Hydroxyl radical probe

Samaha, R.R., Joseph, S., O Brien, B., O Brien, T.W., and Noller, H.F. (1999) Site-directed hydroxyl radical probing of 30S ribosomal subunits by using Fe(II) tethered to an interruption in the 16S rRNA chain. PNAS 96, 366. 

Heilek GM, Noller HF. Site-directed hydroxyl radical probing of the rRNA neighborhood of ribosomal protein S5. Science 1996 272 1659-1662. 

Moine, H., Cachia, G., Westhof, E., Ehresmann, B. and Ehresmann, C. (1997) The RNA binding site of S8 ribosomal protein of Escherichia coli SELEX and hydroxyl radical probing studies. RNA, 3, 255-268. 

Pulse Radiolysis A technique related to flash photolysis pulse radiolysis uses very short (nanosecond) intense pulses of ionizing radiation to generate transient high concentrations of reactive species. See Salmon, G. A. and Sykes, A.G., Pulse radiolysis, Methods Enzymol. 227, 522-534, 1993 Maleknia, S.D., Kieselar, J.G., and Downard, K.M., Hydroxyl radical probe of the surface of lysozyme by synchrotron radiolysis and mass spectrometry. Rapid Commun. Mass Spectrom. 16, 53-61, 2002 Nakuna, B.N., Sun, G., and Anderson, V.E., Hydroxyl radical oxidation of cytochrome c by aerobic radiolysis, Free Radic. Biol. Med. 37, 1203-1213, 2004 BataiUe, C., Baldacchino, G., Cosson, R.P. et al., Effect of pressure on pulse radiolysis reduction of proteins, Biochim. Biophys. Acta 1724, 432-439, 2005. 

Hydroxyl radical induced strand scission is used for probing of the ribose moiety. The probe is insensitive to local conformations and not useful for detailed structural predictions however, the nonselective reactivity and small size makes hydroxyl radical probing a powerful approach for footprinting proteins on RNA. Recently methods have been developed where the hydroxyl radicals are generated locally by attaching the iron ion, which catalyses the radical formation in the Fenton reaction, to specific sites on RNA binding proteins. RNA located in the neighborhood of the tethered Fe2+ will then be modified selectively.7,8... 

Kiselar, J.G. Maleknia, S.D. Sullivan, M. Downard, K.M. Chance, M.R. Hydroxyl radical probe of protein surfaces using synchrotron X-ray radiolysis and mass spectrometry. Int. J. Radial. Biol. 2002, 78,101-114. 

The hydroxyl radical is a small, highly reactive probe that is formed in water and primarily targets hydrophobic residues [109]. This may be an ideal probe for protein-protein interactions because tyrosine, tryptophan and phenylalanine are most likely to be found at an interface [110, 111]. Although protein-DNA interfaces are comprised of charged and hydrogen-bond donor side-chains, even these residues may be probed by hydroxyl radicals [112]. 

Because of its high reactivity, direct observation of hydroxyl radicals is very difficult in natural waters. Most of the evidence for the existence of HO derives from product analysis studies and from studies of relative photolytic reactivities of a series of compounds. Because HO reacts with many organic compounds at nearly diffusion-controlled rates (Fig. 16.3), various organic substrates that do not undergo other photolytic transformations may be used as probe molecules (for more details see Hoigne, 1997 Vaugham and Blough, 1998). 

Kaur H, Halliwell B (1996) Salicylic acid and phenylalanine as probes to detect hydroxyl radicals. In Punchard NA, Kelly FJ (eds) Free radicals, a practical approach. IRL Press at Oxford University Press, Oxford, pp 101-116... 

Sessler, J.L. et al. (2001) Probing the reactivity of the radiation sensitizer motexafin gadolinium (Xcytrin) and a series of lanthanide(III) analogues in the presence of both hydroxyl radicals and aqueous electrons, J. Porphyrins Phthalocyanines 5, 593-599. 

Halliwell B. Use of desferrioxamine as a probe for iron-dependent formation of hydroxyl radicals. Evidence for a direct reaction between desferal and the superoxide radical. Biochem Pharmacol 1985 34(2) 229-233. 

These footprinting analyses, based on enzymic and chemical digestion, are now widely used to define DNA (and RNA) and their complexes with various ligands. Recently active radical probes have been used as footprinting agents in protection assays in a variety of systems (e.g., Tullius and Dombroski, 1986 Chalepakis and Beato, 1989 Hayes and Tullius, 1989 Schickor et al., 1990). Such probes rely on active radical intermediates, most likely hydroxyl radicals, released by Fe(II) in the presence of an electron donor, probably via a Fenton reaction. In addition, hydroxyl radicals also appear to react with DNA in a conformation-specific manner which may allow some prediction of DNA secondary structure (see Burkhoft and Tullius, 1987 Zorbas et al., 1989 Lu et al., 1990). 

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