steady-state and time-resolved fluorescence investigation of 2-pyridone and 3-pyridone in solution...

Steady-state and time-resolved fluorescence investigation of 2-pyridone and 3-pyridone in solution and their specific binding to human serum

albumin

Osama K. Abou-Zied* and Othman I. K. Al-Shihi

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

ABSTRACT

2-pyridone (2Py) and 3-pyridone (3Py) were examined in different solvents and their binding to human serum albumin (HSA) was studied using steady-state spectroscopy and time-resolved fluorescence. Solvation of 2Py and 3Py by water was examined in binary mixtures of 1,4-dioxane and water. Analysis of the absorption and fluorescence data reveals the solvation of the hydrogen bonding center in 2Py by one water molecule and in 3Py by three water molecules. A zwitterionic tautomer of 3Py is formed in water and shows distinct absorption peaks from the absorption of the neutral tautomer. Fluorescence of 3Py was observed in polar solvents only, whereas 2Py is fluorescent in polar and nonpolar solvents. The absorption and fluorescence spectra of 2Py in different solvents indicate less solute-solvent interaction in nonpolar solvents. This observation was confirmed by the measured longer fluorescence lifetime of 2Py in cyclohexane compared to that in water. The mechanism of binding of 2Py and 3Py as probe ligands to HSA was investigated by following the intensity change and lifetime of HSA fluorescence after excitation at 280 nm. The presence of 2Py and 3Py causes a reduction in the fluorescence intensity and lifetime of HSA. This observation indicates that subdomain IIA binding site (Sudlow site I) is the host of the probes and the reduction in the fluorescence of HSA is due to energy transfer from the Trp-214 residue to the probe in each case. The distance between Trp-214 and each of the probes was calculated using Förster theory for energy transfer to be 1.99 nm for HSA/2Py and 2.44 nm for HSA/3Py. The shorter distance in the former complex indicates more efficient energy transfer than in the latter. This was confirmed by estimating the quenching rate constant (kq) in each complex. kq was calculated to be 1.44 x 1012 M-1s-1 for HSA/2Py and 3.45 x 1011 M-1s-1 for HSA/3Py. The calculated distances and the kq values indicate a static quenching mechanism operative in the two complexes. The binding constants were estimated to be K = (3.4 ± 0.4) x 104 M-1 for the HSA/2Py complex and K = (2.3 ± 0.3) x 104 M-1 for the HSA/3Py complex. The number of binding sites of HSA was calculated to be one in both complexes. The latter results, along with the quenching results, indicate that both probes, 2Py and 3Py, bind only in Sudlow site I in subdomain IIA.

Keywords: 2-pyridone, 3-pyridone, Hydroxypyridines, Tautomerization, Human serum albumin, Biological probes, Protein-ligand recognition, Resonance energy transfer

1. INTRODUCTION A large number of low molecular weight compounds bind reversibly to proteins and are widely used as extrinsic

fluorescent probes for the investigation of physicochemical, biochemical and biological systems. The spectral changes observed on the binding of fluorescent probes with proteins are an important tool for the investigation of binding sites, conformational changes and characterization of substrate to ligand binding.1 A very important criterion in the choice of a probe is its sensitivity to a particular property of the microenvironment in which it is located (e.g. polarity, acidity, etc.). There has been an increased interest recently in studying a class of molecules which possess one or more hydrogen bonds in their structure. Due to their extreme sensitivity to solvent polarity and hydrogen bonding with protic solvents, some of these molecules have been suggested as probes for the study of protein conformation and binding sites.2-5 We propose here two such molecules which are 2-pyridone (2Py) and 3-pyridone (3Py).

