- Open Access
Elucidation of binding mechanism of hydroxyurea on serum albumins by different spectroscopic studies
© Naik et al.; licensee Springer. 2014
- Received: 14 February 2014
- Accepted: 5 June 2014
- Published: 15 July 2014
The interaction of hydroxyurea (HU) with serum albumins (SAs) has not been investigated so far. However, it necessitates the interaction study of HU with SAs in phosphate buffer of pH 7.4.
The binding of HU on bovine serum albumin (BSA) and human serum albumin (HSA) was studied in vitro under simulated physiological conditions by spectroscopic methods viz., fluorescence, FT-IR, UV–vis absorption, synchronous fluorescence and three-dimensional fluorescence.
The Stern-Volmer plot indicated the presence of dynamic quenching mechanism in the interaction of HU with SAs. The number of binding sites, n and binding constants, K were obtained at various temperatures according to the double logarithm regression curve. The result of FT-IR spectra, UV–vis absorption, synchronous fluorescence and three-dimensional fluorescence spectra showed that the conformation of SAs has been changed in the presence of HU. The thermodynamic parameters were calculated according to van’t Hoff equation and discussed.
This kind of study of interaction between BSA and HSA with HU would be useful in pharmaceutical industry, life sciences and clinical medicine.
- Bovine serum albumin
- Human serum albumin
The interaction of HU with SAs has not been investigated so far. However, it necessitates the interaction study of HU with SAs in phosphate buffer of pH 7.4. Different aspects of HU-serum albumin interactions viz., quenching mechanism, binding force operating between the drug and proteins, the distance of separation between the protein and HU (based on the theory of fluorescence resonance energy transfer), conformational changes etc. have been studied. This is the first report on the mechanism of interaction of HU with SAs employing fluorescence spectroscopy, UV–vis absorption, FT-IR, synchronous fluorescence and three-dimensional fluorescence spectroscopic methods.
Reagents and chemicals
Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma Chemical Company, St. Louis, USA and used without purification. Hydroxyurea (HU) was obtained from Sigma Aldrich. The solutions of HU, BSA and HSA were prepared in 0.1 M phosphate buffer of pH 7.4 with respect to their molecular weight. All other materials were of analytical reagent grade and Millipore water was used throughout the work.
Fluorescence spectra were recorded using a RF-5301 PC Hitachi spectrofluorometer Model F-2000 (Tokyo, Japan) with a 150 W Xenon lamp, a 1 cm quartz cell and thermostatic cuvette holder. The excitation and emission bandwidths were both 5 nm. The temperature of the sample was maintained by recycling water throughout the experiment. The absorption spectra were recorded on a singlet beam CARY 50-BIO UV–vis. Spectrophotometer (Victoria, Australia), FT-IR Nicolet-5700 (USA) was used to record infrared spectra. All of the pH measurements were performed with an Elico LI120 pH meter (Elico Ltd., India).
Hydroxyurea with protein interaction study
A stock solution of 250 μM of BSA, HSA and HU were prepared in phosphate buffer solution (pH 7.4). An appropriate volume of BSA or HSA to obtain 5 μM and 5 μM HU was mixed and fluorescence spectra were recorded. In the next step fixing the concentration of HSA at 5 μM and drug concentration was varied from 5 to 45 μM. Fluorescence spectra were recorded at three different temperatures (288, 298 and 308 K) in the range 300–450 nm upon excitation at 295 and 280 nm in BSA and HSA case.
The UV measurements of BSA and HSA in the presence and absence of HU were made in the range of 240–340 nm. BSA and HSA concentration was fixed at 5 μM while the drug concentration was varied from 5 to 10 μM in presence of phosphate buffer at 298 K.
The FT-IR spectra of BSA and HSA in presence and absence of HU at 298 K were recorded in the range of 1600 – 3000 cm-1. Serum albumins concentration was fixed at 5 μM while that of HU was 20 μM in presence of phosphate buffer.
Synchronous fluorescence measurements
The synchronous fluorescence characteristics of HU-SAs were noted down at different scanning intervals of Δλ (Δλ = λem - λex). When Δλ = 15 nm, the spectrum characteristics of protein tyrosine residues were observed and when Δλ = 60 nm, the spectrum characteristics of protein tryptophan residues were noticed.
3-D fluorescence studies
3-D fluorescence spectrum was recorded under the following conditions: excitation wavelength range of 250–350 nm and emission wavelength range of 200–500 nm and an increment of 10 nm with other parameters were just the same as that of fluorescence quenching spectra. Cprotein = 5 μM and CHU = 20 μM.
