Phosphorescence emission of 7-azatryptophan and 5-hydroxytryptophan in fluid solutions and in alpha2 RNA polymerase.
The tryptophan analogues 7-azaindole (7-Aza W) and 5-hydroxytryptophan (5-OH W) have a significant absorbance between 310-320 nm, which allows them to act as selective luminescence probes in protein mixtures containing a large number of tryptophan residues. To assess the potential of their phosphorescence emission in probing the nature of protein environments the delayed emission was examined as a function of temperature and solvent viscosity. Whereas in low temperature (135 K) propylene glycol/buffer glasses the phosphorescence of both 7-aza W and 5-OH W is structured, intense and exhibit a lifetime of a few seconds, above the glass transition temperature (180 K) the delayed emission is considerably quenched. Temperature profiles show that as the solvent is more fluid the phosphorescence of 5-OH W becomes red shifted, poorly structured and the triplet lifetime drops steeply reaching 29 micro(s) in buffer at 274 K. For 7-aza W the alterations are more drastic and no phosphorescence could be detected above 193 K. This implies that in fluid aqueous media the excited triplet state of these analogues is deactivated by vary efficient nonradiative processes. The quenching of 5-OH phosphorescence is not prevented even when the chromophore is inserted in a solvent protected protein environment. Indeed, substitution of the single Trp of a2 RNA Polymerase, which according to its relatively low phosphorescence lifetime at ambient temperature is substantially shielded from the solvent, with 5-OH did not inhibit the quenchability of the latter. Knowledge of the quenching mechanisms is therefore needed for this emission to report on the nature of the protein environment.
Cioni
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C.N.R. - Istituto di Biofisica, Via S. Lorenzo, 26, Pisa, 56127, Italy. patrizia@ib.pi.cnr.it
Erijman
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L
Strambini
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GB
D006916:5-Hydroxytryptophan / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D007700:Kinetics; D008163:Luminescent Measurements; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry; D014783:Viscosity
D012997:Solvents; C006657:7-azatryptophan; D014364:Tryptophan; D006916:5-Hydroxytryptophan; D012321:DNA-Directed RNA Polymerases