2-, 3-, and 4-pyridones and their hydroxypyridine (HP) tautomeric forms (2HP, 3HP, and 4HP) are shown in Scheme 1. These molecules have received much interest due to the similarity of their molecular structures with those found in a wide range of drugs of different pharmacological functions.6 Moreover, the Py/HP tautomeric equilibria in * Corresponding author. [email protected]; phone (+968) 2414-1468; fax (+968) 2414-1469

Molecular Probes for Biomedical Applications II,edited by Samuel Achilefu, Darryl J. Bornhop, Ramesh Raghavachari

Proc. of SPIE Vol. 6867, 68670K, (2008) · 1605-7422/08/$18 · doi: 10.1117/12.762218

Proc. of SPIE Vol. 6867 68670K-12008 SPIE Digital Library -- Subscriber Archive Copy

these compounds (shown in Scheme 1) play a relevant role in many biological processes and are greatly influenced by the polarity of the surrounding medium. In 2Py/2HP system, dimers constructed from different tautomeric combinations and linked by hydrogen bonds have been extensively studied experimentally and theoretically and are regarded as models for nucleotide base pairs.7-15

Studies of the above systems in different solvents proved that the 2- and the 4-isomers are distinctly different from the 3-isomer.16-19 The former isomers exist predominantly in the pyridone form in neutral solution, whereas evidence for a zwitterion has been reported for the 3-isomer.19,20 The tautomeric equilibria shown in Scheme 1 have also been shown to depend on environment.7,21-22 While in gas phase or in nonpolar solvents the hydroxypyridine forms dominate, in aqueous solution or in polar solvents the pyridone forms predominate. The change in equilibrium constants may be attributed to the greater stability of pyridone---solvent complexes.23

N OH N O

H

N N

H

OOH

N N

H

OH O

2-hydroxypyridine(2HP)

2-pyridone(2Py)


3-pyridone(3Py)


4-pyridone(4Py)

Scheme 1. Tautomerism of hydroxypyridines and pyridones.

We examine in this paper the steady state absorption and fluorescence spectra and dynamics of 2Py and 3Py in different solvents of varying polarity and hydrogen bonding capability in order to understand the tautomerization mechanisms in these compounds. We exclude from this study 4Py since we found it non-fluorescent in all solvents investigated. We then use 2Py and 3Py as probe-ligands and human serum albumin (HSA) as a prototype protein in order to test the applicability of both molecules as biological probes. HSA constitutes approximately half of the protein found in human blood.24 It recognizes a wide variety of agents and transports these agents in the blood stream. The X-ray crystal structure of HSA25,26 (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 molecule 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. The present study

Proc. of SPIE Vol. 6867 68670K-2

should be useful for understanding molecular recognition in protein-ligand complexes on a molecular level which is crucial to biological function.27

2. MATERIALS AND METHODS 2Py (97%), 3Py (98%) and 4Py (95%) probes were obtained from Aldrich and were used without further

purification. Deionized water (Millipore) was used in all preparations and dilutions. The buffer used was 50 mM sodium phosphate buffer, pH 7.2 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-1.28 The concentration of 2Py and 3Py in all solvents, including the buffer, was 0.1 mM.

Absorption spectra were obtained with an HP 845x Diode Array spectrophotometer. Fluorescence spectra were recorded on a Shimadzu RF-5301 PC spectrofluorophotometer. Lifetime measurements were performed using EasyLife II instrument obtained from Photon Technology International with a pulse width of 1.5 ns and a capability of measuring lifetimes in the range 100 ps-1µs using a Stroboscopic technique. In all the experiments, samples were contained in a 1 cm path length quartz cell and the measurements were conducted at 23 ± 1 °C.

Fig. 1. The crystal structure of HSA and the locations of domain-binding sites. The location of the Sudlow site I and Sudlow site II binding regions are indicated. The position of tryptophan residue (Trp-214) in the middle of helix H2 in subdomain IIA is shown. The structure was recovered from the Protein Data Bank (ID code 1ha2).

