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fulltext41 - Effect of pH on Aqueous Phenylalanine Studied...

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Effect of pH on Aqueous Phenylalanine Studied Using a 265-nm Pulsed Light-emitting Diode A LEXANDER M. M ACMILLAN , a C OLIN D. M C G UINNESS , b K ULWINDER S AGOO , c D AVID M C L OSKEY , c J OHN C. P ICKUP , b AND D AVID J. S. B IRCH a a Photophysics Research Group, Department of Physics, University of Strathclyde, Glasgow, United Kingdom b Unit for Metabolic Medicine, Guy’s, King’s, and St. Thomas’ School of Medicine, Thomas Guy House, Guy’s Hospital, London, United Kingdom c Horiba Jobin Yvon IBH Limited, Glasgow, United Kingdom Recently, we described the characteristics and application of a 265-nm AlGaN light-emitting diode (LED) operated at 1-MHz repetition rate, 1.2-ns pulse duration, 1.32- μ W average power, 2.3-mW peak power, and approximately 12-nm bandwidth. The LED enables the fluorescence decay of weakly emitting phenylalanine to be measured routinely in the condensed phase, even in dilute solution. For a pH range of 1–11, we find evidence for a biexponential rather than a monoexponential decay, whereas at pH 13, only a monoexponential decay is present. These results provide direct evidence for the dominance of two phenylalanine rotamers in solution with a photophysics closer to the other two fluorescent amino acids, tyrosine and tryptophan, than has previously been reported. Although phenylalanine fluorescence is difficult to detect in most proteins because of its low quantum yield and resonance energy transfer from phenylalanine to tyrosine and tryptophan, the convenience of the 265-nm LED may well take protein photophysics in new directions, for example, by making use of this resonance energy transfer or by observing phenylalanine fluorescence directly in specific proteins where resonance energy transfer is inefficient. Key words: 265-nm LED; phenylalanine; rotamer model Introduction Progress in the fabrication of deep ultraviolet (UV) AlGaN light-emitting diodes (LEDs) has been consid- erable in recent years. 1 Recently we demonstrated the first results on exciting protein fluorescence by using tyrosine 2 and tryptophan. 3 This development not only makes the study of protein fluorescence simpler and less expensive than the mode-locked lasers or flash lamps used previously but also offers the potential of new miniaturized implementations, such as lab-on-a- chip technology using immunoassays, thereby opening up new approaches to point-of-care and rapid diag- nostics. In biomolecular research this technology is giving researchers cheaper, more reliable, and simpler means to achieve excitation of not only amino acids and proteins but also other important fluorophores, such as nicotinamide adenine dinucleotide (NADH). Address for correspondence: David J. S. Birch, Photophysics Research Group, Department of Physics, John Anderson Bldg., University of Strath- clyde, 107 Rottenrow, Glasgow G4 0NG, UK. Fax: + 44-141-548 3184.
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