Steady-state spectroscopy of new biological probes

Steady-state spectroscopy of new biological probes

Osama K. Abou-Zied*

Department of Chemistry, Faculty of Science, Sultan Qaboos University, P.O. Box 36, P.C. 123, Muscat, Sultanate of Oman

ABSTRACT

The steady state absorption and fluorescence spectroscopy of 2-(2'-hydroxyphenyl)benzoxazole (HBO) and (2,2'-bipyridine)-3,3'-diol (BP(OH)2) were studied here free in solution and in human serum albumin (HSA) in order to test their applicability as new biological probes. HBO and BP(OH)2 are known to undergo intramolecular proton transfers in the excited state. Their absorption and fluorescence spectra are sensitive to environmental change from hydrophilic to hydrophobic, thus allowing the opportunity to use them as environment-sensitive probes. The effect of water on the steady state spectra of the two molecules also shows unique features which may position them as water sensors in biological systems. For HBO in buffer, fluorescence is only due to the syn-keto tautomer, whereas in HSA the fluorescence is due to four species in equilibrium in the excited state (the syn-keto tautomer, the anti-enol tautomer, the solvated syn-enol tautomer, and the anion species of HBO). Analysis of the fluorescence spectra of HBO in HSA indicates that HBO is exposed to less water in the HBO:HSA complex. For the BP(OH)2 molecule, unique absorption due to water was observed in the spectral region of 400-450 nm. This absorption decreases in the presence of HSA due to less accessibility to water as a result of binding to HSA. Fluorescence of BP(OH)2 is due solely to the di-keto tautomer after double proton transfer in the excited state. The fluorescence peak of BP(OH)2 shows a red-shift upon HSA recognition which is attributed to the hydrophobic environment inside the binding site of HSA. We discuss also the effect of probe-inclusion inside well-defined hydrophobic cavities of cyclodextrins.

Keywords: 2-(2'-hydroxyphenyl)benzoxazole, (2,2'-bipyridine)-3,3'-diol, Cyclodextrins, Human serum albumin, Fluorescent probes, Protein-ligand recognition, Tautomerization

1. INTRODUCTION Fluorescent probes are widely used in biophysical studies in order to explore the structural and dynamical changes in

biomolecules.1,2 These probes are highly sensitive to the environmental changes which are reflected in their fluorescence intensity and/or spectral position. There has been an increased interest recently in a class of molecular probes which are known to undergo excited-state proton transfer. These molecules posses one or more hydrogen bonds in their structure and can be photoinduced to tautomerize in the excited state through an excited-state intramolecular proton transfer (ESIPT) mechanism. Two forms may coexist in the excited state; the normal (N*) and the tautomer (ESIPT product, T*) forms. These two forms are environment-sensitive and fluorescence measurements are usually used to distinguish the two forms. In some probes, both forms are usually highly fluorescent and result in well-separated fluorescence bands. In other probes, the ESIPT process is extremely efficient and fluorescence results only from the tautomer form. The intensities and spectral positions of the fluorescence bands often reflect the physicochemical properties of the microenvironment of the probe.

In this paper, we explore the steady state spectroscopy of two molecules which are proposed as potential biological probes owing to the sensitivity of their absorption and fluorescence spectra to solvent polarity and hydrogen bonding with protic solvents. The two molecules are 2-(2'-hydroxyphenyl)benzoxazole (HBO) and (2,2'-bipyridine)-3,3'-diol (BP(OH)2). We will briefly review the spectroscopy of those two molecules.

The ESIPT process in HBO is known to occur on a femtosecond time scale.3,4 ESIPT dynamics have also been found for analogous compounds, such as 2-(2'-hydroxyphenyl)benzothiazole and 2-(2'-hydroxyphenyl)benzimidazole.5-7 For HBO, the tautomeric and conformational equilibria, in several solvents and both in ground and excited states, have been studied by means of steady-state absorption and fluorescence spectroscopy.3,8-10 In general, two tautomeric forms, the keto and enol forms, coexist. In the ground state, the enol-imine tautomer is more stable, whereas in the first excited

* [email protected]; phone (+968) 2414-1468; fax (+968) 2414-1469

Genetically Engineered and Optical Probes for Biomedical Applications IV, Samuel Achilefu, Darryl J. Bornhop,Ramesh Raghavachari, Alexander P. Savitsky, Rebekka M. Wachter, Eds., Proc. of SPIE Vol. 6449,

64490L, (2007) · 1605-7422/07/$18 · doi: 10.1117/12.703269

Proc. of SPIE Vol. 6449 64490L-1

state the keto-amine tautomer is favored. Depending on the solvent, several enol isomers have been identified: syn-enol (intramolecular O-H---N hydrogen bond), anti-enol (intramolecular O-H---O hydrogen bond), and in polar protic solvents the “solvated syn-enol” (intermolecular hydrogen-bonding with solvent). Only the syn-enol can be converted into the keto form by an ESIPT process. Scheme 1 presents an overview of the tautomeric forms for HBO.