3. RESULTS AND DISCUSSION 3.1 Steady-state spectra and dynamics of 2HP and 3HP in solution

The absorption and fluorescence spectra of 2Py and 3Py in cyclohexane and water are displayed in Figure 2. The first absorption peak in both isomers in cyclohexane is due to a π-π* transition which shifts to a longer wavelength as the solvent polarity increases.19 In 2Py, this behavior is reversed in water and other hydrogen bonding solvents (methanol and ethanol) which indicates that a hydrogen bonding effect in the ground state predominates over solvent polarity effects. The formation of the anion species of 2Py in water was suggested to dominate the ground state and was confirmed by measuring the absorption spectrum of aqueous 2Py in basic medium.29

Comparing the absorption spectra of 2Py in cyclohexane and water, the first absorption band shows more structured absorption transitions in cyclohexane which vanish in water. This observation indicates the less solute-solvent interaction in nonpolar solvents. The two shoulders at 325 and 340 nm of 2Py in cyclohexane can be assigned to the

IA

IB

IIA

IIB

IIIB

IIIA Trp-214

Sudlow ISudlow II

IA

IB

IIA

IIB

IIIB

IIIA Trp-214

Sudlow ISudlow II


dimer formation as suggested in other nonpolar solvents.8,29 The two shoulders are also shown in dioxane and disappear gradually by the addition of water (Figure 3). This observation indicates that the interaction between 2Py and water is strong which prevents the dimer formation.29 It has been shown that in gas phase and in nonpolar solvents the 2HP form dominates, whereas in aqueous solution or in polar solvents the 2Py form predominates.23

Finally, a similar shift in both the absorption and fluorescence spectra of 2Py is shown in Figure 2 in water and cyclohexane. This similarity indicates that the polarity of 2Py in the ground state is close to that in the excited state.

In 3Py, two additional peaks at 247 and 315 nm are obtained in water (Figure 2). These two peaks have been attributed to the zwitterionic form (see Scheme 1), whereas the peak at 277 nm is due to the neutral enol form of 3HP.19,20 By adding water to dioxane (Figure 3), the intensity of the peaks due to the zwitterionic form increases at the expense of that of the neutral form. This indicates the presence of an equilibrium between the neutral and the zwitterionic forms. A relatively strong fluorescence from 3Py was obtained in water only (Figure 2), whereas weak fluorescence was measured in less polar solvents (methanol, acetonitrile, and DMSO) and no fluorescence was measured in cyclohexane. It has been reported that 3Py is only fluorescent in aqueous solvents.19

Fluorescence lifetime measurements were performed on 2Py and 3Py in cyclohexane and water. Since 3Py is not fluorescent in cyclohexane, we could not measure any decay in this case. All fluorescence decays were well-characterized by single-exponential fits. For 2Py, we measured 266 ± 14 ps and 244 ± 30 ps in cyclohexane and water, respectively. The relatively longer lifetime in cyclohexane indicates weaker solute-solvent interaction than in water as mentioned above. Will et al.29 reported lifetimes of 600 ps in n-hexane and 1.5 ns in water which we could not reproduce in the present work. For 3Py, the measured lifetime in water was 300 ± 31 ps.

0.2

0.8

1.4

2.0

Fluo

resc

ence

(nor

mal

ized

)

cyclohexanewater

Wavelength (nm)

200 250 300 350 400 450 500

Abs

orba

nce

(nor

mal

ized

)

0.2

0.8

1.4

2.0

zwitterion3Py

2Py

Fig. 2. Absorption and fluorescence spectra of 2Py and 3Py in cyclohexane and water. λex was 295 nm in 2Py and 315 nm

in 3Py.

In order to mimic biological environments, we studied 2Py and 3Py in binary mixtures of 1,4-dioxane and water. 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.30-32

The absorption spectra of 2Py and 3Py in the 1,4-dioxane/water binary mixtures are shown in Figure 3 for different water content. The spectra of 2Py show a blue shift and a slight increase in intensity of the first absorption peak as water


content increases. The peak also loses its fine structures as the water content increases which is due to more interaction between the 2Py moiety and water (vide supra). In 3Py, the intensity of the first absorption peak at 278 nm (neutral species) decreases as water volume fraction (Vw) increases with a concomitant appearance of two new peaks, a red-shifted peak at 315 nm and a blue-shifted one at 247 (not shown). The latter two peaks are due to the formation of the zwitterionic species as discussed above.

