Abstract
3-(2-Methenylindane-1,3-dione)-1-oxyl-2,2,5,5-tetramethylpyrroline (5-InVSL) is an effective nitroxyl spin label of biological macromolecules, attached rigidly due to its bulky planar side group and the anchoring capability of its oxo groups to protein via hydrogen bonding. Comparing an integral membrane protein, the Ca 2+-ATPase of sarcoplasmic reticulum (M.W. 113 kD) and the water-soluble hemoglobin (M.W. 64 kD) several notable differences were observed in the labeling processes. Hemoglobin has a single sulfhydryl group in the protein interior (β93), whereas Ca 2+-ATPase has two classes of labeling sites which have different accessibilities to the aqueous phase. Since the single labeling site of hemoglobin is not exposed to the aqueous phase the labeling efficiency depends on steric hindrance arising due to the bulky side group which, on the other hand, is rather useful for limiting segmental motion of this label in Ca 2+-ATPase. As a consequence, paramagnetic quenching is proposed for the calibration of spectral parameters used in saturation-transfer ESR of spin-labeled hemoglobin with 5-InVSL, to mask the signals of nitroxyls released via the retro-Michael reaction.
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