H. Olga Hankovszky

A novel reversible thiol-specific spin label: Papain active site labeling and inhibition☆

Abstract A new, highly reactive, thiol-specific spin label, (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)methanethiosulfonate was synthesized. Its unique specificity was demonstrated with the active thiol protease, papain, which was stoichiometrically inhibited within 5 min, resulting in a conformationally sensitive spectrum, which was identical over the pH range 4.5–7.5. The spin-label modification yielded a mixed disulfide between Cys 25 of papain and the 3-methylpyrroline nitroxide which was rapidly and completely reversed by exposing the labeled papain to mild concentrations of dithiothreitol. The concentration of released nitroxide corresponded exactly to the number of reactive thiol groups in the original enzyme. Full enzymatic activity was restored after the spin label was removed. This spin label is useful as a sensitive thiol titrating agent as well as a specific conformational probe of thiol site structure by virtue of its minimal rotational freedom and distance from the covalent disulfide linkage to the macromolecule under study.

A novel spin-label for study of membrane protein rotational diffusion using saturation transfer electron spin resonance. Application to selectively labelled Class I and Class II -SH groups of the shark rectal gland Na+/K+-ATPase

Na+/K+-ATPase in membranous preparations from the rectal gland of Squalus acanthias has been spin-labelled either on Class I -SH groups, which maintain overall ATPase activity, or on Class II -SH groups, for which only phosphorylation activity is preserved. Labelling of the Class I groups requires solubilization of the membranes and subsequent reconstitution by precipitation with Mn2+ in order to remove contaminating peripheral proteins, which are also labelled. Control experiments with preparations in which the Class II groups are labelled demonstrate that the mobility and aggregation state of the enzyme in the reconstituted membranes are similar to those in the native membrane. Both the conventional maleimide nitroxide derivative and a new benzoylvinyl nitroxide derivative have been used for the labelling. The segmental mobility of the labels and the overall rotational diffusion of the labelled protein have been investigated using saturation transfer ESR spectroscopy. The benzoylvinyl spin-label derivative offers particular advantages for the study of the protein rotational mobility in that the segmental mobility is considerably reduced relative to that observed with the maleimide derivative. This is especially the case for the Class I groups, where the maleimide label exhibits pronounced segmental mobility. Comparison of the results from the two labels indicates that the integral of the saturation-transfer spectrum is much more sensitive to segmental motion than are the diagnostic line-height ratios. This fact allows a better level of discrimination between the two types of motion. The results from the benzoylvinyl nitroxide-labelled Class I groups suggest that the Na+/K+-ATPase is probably present as an (alpha beta)2-diprotomer (or higher oligomer) in the native membrane.

Conjugate addition with organometallics and nitration of nitroxide (aminoxyl) free radicals. Synthesis of potentially useful cross-linking spin label reagents

3-Methoxycarbonyl-2,2,5,5-tetramethyl-2,5-dihydropyrrol-1-oxyl (1) was converted with PhMgBr–Cul in a conjugate addition into 3-methoxycarbonyl-2,2,5,5-tetramethyl-4-phenylpyrrolidin-1-oxyl (2). Compound (2) was nitrated with concentrated H2SO4/HNO3 to give the para-nitrophenyl derivative (5). Nitration of other phenyl substituted nitroxides was also investigated. The reaction in strong acids could be carried out in the presence of an acid-sensitive nitroxide free radical. An alkyl nitro compound has also been prepared: a conjugate addition of nitromethane to (1) gave 3-methoxycarbonyl-2,2,5,5-tetramethyl-4-nitromethylpyrrolidin-1-oxyl (20). Both (2) and (20) could be transformed by transfer hydrogenation to amino compounds and then in several steps to potentially useful cross-linking spin label reagents.

Synthesis of Nitroxide Paramagnetic Ketones from Nitroxide Acid Chlorides and Anhydrides by Fiedel-Crafts Acylation

Abstract Friedel-Crafts acylation of benzene or toluene with 5- and 6-membered nitroxide acid chloride, anhydrides derivatives (symmetrical or mixed) and aluminium chloride to aryl nitroxide ketones is described.

Investigation of naturally abundant 17O hyperfine satellites in the electron spin resonance spectrum of 5- and 6-membered cyclic nitroxide (aminoxyl) radicals

Hyperfine satellites of naturally abundant 17O-isotopes have been detected for twelve nitroxide spin labels; line width, saturation, and magnetic parameters are discussed in terms of hyperfine anisotropy and molecular geometry.