We can then write the following equilibrium as a result of solvation by water:

Py + nH2O Keq

Py:(H2O)n (1) Equation 1 assumes that increasing the water content leads to local solvation of the hydrogen-bonding center in 2Py

or 3Py by n water molecules. In this case, water molecules in the first solvation shell will only participate by certain n numbers and the rest of the water molecules will have the same effect as bulk water molecules.

According to Equation 1, the change in the measured absorbance (Aobs) can be related to the change in water concentration [S] as:31,32

n

n

K

Kn

]S[1

]S[AAA

eq

BSeqBobs

+

+= (2)

where AB is the absorbance of the unsolvated Py,

nBSA is the absorbance of the solvated Py, and Keq is the equilibrium

constant.

0.0

0.2

0.4

0.6

0.8

1.0

Wavelength (nm)250 300 350 400

Abs

orba

nce

(rel

ativ

e in

tens

ity)

0.0

0.2

0.4

0.6

Vw

0.0 0.2 0.4 0.6 0.8 1.0

A (r

elat

ive

inte

nsity

)

Vw

Vw

Vw

Vw

0.0 0.2 0.4 0.6 0.8 1.0

A (r

elat

ive

inte

nsity

)

315 nm

278 nm

Fig. 3. Selected absorption spectra of 2Py (upper panel) and 3Py (lower panel) in 1,4-dioxane/water binary mixtures. The

insets show the intensity change of the peak at 295 nm in 2Py and of the peaks at 315 nm and 278 nm in 3Py as a function of water volume fraction (Vw). The solid lines are the best non-linear least-square fits to Equation 2.

The measured changes in the absorbance intensity of 2Py and 3Py as a function of water volume fraction are displayed in the insets in Figure 3. The best fits to Equation 2 are also shown. The calculations from the best fits show


that one water molecule solvates the 2Py molecule (n = 1), whereas three water molecules solvate the 3Py molecule (n = 3). The fit in 2Py was performed for the absorbance increase of the first peak at 295 nm. For 3Py, the fits were performed for the absorbance increase of the peak at 315 nm for the zwitterionic species and for the absorbance decrease of the peak at 278 nm for the neutral species. The fits in the latter cases yielded the same value for n.

The steady state fluorescence spectra were recorded for the same binary mixtures after excitation at 295 nm for 2Py and at 315 nm for 3Py. The spectra are shown in Figure 4. By increasing the water content, an increase in the fluorescence peak intensity is observed accompanied by a slight blue shift. The fluorescence peak of 2Py loses its shoulders as the water content increases. This parallels the same effect in the absorption spectra and is due to a strong interaction between 2Py and water as indicated above. In the case of 2Py, the fluorescence intensity decreases for water volume fractions larger than 0.5. This observation may be attributed to the formation of water aggregates around the 2Py molecule. The behavior of binary mixtures of 1,4-dioxane and water was investigated using Kamlet-Taft parameter α.33-35 Above 25 M concentration of water, the behavior approaches that of pure water. This is manifested in Figure 4 in which the decline in the fluorescence intensity starts at a water volume fraction > 0.5 which is equivalent to a water concentration > 27 M. On the other hand, fluorescence intensity for 3Py increases as the water content increases in the binary mixtures. This behavior is similar to the increase in the absorbance intensity at 315 nm. The changes in the absorbance and fluorescence intensities are due to the formation of the zwitterionic species in water as indicated earlier.