O

NOH

O

NOH

syn-enol

anti-enol

O

NO

H

solvated syn-enol

S

+S

O

N

OH

syn-keto

-S

hν

+S -S

Scheme 1. Equilibrium structures of the ground- and excited-state tautomers of HBO.

We have proposed HBO as a structural mimic of a DNA base pair for which tautomerization may be initiated at a defined time and position within duplex DNA.3,11-13 We characterized the spectroscopy of HBO in time and frequency domains in solution3,13 as well as incorporated in DNA.11-14 In contrast to other model base pairs, such as dimers of 7-azaindole,15 HBO can be incorporated into duplex DNA in a way which aligns the hydrogen bond of HBO with the hydrogen bonds of the flanking bases.11 Therefore, HBO may be used to probe the biologically relevant duplex environment. However, it is important to keep in mind the strengths and weaknesses of HBO as a model. Some structural and electronic properties of HBO in DNA are expected to be different from a Watson-Crick base pair due to the fact that the phenyl and benzoxazole rings of HBO are connected by one covalent and one H-bond, while the natural pyrimidine and purine rings are connected only by H-bonds. However, as a model of a DNA base pair, HBO has several advantages. HBO is easily incorporated into duplex DNA without a strong perturbation of the duplex.11 Additionally, the change in magnitude and orientation of the HBO dipole moment upon phototautomerization is expected to be similar to that which occurs during natural tautomerization.11,12

For HBO in the major groove of DNA, the fluorescence contribution was mainly from the excited-state keto tautomer with a lifetime of a few ns.12,13 This long lifetime was attributed to the incorporation of HBO in a hydrophobic environment, well shielded from the buffer solvent. It is also due to through-space interaction of HBO with flanking bases in which the keto tautomer is preferentially stabilized in the excited state. This is confirmed by the significantly long anisotropy decay times (1.5 ns and 3.6 ns in single- and double-stranded DNA, respectively) which indicate the rigidity of the local structure of HBO attached to DNA.13 On the other hand, fluorescence measurements for HBO in the minor groove of DNA show preferential stabilization of solvated enol which is attributed to the formation of a hydrogen bond between the enol OH group and the O4′ atom of an adjacent nucleotide, an H-bond acceptor that is only available in the minor groove.14

The (BP(OH)2) molecule (shown in Scheme 2) is known to undergo an ultrafast excited-state intramolecular double proton transfer (ESIDPT) in solution.16 At room temperature, BP(OH)2 absorbs in the region of 350 nm yet fluoresces


strongly in the green. Quantum yields of fluorescence in the order of 0.2-0.4 were observed in different solvents at room temperature with lifetimes of a few ns.16-20 Comparison of the absorption and emission properties of BP(OH)2 with related systems possessing only one hydrogen bond reveals that the second hydroxyl group is essential to the observation of the strong green emission. This molecule is also planar in crystalline form21 and is expected to retain its planarity in solutions of non-interacting solvents because of the two strong intramolecular hydrogen bonds. Electro-optical absorption and emission and calculated excited-state dipole moments show that the dipole moments of the di-enol (DE) and the di-keto (DK) tautomers are negligible, whereas it is 4.0-4.9 D for the mono-keto (MK) tautomer.22,23 Several experimental24-28 and theoretical29-32 studies have characterized the ultrafast dynamics in photoexcited BP(OH)2 and the double proton transfer was concluded to occur through both concerted and stepwise mechanisms.

N

N

O

O

H

H

N

N

O

O

H

H

N+

N+O

O

H

H -

-

PT1 PT2

DE tautomer MK tautomer DK tautomer Scheme 2. Tautomerization in BP(OH)2 after single proton transfer (PT1) and double proton transfer (PT2).

