Fluorescence Spectra 159
of HPIP-c in presence of BSA, but the tautomer band is red-shifted from 463 nm in absence of BSA to 471 nm at 200 µM of BSA. The red shift indicates the HPIP-c molecule moves toward the hydrophobic binding site of BSA.
0.1 0.3 0.5 0.7 0.9
0 5 10 15 20 25
[BSA] ( µ M)
Fluorescence Spectra 160
0.0E+0 5.0E+5 1.0E+6 1.5E+6
290 320 350 380 410
Wavelength (nm)
Intensity (a. u.)
2.2 µµµµ M 27 µµµµ M 0 µµµµ M
0.0E+0 7.0E+4 1.4E+5 2.1E+5
290 320 350 380 410
Wavelength (nm)
Intensity (a. u.)
0 µµµµ M 48 µµµµ M
0.0E+0 5.0E+4 1.0E+5 1.5E+5
290 320 350 380 410
Wavelength (nm)
Intensity (a. u.)
0 µµµµ M 23 µµµµ M
Figure 5.25. Fluorescence excitation spectra of (a) HPBI, (b) HPIP-b, and (c) HPIP-c monitored at the tautomer bands in presence of BSA.
(b)
(c) (a)
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Fluorescence Spectra 161
However, in case of HPIP-c the ratio increases with a positive slope upon increase in concentration of BSA. This shows that with increase in concentration of BSA the normal emission becomes more prominent than the tautomer emission. This increase in intensity ratio of HPIP-c indicates that the breaking of cyclic intramolecular hydrogen bonded ring to form trans-enol in HPIP-c is facilitated in BSA. This may be due to hydrogen bonding of the guest molecule with the amino acid residues of BSA where a proton donor or acceptor is available for hydrogen bonding. In other words, the site of BSA at which the molecule HPIP- c binds favors the formation of trans-enol of HPIP-c. Further more, this shows that the hydrophobic sites of BSA at which the molecules HPBI and HPIP-c bind are different. It may be noted that in the former case, cis-enol is the favored conformer.
The fluorescence excitation spectra of the fluorophores monitored at the tautomer emission as the function of BSA concentration is more interesting (Figures 5.25) than that of normal emission (not shown). The excitation spectra of HPBI monitored at the tautomer band are different from those of HPIP-b and HPIP-c in the sense for initial addition of BSA up to 2.2 µM, the intensity of the band in HPBI decreases (Figures 5.25a). For further addition of BSA, the intensity increases with increase in BSA concentration. However, the fluorescence excitation spectral intensities of HPIP-b and HPIP-c gradually increase with increase in BSA concentration (Figures 5.25b and 5.25c). Even in the absence of BSA the excitation spectra of HPBI has a shoulder at ~ 360 nm and its intensity increases with increase in BSA concentration. On the other hand, in the excitation spectra of HPIP-b and HPIP-c no such shoulder are observed in absence of BSA, but a shoulder emerges on red side of the spectrum in both HPIP-b and HPIP-c with addition of BSA. The new band gains intensity with addition of BSA.
As the main excitation band is due to cis-enol, to understand the red end shoulder, the fluorescence spectra were obtained by excitation at the red end shoulder (Figure 5.26). HPBI has a new emission band at 440 nm in water and the intensity of the band increases with increase in BSA concentration. Addition of BSA shifts the emission maximum of HPIP-b to red side and new band appears at 443 nm and its intensity enhances with increase in BSA quantity. In HPIP-c, no band is observed in absence of BSA and a band emerges with addition of BSA and its intensity increases with further addition of BSA.
The red side excitation bands and the corresponding fluorescence bands in HPBI can be assigned to zwitterions and in HPIP-b and HPIP-c, they can be assigned to corresponding monoanions. These assignments are based on the following facts: (i) The red side
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Fluorescence Spectra 162
0.0E+0 3.0E+4 6.0E+4 9.0E+4 1.2E+5 1.5E+5
370 420 470 520 570 620
Wavelength (nm)
Intensity (a. u.)
4
µµµµ
M 200µµµµ
M0
µµµµ
M0.0E+0 3.0E+4 6.0E+4 9.0E+4 1.2E+5
380 430 480 530 580 630
Wavelength (nm)
Intensity (a. u.)
0
µµµµ
M 200µµµµ
M0.0E+0 3.0E+4 6.0E+4 9.0E+4 1.2E+5
380 430 480 530 580 630 680 Wavelength (nm)
Intensity (a. u.)