0

50

100

150

200

Wavelength (nm)320 380 440 500 560

Fluo

resc

ence

(rel

ativ

e in

tens

ity)

30

100

170

240Vw

Vw = 0.5

Vw

0.0 0.2 0.4 0.6 0.8 1.0

F (r

elat

ive

inte

nsity

)

Vw

0.0 0.2 0.4 0.6 0.8 1.0

F (r

elat

ive

inte

nsity

)

Fig. 4. Selected fluorescence spectra of 2Py (upper panel, λex = 295 nm) and 3Py (lower panel, λex = 315 nm) in 1,4-dioxane/water binary mixtures. The insets show the intensity change of the peak at 368 nm in 2Py and of the peak at 390 nm in 3Py as a function of water volume fraction (Vw). The solid lines are the best non-linear least-square fits to Equation 2. See text for details.

Finally, fitting the changes in the fluorescence intensities of 2Py and 3Py as a function of water concentration to Equation 2 (replacing A for absorbance by F for fluorescence) yields the same stoichiometries as shown above from the absorption spectra (n = 1 in the case of 2Py and n = 3 in the case of 3Py). The fits are depicted in the insets in Figure 4.

3.2 Binding mechanisms of 2Py and 3Py to human serum albumin

Binding of small molecules such as 2Py and 3Py to HSA may result in a conformational change of the HSA protein. This change is a consequence of altering the intramolecular forces involved in maintaining the secondary structure of the protein.36 Fluorescence measurements are used to obtain information related to the mechanisms of binding of small molecules to proteins such as binding modes, binding constants, binding sites, and intermolecular distances. For 2Py and 3Py, the absorption spectra overlap with that of HSA. This makes selective excitation of the probe impossible without


disturbing the HSA protein. For this reason, we proceed with excitation in the HSA maximum absorption at 280 nm and follow the change in its fluorescence in the presence of 2Py and 3Py. In this case, we can measure the quenching effects of the small molecules on the fluorescence of HSA.

HSA has three fluorophores which are tryptophan, tyrosine and phenylalanine. The intrinsic fluorescence of HSA is due mainly to tryptophan alone, because phenylalanine has a very low fluorescence quantum yield and the fluorescence of tyrosine is almost totally quenched if it is ionized or near an amino group, a carboxyl group, or a tryptophan.37 HSA has only one tryptophan residue (Trp-214) which lies in subdomain IIA as shown in Fig. 1. Two binding sites are located in hydrophobic cavities in subdomains IIA and IIIA and are known as Sudlow sites I and II, respectively.38 Accordingly, if the 2Py or 3Py probes bind in Sudlow site I, this may enhance fluorescence quenching of HSA. Fluorescence quenching mechanism is not expected to operate if binding of the probes is located in Sudlow site II due to its far location from Trp-214.

Figure 5 shows the overlap between the UV absorption spectra of 2Py and 3Py with the fluorescence spectrum of HSA. As can be seen from the graphs, the overlaps are quite large which enhance resonance energy transfer. The molar extinction coefficient of 2Py in a buffer containing HSA was estimated at the maximum of the absorption peak to be ε295 = 6965 M-1cm-1. For 3Py, the maximum absorption at 315 nm in buffer containing HSA was estimated to be ε315 = 3257 M-1cm-1. The ε values indicate that 2Py may show more fluorescence quenching effect than 3Py. This indeed was the observation as shown in Figure 6. In this Figure, fluorescence of HSA was measured after excitation at 280 nm which represents the maximum absorption of the protein. Fluorescence of HSA was quenched more in the presence of 2Py than in the presence of 3Py as shown in the Figure.

Fig. 5. Overlap of the fluorescence spectrum of HSA (λex = 280 nm) with the absorption spectrum of 2Py (upper panel, ε295 = 6965 M-1cm-1) and with the absorption spectrum of 3Py (lower panel, ε315 = 3257 M-1cm-1)). All solutions were prepared in a pH 7.2 sodium phosphate buffer.

