Spin-labelling of phosphorylase b using a paramagnetic 1-fluoro-2,4-dinitrobenzene derivative

Phosphorylase b (1,4-alpha-D-glucan:1,6-alpha-D-glucan 6-alpha-glucosyltransferase, EC 2.4.1.1) can be specifically spin-labelled at a site essential for the catalytic action of the enzyme. A paramagnetic analogue of 1-fluoro-2,4-dinitrobenzene was synthesized and used as a dinitrophenylating agent. Reaction of phosphorylase b with the paramagnetic probe combined with the thiolysis method, leads to spin-labelling of a single -NH2 group (0.75 groups per subunit) with concomitant loss of 50% of the catalytic activity. Dinitrophenylation does not change the sedimentation profile of the enzyme. The ESR spectrum of modified phosphorylase b indicates that the attached label has rather limited segmental mobility and its environment is slightly hydrophobic. Small but subtle conformational changes induced by ligands in this critical site of the macromolecule can be directly detected by the spin-label. Also, sulfhydryl group modification of the spin-labelled enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) has a pronounced effect on the resonance spectrum.