0
µµµµ
M 200µµµµ
MFigure 5.26. Fluorescence spectra of (a) HPBI (λexc = 365 nm), (b) HPIP-b (λexc = 375 nm), and (c) HPIP-c (λexc = 375 nm) in presence of BSA.
fluorescence spectral band maximum (440 nm) is red shifted compared to monoanion of HPBI. (ii) Sinha and Dogra reported the formation of zwitterions in aqueous medium at pH 6,95 and the fluorescence maximum of the observed zwitterions agree well with that of
(a)
(b)
(c)
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Binding Constants 163
Table 5.3. Fluorescence excitation (λmaxexc nm) and emission (λmaxfl nm) band maxima of the red shifted band.
Solution λmaxexc λmaxfl
HPBI
0 µM 362 441
27 µM 360 440
Monoanion in water95 304, 228 410
Zwitterion95 440
HPIP-b
0 µM - -
48 µM ~ 365 443
Monoanion in water6 360 461
Monoanion in DMSO 369 421
HPIP-c
0 µM - -
23 µM 366 439
Monoanion in water 351 438
Monoanion in DMSO 363 433
fluorescence spectrum obtained by excitation at 365 nm in case of HPBI and 375 nm in case of HPIP-b and HPIP-c (Table 5.3). (iii) However, formation of zwitterions was not observed for pyridine nitrogen substituted analogues in any environment.6,174,175 (iv) The pKa value for neutral-monoanion equilibrium of HPBI is very high (~ 10), whereas those of HPIP-b (8.6) and HPIP-c (9.3) are relatively small. The pKa value is expected to decrease inside the protein, due to interaction of phenolic –OH group with amino acid residues of proteins, as found in β-CD (Section 5.1). (v) The fluorescence excitation and emission spectral maxima of the new red side band of HPIP-b and HPIP-c also in reasonable agreement with those of corresponding monoanions (Table 5.3). Small discrepancy in the excitation and emission spectral maxima may be due to effect from the environment.
5.3.3. Binding Constants
The association constants for the binding of the ligands with BSA protein were determined from the fluorescence emission data by using the Benesi-Hildebrand equation (Equation 5.1).366,367 The emission intensities of the tautomer emission at 455 nm, 484 nm and 472 nm are used for the Benesi-Hildebrand plot of HPBI, HPIP-b and HPIP-c, respectively. The Benesi-Hildebrand plots (Figure 5.27) obtained with good linear correlations of R2 = 0.99 confirm the formation of 1:1 inclusion complexes between the fluorophores and BSA protein. The binding constants of HPBI, HPIP-b and HPIP-c with BSA protein are 14.3 x 103 M-1, 14.9 x 103 M-1 and 4.0 x 103 M-1, respectively. It is difficult to explain the trend in the binding constant values due to the complicacy of the complexes.
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Conclusion 164
However, the possible reason could be that HPBI is more hydrophobic than HPIP-c; and the hydrophobicity plays greater role than hydrogen bonding at the hydrophobic binding sites at which HPBI and HPIP-c bind. On the other hand, HPIP-b binds strongly with the binding site through hydrogen bonding which has greater impact than hydrophobicity.
y = 502x + 2.01 R2 = 0.99 y = 174x + 2.59
R2 = 0.99 y = 105x + 1.50
R2 = 0.99
0 30 60 90
0 0.03 0.06 0.09 0.12 0.15 0.18 1 / [BSA] (x 10
6M
-1)
1 / ( I- I
o)
HPBI
HPIP-b
HPIP-c
Figure 5.27. Benesi-Hildebrand plot for the HPBI (●), HPIP-b (■), and HPIP-c (▲).
5.3.4. Conclusion
Study on interaction of the fluorophores with BSA is reported. All three fluorophores form 1:1 complexes with BSA. The fluorescence of both normal and tautomer bands increase substantially in BSA. However, the intensity ratios of normal to tautomer bands of HPBI and HPIP-b decrease, but that of in HPIP-c increases in BSA. HPBI and HPIP-c bind in the hydrophobic site of BSA. On the other hand, HPIP-b binds at the hydrophilic site of BSA.
Although both HPBI and HPIP-c bind at hydrophobic sites, their binding pockets and interaction with BSA are different. Further docking studies are required to identify the exact location of the fluorophores. In addition to cis- and trans-enolic forms, HPBI is present in the zwitterionic form, whereas HPIP-b and HPIP-c are present as monoanion in addition to cis- and trans-enols in the ground state. In the excited state also along with tautomer and enol forms, HPBI and its pyridine nitrogen analogues are present in the zwitterionic form and the monoanionic form, respectively.
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