Fluorescence of sanguinarine: spectral changes on interaction with amino acids.

The quaternary isoquinoline alkaloid, sanguinarine (SG) exhibits a wide range of biological activities. This study examines spectral changes expected from SG binding to proteins. Fluorescence spectra of the cationic form of sanguinarine (SG(+)) are sensitive to environment polarity. On the other hand, spectra of the neutral form of sanguinarine, pseudobase (SGOH) and dihydrosanguinarine (DHSG, the first metabolite of SG) exhibit higher sensitivity to the ability of solvent to form a solute-to-solvent hydrogen bonds. Interaction with cysteine has been the only mode of SG binding to enzymes that has been considered so far. In reality, our experiments have revealed spectral changes on specific interactions of SG(+) with Cys, Glu and Tyr in the protic environment and with Arg and Glu in the aprotic environment. We have also detected interactions of SGOH with Cys in the protic environment and with Cys, Glu and Lys in the aprotic environment. The DHSG spectra were only altered in the presence of the Cys analog in the protic environment. We have also demonstrated that spectral change analysis can aid investigation of SG/DHSG interactions with proteins and we were able to identify SG(+)-binding site on Na(+)/K(+)-ATPase.

Interaction of sanguinarine and its dihydroderivative with the Na+/K+-ATPase. Complex view on the old problem.

The effects of sanguinarine (SG) and its metabolite dihydrosanguinarine (DHSG) on Na(+)/K(+)-ATPase were investigated using fluorescence spectroscopy. The results showed that the enzyme in E1 conformation can bind both charged and neutral (pseudobase) forms of SG with a K(D)=7.2+/-2.0 microM or 11.7+/-0.9 microM, while the enzyme in E2 conformation binds only the charged form of SG with a K(D)=4.7+/-1.1 microM. Fluorescence quenching experiments suggest that the binding site in E1 conformation is located on the surface of the enzyme for both forms but the binding site in E2 conformation is protected from the solvent. We found no evidence for interaction of Na(+)/K(+)-ATPase and DHSG. This implies that any in vivo effect of SG attributable to inhibition of Na(+)/K(+)-ATPase can be considered only prior to SG-->DHSG transformation in the gastro-intestinal tract and/or blood. Hence, Na(+)/K(+)-ATPase inhibition will be effective in SG topical application but its duration will be very limited in SG oral or parenteral administration.

Changes in electrostatic surface potential of Na+/K+-ATPase cytoplasmic headpiece induced by cytoplasmic ligand(s) binding.

A set of single-tryptophan mutants of the Na(+)/K(+)-ATPase isolated, large cytoplasmic loop connecting transmembrane helices M4 and M5 (C45) was prepared to monitor effects of the natural cytoplasmic ligands (i.e., Mg(2+) and/or ATP) binding. We introduced a novel method for the monitoring of the changes in the electrostatic surface potential (ESP) induced by ligand binding, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. This approach opens a new way to understanding the interactions within the proteins. Our experiments revealed that the C45 conformation in the presence of the ATP (without magnesium) substantially differed from the conformation in the presence of Mg(2+) or MgATP or in the absence of any ligand not only in the sense of geometry but also in the sense of the ESP. Notably, the set of ESP-sensitive residues was different from the set of geometry-sensitive residues. Moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. This property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain.

Fluorescence of sanguinarine: fundamental characteristics and analysis of interconversion between various forms.

The quaternary isoquinoline alkaloid, sanguinarine (SG) plays an important role in both traditional and modern medicine, exhibiting a wide range of biological activities. Under physiological conditions, there is an equilibrium between the quaternary cation (SG+) and a pseudobase (SGOH) forms of SG. In the gastrointestinal tract, SG is converted to dihydrosanguinarine (DHSG). All forms exhibit bright fluorescence. However, their spectra overlap, which limited the use of powerful techniques based on fluorescence spectroscopy/microscopy. Our experiments using a combination of steady-state and time-resolved techniques enabled the separation of individual components. The results revealed that (a) the equilibrium constant between SG+ and SGOH is pKa = 8.06, while fluorescence of DHSG exhibited no changes in the pH range 5-12, (b) the SGOH has excitation/emission spectra with maxima at 327/418 nm and excited-state lifetime 3.2 ns, the spectra of the SG+ have maxima at 475/590 nm and excited-state lifetime 2.4 ns. The DHSG spectra have maxima at 327/446 nm and 2-exponential decay with components 4.2 and 2.0 ns, (c) NADH is able to convert SG to DHSG, while there is no apparent interaction between NADH and DHSG. These techniques are applicable for monitoring the SG to DHSG conversion in hepatocytes.

ATP and magnesium drive conformational changes of the Na+/K+-ATPase cytoplasmic headpiece.

Conformational changes of the Na(+)/K(+)-ATPase isolated large cytoplasmic segment connecting transmembrane helices M4 and M5 (C45) induced by the interaction with enzyme ligands (i.e. Mg(2+) and/or ATP) were investigated by means of the intrinsic tryptophan fluorescence measurement and molecular dynamic simulations. Our data revealed that this model system consisting of only two domains retained the ability to adopt open or closed conformation, i.e. behavior, which is expected from the crystal structures of relative Ca(2+)-ATPase from sarco(endo)plasmic reticulum for the corresponding part of the entire enzyme. Our data revealed that the C45 is found in the closed conformation in the absence of any ligand, in the presence of Mg(2+) only, or in the simultaneous presence of Mg(2+) and ATP. Binding of the ATP alone (i.e. in the absence of Mg(2+)) induced open conformation of the C45. The fact that the transmembrane part of the enzyme was absent in our experiments suggested that the observed conformational changes are consequences only of the interaction with ATP or Mg(2+) and may not be related to the transported cations binding/release, as generally believed. Our data are consistent with the model, where ATP binding to the low-affinity site induces conformational change of the cytoplasmic part of the enzyme, traditionally attributed to E2-->E1 transition, and subsequent Mg(2+) binding to the enzyme-ATP complex induces in turn conformational change traditionally attributed to E1-->E2 transition.