The fluorophores used in this work were purchased in a form of powder, that then were dissolved in the appropriate buffer, and some of them were used in buffered aqueous solution received from supplier. For the molecules used in powder form the buffer solution was prepared by dissolving buffer concentrates into distilled water. PH was controlled afterwards using pH paper. The list of fluorophores used in this work is provided in Table 3.2.
Table 3.2:Concentration of solutions. ε: molar extinction coeffi-cient.
substance M [g/mol] Buffer ε[M−1cm−1]
Trp 204.23 1 mM 5,479at 278 nm
Ala-trp 275.30 1mM
-Cyclo(-gly-trp) 204.23 1 mM Cyclo(-leu-trp) 275.30 1mM
IgG 150.000 0.01-0.13 mM 219,000at 280 nm
IgM 970.000 1.55µM
-HSA 67.000 0.1-0.8 mM 36,500at 280 nm BSA 67.000 0.1-0.8 mM 43,824at 280 nm
Immunoglobulin G and Immunoglobulin M were obtained from Sigma Aldrich in liquid form, supplied as a liquid in 0.01 M phosphate buffered saline, pH 7.2, containing 15 mM sodium azide. Human serum albumin (solid) was obtained also from Sigma-Aldrich and diluted in sterile PBS.
Flow cells
All the experiments biomolecules were carried out in liquid phase. The solutions containing trp and trp-containing dipeptides study are circulated in closed-circuit through a flow cell, where the interaction with the laser took place. The experi-ments with serum blood proteins: IgG, IgM, HSA are handled using quartz cells 0.1 mm path length, obtained fromStarna Scientific. Cells are constantly moving to avoid photobleaching.
Home-made flow cells with thin windows were used during the coherent control experiments. It is manufactured in the Physics Section workshop of the University of Geneva. Light path is 1 mm length, the housing is made of Aluminum, and the windows (Sapphire plates, 0.5" diameter, 0.01" thickness) are glued to the housing with epoxy resin.
Sample solution are circulated through special tube made of teflon (PTFE). The pump is to flow the solutions through the flow cells is a 2-channel peristaltic pump from Masterflex (pump head model 07536-02, driver model 07553-77). The solu-tion is flown through the cell by letting it fall by gravity, from a flask placed higher than the flow cell, to a lower flask, from which it is pumped back to the upper flask.
This ensured a smooth circulation into the cell.
Optimal Dynamic Discrimination of Trp-containing dipeptides
In this chapter we describe application of optimal dynamic discrimination (ODD) for discrimination of tryptophan-containing dipeptides, which is the first step to-wards protein identification. Protein fluorescence is defined by the presence of aromatic amino-acids (tryptophan, tyrosin, phenylalanin) which can provide very sensitive probe. Tryptophan is the most abundant amino-acid. Due to high sensi-tivity to the local environment it is widely used as a probe for protein study such as conformation changes, binding, denaturation, etc. Tryptophan fluorescence alone cannot be efficiently used for discrimination among proteins in ensemble, because of the strongly overlapping spectra with other multiple fluorescent amino-acids.
ODD can be a very efficient technique in discrimination of very similar molecules even if their absorption and fluorescence spectra are identical. Rabitz and co-workers demonstrated capabilities of ODD of discriminating flavins in visible by coherently manipulating the evolution of molecular wavepacket, excited by an op-timally shaped laser pulse [11]. The lack of the device capable for efficient pulse-shaping in deep UV (DUV, 240-300 nm) limited its application for proteins and amino-acids which absorption bands lay in this spectral region. Discrimination was recently achieved among the two main fluorescence probes, tryptophan (trp) and tyrosin (tyr) [10]. Here we demonstrate the extension of this approach for discrimination among small peptides- trp-containing dipeptides: ala-trp, cyclo(-gly-trp), cyclo(-leu-trp). The choice of dipeptides was also made because of the possible applications for cancer treatment and cancer vaccination using small
pep-73
tides. Their label free-detection and imaging is very important for biologists and biomedical researches.
4.1 Preliminary measurements
We investigate samples of trp, ala-trp, cyclo(-gly-trp) and cyclo(-leu-trp), des-olved in distilled water (PH=7), by steady state spectroscopy. Figure 4.1 shows fluorescence and absorption spectra of the samples. As already mentioned, since trp is the optically active element in ala-trp, cyclo(-gly-trp) and cyclo(-leu-trp), their absorption and emission spectra (in water, PH=7) are dominated by theS0→ S1excitation of the indole ring, and strongly overlap. Upon DUV excitation at 266 nm, the largest fluorescence Stokes shift is observed for ala-trp, while cyclo(-gly-trp) and cyclo(-leu-cyclo(-gly-trp) exhibit smaller shifts as compared to free trp in water. Both exposition to water and the link to the second amino-acid contribute to these shifts [104]. The spectra consist of the weighted contributions from all the conformers associated with each dipeptide [130,131]. The shifts induced by the peptide bond and the trp nano-environement amount only to some nm and discrimination within a mixture of dipeptides using linear fluorescence is clearly out of reach.
After the preliminary measurements with steady-state spectroscopy, we perform pump-probe depletion (PPD) approach, adopted for this experiment. The success-ful implementation of PPD has been already performed for other molecules, such as flavins and free amino-acids. The basic principle consists in the time-resolved observation of the competition between excited state absorption (ESA) into higher lying excited states and fluorescence into the ground state. This approach makes use of two physical processes beyond that available in the usual linear fluores-cence spectroscopy: (1) the molecular wavepacket dynamics in the intermediate state (S1) and (S2) the transition dipole strength to higher lying excited states (Sn).
More precisely, a first femtosecond pump pulse set at 266 nm, coherently excites trp from the ground stateS0to a set of vibronic levelsS1. A molecular wavepacket is formed by coherent superposition of the S1 states, the evolution of which is probed by a delayed 800 nm pulse. The second 800 nm femtosecond probe pulse transfers part of theS1population to higher lying electronic statesSn. AsSnstates are likely to ionize [132,133] (autoionization yield: 0.2), the population ofS1is ir-reversibly depleted and thus fluorescence to the ground state. Figure 4.2 illustrates typical fluorescence depletion trace for trp pumped in DUV at 266 nm and probed
Wavelength, [nm]
Normalized ab sorption, [a.u.]
trp ala-trp
cyclo(-leu-trp) cyclo(-leu-trp)
Normalized fluorescence, [a.u.]
Figure 4.1:Normalized absorption and fluorescence spectra, mea-sured for the four molecules under consideration at the same molar concentration (1 mM)
at 800 nm, obtained with PPD technique.