We have recently proposed BP(OH)2 as a model for natural base pairs to study tautomerization in duplex DNA.33 Molecular dynamics simulations were performed for a dodecamer duplex DNA containing BP(OH)2 as a model base pair in the center of the duplex. The results of the simulations indicate that BP(OH)2 can serve as a good mimic of a natural base pair with no major perturbation to the helical structure’s stability. One of the two hydrogen bonds in BP(OH)2 resides in the major groove of the duplex DNA, whereas the other one is situated in the minor groove. Similar biological environments were then tested by studying the steady-state absorption and fluorescence spectra of BP(OH)2 in solvents of varying polarity and hydrogen bonding capability, and in binary mixtures of p-dioxane/water. Unique absorption due to water solvation was observed in the region of 400-450 nm. A large blue shift in the fluorescence band due to intermolecular hydrogen bonding was observed in polar, protic solvents. The shift increases with increasing solvent polarity. The geometry of BP(OH)2 inside the DNA duplex and its unique spectroscopic characteristics in water suggest that this molecule can serve as a possible water sensor in DNA.

As an extension to the above studies, we examine in this paper the steady state spectroscopy of HBO and BP(OH)2 in human serum albumin (HSA). In this study, both molecules are used as probe ligands and HSA was chosen as a prototype protein. The aim of the study is to try to understand molecular recognition in protein-ligand complexes on a molecular level which is crucial to biological function and of practical importance in the discovery of new drugs and in phototherapy.34 HSA constitutes approximately half of the total blood protein.35 It recognizes a wide variety of agents and transports these agents in the blood stream. The X-ray crystal structure of HSA36,37 (Figure 1) indicates an asymmetric heart-shaped molecule that can be roughly described as an equilateral triangle. The two heart lobes contain the molecule’s hydrophobic binding sites while the outside of the molecules contains most of the polar groups. As shown in Figure 1, the binding sites in HSA are classified into three domains. Each domain is a product of two subdomains, A and B, with common structural motifs.

In order to understand the nature of the binding between the probes (HBO and BP(OH)2) and HSA, the spectral changes of the probes upon recognition by HSA will be compared to the spectral changes upon inclusion of the probes inside well-defined nanocavities of cyclodextrins (CDs). CDs are linked glucopyranose rings forming doughnut-shaped structures38-40 which quite often provide nanoenvironments very similar to biological environments.40,41


I B

I A

III B

III A

II A

II B

Fig. 1. The crystal structure of HSA and the locations of domain-binding sites. The structure was obtained from the Protein

Data Bank (ID code 1ha2).

2. MATERIALS AND METHODS

HBO (98%) and BP(OH)2 (98%) probes were obtained from Aldrich and were used without further purification. α-CD (≥ 98%) was purchased from Fluka and used as supplied. Deionized water (Millipore) was used in all preparations and dilutions. The buffer used was 50 mM sodium phosphate buffer, pH 7.0 and was obtained from Aldrich. HSA (essentially fatty acid free) was purchased from Sigma. Concentration of HSA in the buffer was determined spectrophotometrically by using ε280 = 36.6 mM-1.cm-1.42 Accordingly, the concentration of HSA in the samples was estimated to be ≈ 0.5 mM. The concentration of HBO and BP(OH)2 in all solvents, including the buffer, was ≈ 0.01 mM. Owing to the insolubility of HBO in water, a stock solution in 1,4-dioxane was prepared and an aliquot was added to HSA in buffer to a final concentration of 0.01 mM. The final 1,4-dioxane concentration in the solution was <1.0%.

Absorption spectra were obtained with an HP 845x Diode Array spectrophotometer. Fluorescence spectra were recorded on a Shimadzu RF-5301 PC spectrofluorophotometer. In all the experiments, samples were contained in a 1 cm path length quartz cell and the measurements were conducted at 23 ± 1 °C.

3. RESULTS AND DISCUSSIONS 3.1 Steady-state spectra of HBO in human serum albumin

We have reported elsewhere the absorption spectra of HBO in different solvents of varying polarity and hydrogen-bonding capability.3,43 HBO shows two low-energy absorption peaks in the region of 316-335 nm. From molecular orbital calculations,44 the two peaks were attributed to the ππ* transitions of mainly the anti-enol at 318 nm and the syn-enol at 334 nm.

