doi:10.1016/j.jphotobiol.2006.01.004 
Copyright © 2006 Elsevier B.V. All rights reserved.
Ultrafast photoinduced deligation and ligation dynamics: DCM in micelle and micelle-enzyme complex
Rupa Sarkar, Ajay Kumar Shaw, Manoranjan Ghosh and Samir Kumar Pal
, 
Unit for Nano Science and Technology, S.N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India
Received 30 July 2005;
revised 28 December 2005;
accepted 2 January 2006.
Available online 20 February 2006.
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Abstract
We report studies on diffusion controlled deligation and ligation dynamics of a probe ligand 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl) 4H-pyran (DCM) with cationic cetyltrimethylammonium bromide (CTAB) micelles. In order to investigate the effect of spatial heterogeneity on the dynamics we study the DCM labeled micelle upon complexation with an enzyme α-chymotrypsin (CHT). The variation of fluorescence line-width (Γ(t)) of DCM in the complex and also in the micelle indicates the diffusion dynamics of DCM through various environments of different polarities. The temporal behavior of Γ(t) reveals that at 50 mM CTAB concentration the excited DCM traverses 6.5 Å distance from the surface of a host micelle (deligation) before entering to a stern layer of another adjacent micelle (ligation). From neutron scattering experiment the distance 6.5 Å is found to be the thickness of a stern layer of CTAB micelle. No indication of ligation has been found at 2 mM CTAB concentration as the intermicellar distance is estimated to be very large (416 Å) compared to the previous case. The dynamical behavior of Γ(t) is also indicative of significantly slower diffusion of the ligand molecules (DCM) at the surface of the micelle in presence and absence of the enzyme compared to that in the bulk buffer. We have also studied the dynamics of solvation and local geometrical restriction on the probe DCM at the micellar surface with and without CHT. With picosecond time resolution, we found time constants of the solvation relaxation processes of the DCM labeled enzyme-micelle complex to be 230 ps (45%) and 870 ps (55%), which were comparable to those of the micelle without the enzyme. The time dependent anisotropy revealing local orientational motions of the probe in the complex was also found to be similar to that of DCM at the micellar surface in absence of CHT. These studies attempt to link the dynamical features for insight into the ligand mediated intercellular communication and the biological function of the enzyme α-chymotrypsin upon complexation with the CTAB micelle.
Keywords: Deligation and ligation dynamics; Solvation; DCM; Temporal FWHM; Anisotropy; Picosecond resolved fluorescence transients; CTAB micelle
Fig. 1. A schematic representation of diffusion controlled ligand mediated intercellular communication. The efficiency of the communication decreases with the effective intercellular separation.
Fig. 2. Upper. A schematic representation of spectral diffusion on the excited state potential surface. Emission spectra 1, 2 and 3 reflect the evolution of the solvation free- energies along the solvation coordinate. Lower. Time integrated fluorescence emission spectrum. The solid line is convolution of three representative emission spectra 1, 2 and 3 (dotted lines) that results from solvation of the probe ligand.
Fig. 3. A schematic representation of the effect of diffusion controlled dynamic heterogeneity on the emission spectrum of a probe at various time-interval after the excitation (t1 < t2 < t3). The probe acquires significant dipole moment upon excitation and hence becomes more polar in the excited state. Left panel indicates the population of the probe molecules in three representative environments with different polarities (environment-1 < environment-2 < environment-3) at various times. As time progresses the population of the excited polar probe molecules diffuse to the environment-3. The fluorescence intensity of the spectra (dotted line) as indicated by number 1, 2 and 3 represents population of the probe molecules in the environments 1, 2 and 3 respectively. The solid line is the convolution of the three representative spectra. Note the decrease in fluorescence line-width of the convoluted spectrum, which in turn reflect the temporal diffusion of the probe molecules.
Fig. 4. Upper. Solubility of DCM (μM) vs CTAB concentration (mM). Inset shows extinction coefficient (ε) of DCM in 50 mM CTAB. Lower. Plot of normalized fluorescence of DCM in various CTAB concentrations.
Fig. 5. Steady-state emission spectra of DCM in various environments. The absorption spectrum of DCM in the CTAB micelle is also shown. The arrow indicates the wavelength of excitation (405 nm).
Fig. 6. Normalized fluorescence transients of DCM in various environments. The Instrument response function (IRF) is shown for comparison (excitation at 405 nm).
Fig. 7. Time resolved normalized fluorescence transients of DCM in the micelle at 50 mM CTAB concentration in absence (upper) and presence (lower) of the enzyme CHT at three characteristic wavelengths. The instrument response function (IRF) is shown for comparison (excitation at 405 nm).
Fig. 8. Time resolved emission spectra (TRES) of the micelle-bound DCM at 50 mM CTAB concentration in absence (upper) and presence (lower) of the enzyme CHT. The dotted curves indicate steady-state emission spectra of DCM in the corresponding sample solutions.
Fig. 9. Solvation correlation function, C(t) of the micelle-bound DCM in absence (upper) and presence (lower) of the enzyme CHT. Insets show time resolved anisotropy, r(t) of DCM in the corresponding samples.
Fig. 10. Full width at half maxima (FWHM, Γ(t)) of time resolved fluorescence spectra of the micelle-bound DCM at 50 mM CTAB concentration in absence and presence of the enzyme CHT.
Fig. 11. Schematic diagram showing the micellar packing at 50 mM CTAB concentration.
Fig. 12. Upper. Solvation correlation function, C(t) of the micelle-bound DCM at 2 mM CTAB concentration. Inset shows time resolved anisotropy, r(t) of DCM in the sample. Lower. Full width at half maxima (FWHM, Γ(t)) of time resolved fluorescence spectra of the micelle-bound DCM at 2 mM CTAB concentration.
Table 1.
The results of the time dependent solvation and anisotropy measurements of the probe DCM in various environments
Table 2.
Numerical fitting parameters of the time dependent line-width of DCM emission (50 mM CTAB concentration)
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