Interaction of native and modified Naja melanoleuca phospholipases A2 with the fluorescent probe 8-anilinonaphtalene-1-sulfonate

Abstract

The fluorescent probe 8-anilinonaphtalene-1 -sulfonate (ANS) binds at the active site of the Nuja rnelanoleuca snake venom phospholipase A,, thus protecting the enzyme against active-site-directed chemical modification. Both hydrophobic and electrostatic interactions are involved in the binding. At pH 7.5, a binding constant of 100pM was determined, which improved twofold upon addition of the enzymatic cofactor Ca2 +. The pH dependence of the ANS binding in the absence and presence of Ca2+ ions showed a perturbation of a group with a pK, value of 5.2, which could be assigned to the carboxylate group of the Ca2+-binding ligand Asp49 at the active site of the protein.Monomeric concentrations of the substrate analog n-decylphosphocholine displace ANS from the protein, indicating again that both ligands bind at the active site. Binding studies with several modified N . rnelanoleuca enzymes showed that a loss of enzymatic activity on aggregated substrates was correlated with a loss of affinity for the active site bound ANS molecule. It is suggested therefore, that the fluorescent ANS probe can detect structural rearrangements at the active site, which are important for enzymatic activity.8-Anilinonaphtalene-1-sulfonate (ANS) has been used extensively as a fluorescent probe to monitor conformational changes in proteins. Whereas in aqueous solution the probe shows only a weak green fluorescence, an intense blue-shifted fluorescence is observed upon interaction with several biological macromolecules or membranes. A decreased polarity of the probe's environment is thought to be the main factor contributing to the observed fluorescence enhancement upon binding of ANS to a protein [l].Phospholipase A, catalyzes the hydrolysis of the 2-acyl ester bonds in diacylphosphoglycerides. The enzyme needs Ca2+ as an absolute cofactor, which is bound at the active site of the protein. Pieterson et al. [2] showed that the fluorescence of ANS is increased and blue-shifted upon addition of the pig pancreatic phospholipase A,, indicating that this enzyme binds one or more ANS molecules. Since the addition of the cofactor Ca2+ to the ANS-protein complex results in a further increase of the ANS fluorescence, the probe could be used to determine a Ca2+-binding constant for this enzyme. Enzyme. Phospholipase A, (EC 3.1.1.4).Recently [6, 71, we have reported that certain chemical modifications in the N-terminal region of the polypeptide chain inactivate the Naja rnelanoleuca phospholipase A,. As the affinity for lipid-water interfaces of the modified proteins is hardly affected, the loss of enzymatic activity might be attributed to changes in the active site of the protein. It will be demonstrated in the present study that ANS binds at the active site, where the cofactor Ca2 + and monomeric substrates are bound. Moreover, it will be shown that ANS can be used as a fluorescent probe to detect conformational changes at the active site of the modified proteins. MATERIALS AND METHODS EnzymesPhospholipase A, was purified from Naja rnelunoleuca sna...

Effects of salts on the interaction of 8-anilinonaphthalene 1-sulphonate and thermolysin

“…23) ANS is widely used for the analysis of proteins. [24][25][26][27][28] We previously reported the interaction of ANS and human matrix metalloproteinase 7. 29) In the present study, to explore the mechanism of salt-induced activation and stabilization of thermolysin, we analyzed the interaction of ANS and thermolysin.…”