Fluorescence spectra of HBO in HSA are shown in Figure 2. Fluorescence from HBO in buffer shows only one peak with a maximum at 490 nm. After excitation at 334 nm, the fluorescence of HBO in HSA shows an enhancement in the peak intensity by more than 100% with a slight blue shift, and the appearance of two new peaks at 430 nm and 380 nm. We have previously studied the fluorescence of HBO in different solvents and in binary solvents and we characterized the species responsible for each of the fluorescence peaks.43 The peak at 490 nm is due to the syn-keto tautomer which is formed in the excited state after ESIPT. The formation of the keto tautomer in the buffer solution is due to water-assisted tautomerization in the excited state which dominates in bulk water (species I in Scheme 3). On the other hand, the peak at 430 occupies the region of the anion species of HBO (species II in Scheme 3) and the peak at 380 nm is due to contribution from both the solvated syn-enol tautomer and the anti-enol tautomer (see Scheme 1).


0

20

40

60

80

100HBO-HSA / BufferHBO / Buffer

λ (nm)

350 400 450 500 550 600 650

Fluo

resc

ence

(rel

ativ

e in

tens

ity)

0

20

40

60

(a) λex = 334 nm

(b) λex = 318 nm

Fig. 2. Fluorescence spectra of HBO free in buffer and in buffer containing HSA at two different excitation wavelengths.

In order to mimic biological environments, we studied HBO in binary mixtures of 1,4-dioxane and water.43 1,4-dioxane and water are miscible in all proportions and thus provide an opportunity to study the effect of a broad range of solvent polarity. Their mixtures are proposed as media to study probes in microenvironments similar to those encountered in vesicles and at interfaces.33,45 We observed only the syn-keto tautomer in binary mixtures with water contents >80%. At such high water contents, the behavior of the binary mixtures approaches that of pure water.46,47 Fitting the change in the fluorescence intensity of HBO as a function of the water content in the binary mixtures indicates the participation of two water molecules in the process of water-assisted tautomerization (species I in Scheme 3) which was confirmed by ab initio calculations.43 On the other hand, in mixtures with water contents between 20% and 80%, we observed the anion species of HBO. The stabilization of the HBO anion is due to the higher ionizing strength in mixtures containing high water contents.48 The formation of ionic species of HBO in solvents of high water contents is due to the hydrogen-bonding characteristics of water and its ability to accept the proton from HBO by first forming an intermolecular hydrogen bond. The formation of the HBO anion was also proposed in the complex formation between HBO and cyclodextrins.10 Cyclodextrin cavities resemble biological media in which the included guest is buried in a hydrophobic interior, while the hydrophilic exterior tends to stabilize hydrogen bonding between the OHs of CDs and the phenolic OH of HBO which leads to the formation of the HBO anion.

The increase in fluorescence intensity and the slight blue shift of the syn-keto tautomer of HBO in HSA indicate the change in the local environment of HBO upon recognition by HSA and confirm the binding of HBO to HSA. We estimated the binding constant of HBO:HSA complex to be 50,000 ± 10,000 M-1 by measuring the spectroscopic changes as a function of HSA concentration. The formation of this complex tends to increase the fluorescence quantum yield of HBO due to the reduced nonradiative decay rate as HBO binds inside one of the HSA domains and is exposed to less water. Also, the appearance of the anion peak at 430 nm is indicative of a reduced water environment around HBO as discussed above for the results in binary mixtures. The regions for ligand bindings to HSA are located in hydrophobic cavities in subdomains IIA and IIIA.36 Binding site IIA is dominated by the strong hydrophobic interactions with most neutral, bulky, heterocyclic compounds. Since an aromatic anion molecule of warfrin is known to be bound to HSA in subdomain IIA by hydrophobic interaction,36 HBO is also expected to be bound in subdomain IIA. Finally, the


appearance of the peak at 380 nm indicates the formation of both the anti-enol and the solvated syn-enol tautomers (Scheme 1). The formation of the anti-enol tautomer is supported by measuring the fluorescence after excitation at 318 nm (Figure 2) which shows an increase in the peak intensity at 380 nm with a concomitant decrease in the peak intensity at 490 nm. As mentioned above, absorption at 318 nm is dominated by the anti-enol tautomer.3,8 We measured a small contribution form the anti-enol tautomer when HBO is incorporated in the major groove of DNA.12 This contribution becomes appreciable when HBO is positioned in the minor groove of DNA, along with equal contribution from the solvated syn-enol.14

O

N-O

O

NO

H

O

H

H O

H

H

III Scheme 3. Water-assisted tautomerization in HBO (I), and HBO anion (II).

