J. Biol. Chem., Vol. 260, Issue 17, 9636-9641, 08, 1985
Labeling of a thiol residue in sarcoplasmic reticulum ATPase by pyrene maleimide. Solvent accessibility studied by fluorescence quenching
E Kurtenbach and S Verjovski-Almeida
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.
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