Binding of a spin-labeled phenylalanine analog to sickle hemoglobin: EPR and NMR studies

We have synthesized a spin-label analog of phenylalanine as a competitive inhibitor probe of the sickle hemoglobin aggregation process. Sickle hemoglobin gelation measurements indicate that the spin-label phenylalanine analog is a potent inhibitor of deoxy sickle hemoglobin aggregation. We have also used spin label EPR and high-resolution proton NMR to study the interaction of the phenylalanine analog with hemoglobin, and find that the kinetic off-rate is comparable to, or slower than the hemoglobin rotational rate (i.e., greater than or equal to 10(8) s-1), and that at least one, and perhaps two significant localized interaction region(s) exist within a few angstroms of the beta chain N- and C-termini. Correlation with other known structural information suggests that the observed interaction sites may be relevant to the mechanism for inhibition of sickle hemoglobin aggregation.     

Analysis of the reduction of nitroxides by flavin mononucleotide

This article describes a simle method to prepare hydroxylamines from nitroxides by photo-activated flavin mononucleotide. The half-time of reduction varied from 2 to 38.4 s for a series of nitroxides. For most nitroxides short exposures to light (min) were sufficient to produce significant amounts of hydroxylamine; longer periods of exposure increased the yields of other products. Proxyl (2,2,5-trimethyl-5-alkylpyrrolidine-N-oxy) nitroxides were unsually reactive with a much higher yield of products which could not be reoxidized by ferricyanide to the nitroxides. Optimum conditions for reversible reduction depend on the nitroxide and the amounts of other reducible substances such as oxygen and ferricyanide that may be present.