3.2 Steady state spectra of BP(OH)2 in cyclodextrins and human serum albumin

The BP(OH)2 molecule shows unique spectroscopic features in water that may position it as a new biological probe. We reported the steady state absorption and fluorescence spectra of BP(OH)2 in solvents of varying polarity and hydrogen bonding capability, and in binary mixtures of p-dioxane/water in order to test its applicability as a model DNA base pair.33 In water, two new absorption peaks were observed in the region of 400-450 nm which are attributed to the solvation of the hydrogen bonding centers of BP(OH)2. A blue shift in the fluorescence peak was only observed in polar, protic solvents due to intermolecular hydrogen bonding between BP(OH)2 and the solvent. This blue shift is at its maximum in water and was measured to be ca. 36 nm from the peak position in cyclohexane.

We explore here the caging effects of cyclodextrins on the steady state spectra of BP(OH)2. We compare the caging effect of α-CD with our previous results on the effects of other CDs.49 Figure 3 depicts the absorption and fluorescence spectra of BP(OH)2 in different CDs. The spectra of BP(OH)2 in water and cyclohexane are also shown for comparison. The peak at 344 nm in the absorption spectra represents the transition to the lowest 1(π,π*) state.16 The double-peak absorption in the region of 400-450 nm is due to water solvation as mentioned above, and was suggested to be due to the stabilization of the DK tautomer in the ground state (see Scheme 2).31

As shown in Figure 3, the intensity of the 344 nm peak in the absorption spectra increases as the cavity size of the CD decreases (going from γ-CD to β-CD to α-CD),38,39 with a concomitant decrease in the peak intensities in the region of 400-450 nm. This result is due to the caging effect inside the CD cavity. As the cavity size decreases, the guest molecule is buried inside a more hydrophobic environment where water is expelled outside.40 This is reflected in the values of the calculated binding constants for the complexes estimated from the changes in the absorbance intensity as a function of CD concentration.49 The calculated binding constants (K) are 385 ± 100 M-1 for γ-CD, 860 ± 95 M-1 for β-CD, and (9.3 ± 0.9) x 103 M-2 for α-CD. The stoichiometry for the complexes between BP(OH)2 and both γ-CD and β-CD was calculated to be 1:1,49 whereas for the case of BP(OH)2 with α-CD the stoichiometry was found to be 1:2 (one molecule of BP(OH)2 and two molecules of α-CD). The latter result suggests that the BP(OH)2 molecule is completely buried inside the cavities of the two α-CDs which is supported by the high intensity of the 344 nm peak and the disappearance of the peaks due to water. The absorption spectrum of BP(OH)2 in aqueous α-CD resembles that in cyclohexane in which the environment is hydrophobic.


λ (nm)

250 300 350 400 450 500

ε (m

M-1

cm-1

)

0

5

10

15

20

25water0.01 M γ-CD0.01 M β-CD0.1 M α-CDCyclohexane

λ (nm)

400 450 500 550 600 650

Fluo

resc

ence

(nor

mal

ized

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

(a) (b)

Fig. 3. Absorption (a) and fluorescence (b) of BP(OH)2 in water, cyclohexane, and in different aqueous cyclodextrins. λex = 344 nm.

From the fluorescence spectra of BP(OH)2 in CDs (Figure 3), a red shift in the fluorescence peak was observed compared to the peak position in water. The spectra are normalized for clarity. As the cavity size of the CD decreases, the red shift is more pronounced. The maximum red-shift from the fluorescence peak in water is observed for BP(OH)2 in α-CD which is close to that in cyclohexane. The fluorescence results reflect the caging effect which is attributed to the increased hydrophobic environment around the BP(OH)2 molecule as the cavity size of the CD gets smaller.

The absorption and fluorescence spectra of BP(OH)2 in HSA are shown in Figures 4 and 5, respectively. The corresponding spectra of BP(OH)2 in buffer only are also shown for comparison. In HSA, the absorbance in the region of 400-450 nm is reduced whereas that at 344 nm is enhanced. According to the results obtained above in CDs, the change in the absorption of BP(OH)2 in HSA is due to the change in the local environment of BP(OH)2 upon recognition by HSA which confirms the binding of BP(OH)2 to HSA. We estimated the binding constant of BP(OH)2:HSA complex to be 43,000 ± 7,000 M-1 by measuring the spectroscopic changes as a function of HSA concentration.

λ (nm)

250 300 350 400 450 500

ε (m

M-1

cm

-1)

0

2

4

6

8

10BP(OH)2-HSA / BufferBP(OH)2 / BufferHSA / Buffer

Fig. 4. Absorption spectra of BP(OH)2 free in buffer and in buffer containing HSA. The spectrum of HSA in buffer is

included for comparison.


