatin C all gave inhibitor/enzyme stoichiometries of close to 1.0. A dissociation constant of 1.8 microM for the reaction of chicken cystatin with a papain derivative, S-[N-(3-carboxypropyl)succinimidyl]-papain, was also determined by the same technique. These results show the usefulness of the fluorescent papains for the characterization of interactions between cysteine-proteinase inhibitors and their target enzymes.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.