We confirmed the steady-state observation by measuring the lifetimes of HSA in the absence and presence of the probes. Figure 7 displays the decay curves. All the decay curves were best fitted using a monoexponential-decay function. The lifetime was calculated to be 6.41 ± 0.03 ns for HSA. In the presence of 2Py, the HSA lifetime was

ε/ε 3

15

0.2

0.5

0.8

1.1

Fluo

resc

ence

(nor

mal

ized

)

0.2

0.5

0.8

1.12Py/absorptionHSA/fluorescence

Wavelength (nm)

250 300 350 400 450

ε/ε 2

95

0.2

0.5

0.8

1.1

0.2

0.5

0.8

1.13Py/absorptionHSA/fluorescence


calculated to be 6.05 ± 0.01 ns and in the presence of 3Py, the calculated HSA lifetime was 6.20 ± 0.03 ns. A shorter lifetime in the presence of 2Py is a manifestation of a stronger quenching mechanism than that in the presence of 3Py.

Wavelength (nm)

285 325 365 405 445

Fluo

resc

ence

inte

nsity

0

50

100

150

200

250

HSA2Py+HSA3Py+HSA

Fig. 6. Quenching effect of 2Py and 3Py on the fluorescence spectrum of HSA. Concentrations of HSA, 2Py and 3Py were

0.1 mM in phosphate buffer. λex = 280 nm.

Time (ns)

-2 -1 0 1 2 3 4 5

Fluo

resc

ence

inte

nsity

0

150

300

450

600

750

900

IRFHSA2Py+HSA3Py+HSA

Fig. 7. Fluorescence decays of HSA and of HSA after adding 2Py and 3Py as indicated in the graph. Concentrations of

HSA, 2Py and 3Py were 0.1 mM in phosphate buffer. λex = 280 nm. IRF is shown by the dashed line.

Quenching of the HSA fluorescence by 2Py or 3Py indicates that the probe molecules are binding in Sudlow site I in subdomain IIA (see Figure 1). The distance between the donor (HSA) and acceptor (probe) can be calculated according to Förster’s theory for resonance energy transfer (FRET).39 The efficiency of energy transfer, E, is related to the distance (rAD) between the donor (HSA) and acceptor (2Py or 3Py) by


⎟⎟⎠

⎞⎜⎜⎝

⎛−=

+=

066

0

60 1

FF

rRRE

DA

(3)

where R0 is the Förster distance (critical distance) when the efficiency of energy transfer is 50%. F and F0 are the fluorescence intensities of HSA in the presence and absence of the quencher, respectively. The value of R0 can be calculated from

6/142

0 )(211.0 JnR Dφκ −= (4)

where κ2 is the spatial orientation factor between the emission dipole of the donor and the absorption dipole of the acceptor, n is the refractive index of the medium, φD is the fluorescence quantum yield of the donor, and J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor and is given by

∑ ∑ ∆∆

=λλ

λλλελ)(

)()( 4

FFJ (5)

where F(λ) is the fluorescence intensity of the donor at wavelength λ, and ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ.

J can be evaluated by integrating the overlapped portion of the spectra in Figure 5. If both the donor and acceptor are tumbling rapidly and free to assume any orientation, then the dipole orientation factor, κ2, equals 2/3.40 In the present case, n = 1.36 and φD = 0.15.41 Using the aforementioned values, we calculated R0 = 1.75 nm, E = 0.315, and rAD = 1.99 nm for HSA/2Py complex. The corresponding values for the HSA/3Py complex are: R0 = 1.81 nm, E = 0.216, and rAD = 2.24 nm. The donor-to-acceptor distance in both complexes is less than 7 nm, indicating a static quenching interaction between the donor and acceptor according to Förster’s nonradiative energy transfer theory.39 Comparing the calculated values for HSA/2Py with those for HSA/3Py, the smaller E value and the larger rAD value in the latter indicate lower energy transfer efficiency than in the former.