0

50

100

150

200

250 BP(OH)2-HSA / BufferBP(OH)2 / Buffer

λ (nm)

350 400 450 500 550 600 650

Fluo

resc

ence

(rel

ativ

e in

tens

ity)

0

50

100

150

(a) λex = 344 nm

(b) λex = 409 nm

Fig. 5. Fluorescence spectra of BP(OH)2 free in buffer and in buffer containing HSA at two different excitation

wavelengths.

In the BP(OH)2:HSA complex, the BP(OH)2 molecule is exposed to less water which is supported by the decrease in the absorption intensity in the region of 400-450 nm. This is also reflected in the fluorescence curves shown in Figure 5. Excitation at 344 nm results in a red shift of the fluorescence peak in HSA by ca. 22 nm compared to that in buffer only. The increase in the peak intensity is due to the increase in the absorbance at 344 nm in HSA compared to the absorbance in buffer. The increase in fluorescence intensity is also attributed to the reduced nonradiative decay expected inside the hydrophobic cavity of HSA. The fluorescence results after excitation at 409 nm show a decrease in intensity in HSA due to the decreased absorbance at 409 nm upon recognition by HSA. Fluorescence of BP(OH)2 after excitation in the region of 400-450 nm reflects the degree of exposure to water (or buffer in this case) and involves only a change in intensity with no peak shift.33,49

4. SUMMARY AND FINAL REMARKS The HBO and BP(OH)2 molecules were examined as potential probes in biology. HSA was used as the biological

system. In the ground state, HBO exists in equilibrium between the syn- and anti-enols. The syn-conformer exists in equilibrium between an internally H-bonded “closed” syn-enol and the solvated syn-enol. Only the “closed” syn–enol efficiently undergoes ESIPT upon photoexcitation to yield an excited syn-keto tautomer. Both the anti-enol and the solvated syn-enol fluoresce at 380 nm, while the keto tautomer fluorescence occurs at 490 nm. In HSA, fluorescence from HBO indicates the presence of four species in the excited state: the syn-keto tautomer, the anti-enol tautomer, the solvated syn-enol tautomer, and the anion species of HBO. The latter fluoresces at 430 nm. The presence of these species in HSA compared with only the presence of the syn-keto tautomer in the buffer solution indicates the environmental change around HBO due to recognition by HSA. Comparison of the fluorescence spectra of HBO in HSA with those in binary mixtures of 1,4-dioxane/water and in CDs indicates that HBO is exposed to less water in the HBO:HSA complex.

For the BP(OH)2 molecule, efficient double-proton transfer in the excited state causes a complete conversion of the DE form to the DK tautomer. Complex formation between BP(OH)2 and HSA shows a dramatic change in the


molecule’s absorption and fluorescence spectra due to environmental changes inside the HSA protein. Inside HSA, water is less accessible to BP(OH)2 and the environment is more hydrophobic. The hydrophobic interior of HSA was revealed in the absorption and fluorescence spectra in terms of a decrease in the intensity of the absorption peaks of BP(OH)2:water complex at 400-450 nm and a red-shift in the fluorescence peak. The latter indicates that the BP(OH)2 molecule is in a hydrophobic environment. The results in HSA were confirmed by measuring the spectroscopic changes due to inclusion of BP(OH)2 inside CD cavities. The changes in the absorption and fluorescence spectra of HBO and BP(OH)2 molecules in HSA suggest that both molecules bind in the hydrophobic cavity in subdomain IIA of HSA.

The spectral changes due to HSA recognition of HBO and BP(OH)2 reveal important features for both molecules as potential biological probes. The change in the fluorescence peak intensity and position can be used to explore the environment inside a given biological system in which the two molecules can be used as fluorescent probes. For BP(OH)2, the unique absorption peaks due to water solvation in the ground state may act as a probe of how BP(OH)2 is shielded from water in solution. Since the two peaks in the spectral region of 400-450 nm are due to water complexes with BP(OH)2, these peaks can be used as a probe of the accessibility of water to the BP(OH)2 molecule. This is important in biological systems in which molecular probes are used to detect changes in environment due to certain mechanisms such as protein unfolding and DNA unwinding.

5. ACKNOWLEDGEMENT This work was supported by the Sultan Qaboos University (grant no. IG/SCI/CHEM/05/03).

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