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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Labeling of a thiol residue in sarcoplasmic reticulum ATPase by pyrene maleimide. Solvent accessibility studied by fluorescence quenching.

Sarcoplasmic reticulum ATPase was specifically labeled by the fluorescent probe N-(1-pyrene)maleimide which modified 1 mol of a highly reactive thiol residue per mol of ATPase under appropriate conditions, when the probe concentration was varied in the range 0.1-1.5 microM. Addition of inorganic phosphate to the labeling medium increased both the rate of labeling and the number of modified thiol residues. Addition of ATP gave a marked kinetic protection from labeling, suggesting that the label was attached to a protein domain which is sensitive to changes at the catalytic site. Quenching of pyrene fluorescence emission of labeled ATPase by acrylamide and cesium chloride gave linear Stern-Volmer plots. The Stern-Volmer quenching constants of pyrene-ATPase fluorescence were 10 times lower than the constant obtained for acrylamide quenching of the fluorescent adduct of pyrene-maleimide-cystein used as a control, indicating that the pyrene moiety of the probe was considerably shielded from the medium solvent when covalently attached to the ATPase. The efficiency of quenching of pyrene-ATPase fluorescence increased by a significant amount upon addition of 100 microM Ca2+, when compared to the quenching in the presence of a Ca2+ chelator. It suggests that occupancy of the high affinity Ca2+ sites of the ATPase increases the accessibility of medium solvent into hydrophobic domains of the enzyme. The fluorescence lifetime of the solubilized pyrene-ATPase emission was 144-149 ns. The fluorescence polarization of pyrene-ATPase solubilized by nonionic detergent C12E8 was rho = 0.10 and it increased with an increase in the viscosity of the medium yielding a linear Perrin plot. The rotational correlation time for the soluble ATPase was 532 ns, corresponding to the overall rotation of a detergent-pyrene-ATPase particle with radius of 87A.[1]


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