The operative quenching mechanism in the above complexes can be described by the Stern-Volmer quenching equation

][1][1 00 QKQk

FF

SVq +=+= τ (6)

Where kq is the quenching rate constant of the biomolecule, KSV is the Stern-Valmer constant, τ0 is the lifetime of the biomolecule without the quencher, and [Q] is the quencher concentration. By varying the concentrations of 2Py and 3Py and keeping the HSA concentration fixed, we calculated the quenching rate constants using Equation (6). For HSA/2Py, kq = 1.44x1012 M-1s-1, and for HSA/3Py, kq = 3.45x1011 M-1s-1. The calculated values of kq are greater than the maximum dynamic collisional quenching constant of various kinds of quenchers with biopolymers.42 The results confirm a static quenching mechanism is operative in the present complexes. The larger value of kq for the HSA/2Py complex compared to that for the HSA/3Py complex again indicates less quenching in the latter.

For small molecules that bind independently to a set of equivalent sites in a macromolecule, the equilibrium between free and bound molecules is given by the equation43


]log[loglog 0 QmKF

FF+=⎥

⎦

⎤⎢⎣

⎡ − (7)

where K and m are the binding constant and the number of binding sites, respectively, which can be calculated by plotting log(F0-F/F) versus log[Q]. The calculated values are K = (3.4 ± 0.4) x 104 M-1 and m = 1.1 for HSA/2Py and K = (2.3 ± 0.3) x 104 M-1 and m = 1.1 for HSA/3Py. The values of m indicate that in each case the probe is located in one binding site. The results, along with the quenching results, indicate that both probes, 2Py and 3Py, bind only in Sudlow site I in subdomain IIA and not in Sudlow site II (subsodmain IIIA).

4. SUMMARY AND FINAL REMARKS The 2Py and 3Py molecules were examined as potential probes to explore the binding sites in HSA. The steady state

absorption and fluorescence spectra of 2Py and 3Py were examined in cyclohexane, water and in binary mixtures of 1,4-dioxane and water. A blue shift in the absorption and fluorescence spectra of 2Py in water is due to the formation of an anion. In 3Py, the zwitterionic species is stable in water and shows absorption peaks distinct from those of the neutral species. Fluorescence from 3Py was observed clearly in water and other less polar solvents, but no fluorescence was measured in cyclohexane. A quantitative analysis of the spectra in the binary mixtures of 1,4-dioxane and water indicates that one water molecule solvates the hydrogen bonding center of 2Py, whereas three water molecules are needed to stabilize the zwitterionic species. Picosecond fluorescence decays were measured for the two probes. For 2Py in cyclohexane and water, a slightly longer lifetime was observed in cyclohexane which was attributed to a weaker solute-solvent interaction than in water.

The mechanism of binding of the probes to HSA was investigated using steady state and lifetime measurements. The presence of 2Py and 3Py causes a reduction in the fluorescence intensity and lifetime of HSA. This observation indicates that subdomain IIA binding site (Sudlow site I) is the host of the probes and the reduction in fluorescence of HSA is due to energy transfer from the Trp-214 residue to the probe in each case. The distance between Trp-214 and each of the probes was calculated using Förster theory for energy transfer to be 1.99 nm for HSA/2Py and 2.44 nm for HSA/3Py. The shorter distance in the former complex indicates more efficient energy transfer than in the latter. This was confirmed by estimating the quenching rate constant in each complex. kq was calculated to be 1.44x1012 M-1s-1 for HSA/2Py and 3.45x1011 M-1s-1 for HSA/3Py. The calculated distances and the kq values both indicate a static quenching mechanism operative in the two complexes. The binding constants and the number of binding sites of both complexes were also estimated. The calculations show that the binding constant for HSA/2Py is larger than that for HSA/3Py. One binding site in each complex was estimated from the calculations which indicates that both probes bind only in Sudlow site I in subdomain IIA.

The results obtained in this paper suggest that 2Py and 3Py can be used as potential probes to explore binding sites in proteins. The small molecular sizes of both molecules are not expected to cause any deformation to the interior of the proteins. The reversible nature of the binding mechanism between small molecules such as 2Py and 3Py and large proteins such as HSA gives a good opportunity to study the origin of interaction inside the binding sites and operative mechanisms inside the proteins such as energy transfer.

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

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steady-state and time-resolved fluorescence investigation of 2-pyridone and 3-pyridone in solution...

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