Ultrafast excited-state dynamics of tryptophan in water observed by transient absorption spectroscopy Divya Sharma, Jérémie Léonard, Stefan Haacke * Institut de Physique et Chimie des Matériaux de Strasbourg, UMR 7054,Université de Strasbourg – CNRS, 23,Rue du Lœss, F-67034 Strasbourg cedex, France a r t i c l e i n f o Article history: Received 26 November 2009 In final form 19 February 2010 Available online 23 February 2010 a b s t r a c t The excited-state dynamics of tryptophan in water is resolved by femtosecond transient absorption (TA) experiments covering the near-UV and Vis range of wavelengths. After a 1 ps solvent relaxation time, the excited-state decays non-radiatively, while an absorption band rises simultaneously at 425 nm on the nanosecond time scale. The 425 nm induced absorption appears to be the signature of the primary pho- toproduct resulting from the quenching of Trp excited-state in water. While time-resolved Trp fluores- cence spectroscopy has characterized Trp’s excited-state lifetime in detail, the present TA experiments give first evidence for the spectroscopic signatures of the quenching-induced photoproduct. Ó 2010 Elsevier B.V. All rights reserved. Tryptophan (Trp) – the most important intrinsic protein fluoro- phore among amino acids – has been extensively used as a molec- ular reporter for structural dynamics of proteins [1–6]. As a matter of fact,the mechanisms of Trp’s excited-state (S 1 ) quenching de- pend sensitively on the environment and on the molecular config- uration. However,their precise understanding is far from trivial [7,8] and has long been debated. In aqueous solution, besides sub-ps photo-ionization [9–11] and intersystem crossing [12–14] at long times,the intermediate, nanosecond time scale is charac- terized by a multi-exponential fluorescence decay. The latter has been rationalized on the basis of different rotameric configurations [15],and is thought to involve an excited-state proton [15–18] or charge-transfer [19,20], eventually leading to a primary photo- product whose spectroscopic signature has not been identified so far. Nanosecond photolysis studies have reported an absorption band (k peak = 400–440 nm, s 20–45 ns) – termed as ‘T 1 ’ [12–14], that could qualify for being the primary photoproduct’s signature. It has been observed only in conditions in which Trp’s side chain ammonium group is protonated (NH þ 3 ), which seems to be the key side chain group for S 1 quenching [17,20], but due to insuffi- cient time-resolution,a direct correlation of its formation with the S 1 quenching time is lacking.Furthermore,T 1 is usually as- signed to a triplet state of the protonated indole moiety [12–14], without a clear experimental proof though, since the usual quench- ing assays fail or give imprecise results for this short-lived species [13,14]. As recognized in a few reports [7,20], ultrafast absorption experiments are lacking that would give evidence for the forma- tion dynamics and the nature of the photoproduct resulting from S 1 quenching. We therefore performed femtosecond transient pump–probe spectroscopy studies on aqueous Trp. A 266 nm pump beam is obtained by third harmonic generation of 800 nm,40 fs pulses from a commercial5 kHz regeneratively amplified Ti:sapphire laser system. A white-light continuum (WLC) is generated by focussing 2l J of the fundamental in a 2 mm CaF 2 crystal,and used as the probe. The WLC extends down to 280–290 nm in the UV range. It is split into probe and reference beams. The former is focussed to a diameter of 50 l m, and over- laps in the sample with the pump beam with a diameter at least twice as large. Both probe and reference beams are detected simul- taneously with a spectrograph equipped with the CCD camera cooled at 60 °C. The probe and reference acquisition rate is 200 Hz. Data from consecutive runs measuring separate spectral windows are merged and analyzed. To this end,the WLC is spec- trally ‘shaped’ in the 290–400 and 350–700 nm range with colour filters placed in front of the spectrometer. The linear polarizations of both beams form the ‘magic’ angle of 54.7° and the time-resolu- tion is 120 fs.The maximum pump–probe delay time is 1.3 ns in this experiment. L-Tryptophan of 99.5% purity was used as received from Fluka. Tryptophan was dissolved to a concentration of 3 mM (corresponding to absorbance of 1.4 mm 1 at 266 nm) in solution of 0.01 M phosphate saline (pH 7.4). For transient absorption mea- surements,samples were circulated in a 0.5 mm-thick quartz cap- illary using a peristaltic pump. The pump beam excitation density was maintained below 2 mJ/cm 2 , meaning less than 5% excitation probability for Trp pumped at 266 nm ( e 4800/M/cm), hence in the linear excitation regime. In addition, we carefully checked that the tryptophan spectral signatures below 450 nm do not depend significantly on the pumping intensity in the range 0–5 mJ/cm 2 nor on the concentration in the range 0.15–3 mM. Above 0009-2614/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2010.02.057 * Corresponding author. E-mail address: stefan.haacke@ipcms.u-strasbg.fr (S. Haacke). Chemical Physics Letters 489 (2010) 99–102 Contents lists available at ScienceDirect Chemical Physics Letters j o u r n a l h om e pa ge : w w w . e l s e v i e r . c o m / l o c a t e / c p l e t t