Effects of some common ions
The effects of some common ions viz., Co2+, Cu2+, Ni2+, Ca2+ and Zn2+ were investigated on HU with BSA and HSA interactions. The fluorescence spectra of HU with BSA or HSA system were recorded in presence of above ions at excitation at 295 and 280 nm for BSA and HSA at 298 K. The overall concentration of BSA or HSA and that of the common ions were fixed at 5 μM, while the concentration of HU was varied from 0 to 45 μM at 298 K.
Analysis of fluorescence quenching of serum albumins by hydroxyurea
Binding parameters and mechanism
Interaction parameters of HU-BSA system at different temperatures
Ksv × 10-4M-1
Kq × 10-12M-1S-1
Binding constant K × 10-4M-1
No. of binding sites n
ΔH0(k J mol-1)
ΔG0(k J mol-1)
58.08 ± 2.0
281.47 ± 13.0
-25.79 ± 3.0
Interaction parameters of HU-HSA system at different temperatures
Ksv × 10-4M-1
Kq × 10-12M-1S-1
Binding constant K × 10-4M-1
No. of binding sites n
ΔH0(k J mol-1)
ΔG0(k J mol-1)
102.94 ± 3.0
434.95 ± 12.0
-26.67 ± 2.0
Since the fluorescence lifetime of the biopolymer (Chen et al. 1990) is 10-8 s, the quenching rate constant, Kq can be calculated using the above equation. The values of Kq are given in Tables 1 and 2. The maximum scatter collision quenching constant, Kq of various quenchers with the biopolymer (Lakowicz & Weber 1973) is reported to be 2 × 10 (Donehower 1990) LM-1 S-1. The order of magnitude of Kq was calculated to be 10 (Heerenberg 1992) for both BSA and HSA with HU systems in the present study. So, the rate constants of the protein quenching procedure initiated by HU are greater than the value of Kq for the scatter mechanism.
Thermodynamic parameters and the nature of binding forces
Absorption spectroscopic studies
FT-IR spectroscopic studies
Synchronous fluorescence spectra
It can be seen from the Figure 8, the emission strength of tryptophan residues decreased faster than that of tyrosine residues, which revealed that tryptophan residues contributed more to the quenching of intrinsic BSA and HSA florescence in both the system. In addition, a slight red shift observed in both the tyrosine and tryptophan residues, indicated the less hydrophobic environment and more exposed to the solvent molecules during the binding process in both the systems. So both the microenvironment of tyrosine and tryptophan residues was changed, resulting in conformational changes of BSA and HSA during the binding process (Liu et al. 2001; Zhang et al. 2008).
Three-dimensional fluorescence spectra
As referred to peak 1, it mainly reveals the spectral characteristic of tryptophan and tyrosine residues. The reason is that when serum albumin is excited at 280 nm for BSA and 280 nm for HSA, it mainly reveals the intrinsic fluorescence of tryptophan and tyrosine residues, while fluorescence by the phenylalanine (Phe) residue is negligible. Compared with UV absorption spectrum of SAs (Figure 6), there is an absorption peak at around 295 nm for BSA and 280 nm for HSA, which is mainly induced by the π → π * transition of an aromatic amino acid. The Trp, Tyr and Phe residues in the binding cavity of protein have conjugated π - electrons and thus easily form charge transfer compounds with electron deficient species or other π - electron systems (Kang et al. 2004). The fluorescence intensity of the peak 1 decreased markedly and the maximum emission wavelengths of the peak 1 have obvious blue shift following the addition of HU, indicating that the conformations of the tryptophan and tyrosine residues of BSA and HSA were altered. Therefore, we can conclude that the binding of HU-SAs induced some micro environmental and conformational changes in BSA and HSA, a complex between HU-SAs has been formed.
Effect of metal ions on the interactions of serum albumins by hydroxyurea
Effect of common ions on binding constant of HU-BSA and HU-HSA systems
Binding constant (M-1)
3.1754 × 104
4.3863 × 104
HU + Co2+
2.08 × 105
4.18 × 104
HU + Ni2+
2.74 × 104
3.74 × 104
HU + Ca2+
3.13 × 104
3.83 × 104
HU + Zn2+
2.98 × 104
4.08 × 104
HU + Cu2+
2.86 × 104
3.26 × 105
Comparison of two systems
The values of binding constants (Table 1) suggest the interaction of HU with BSA and HSA are almost similar fashion. The quenching mechanism is also similar in both the system i.e., dynamic quenching. The binding sites were unity in both the systems. The thermodynamic parameters revealed that the HU and BSA and HSA undergo hydrophobic interaction. The synchronous fluorescence spectra and three dimensional fluorescence spectra reveals that the microenvironment of tyrosine and tryptophan residues was changed, resulting in conformational changes of SAs during the binding process.
The present work provides an approach for studying the interactions of BSA and HSA with hydroxyurea using absorption, fluorescence, FT-IR, synchronous and 3-D fluorescence techniques under physiological conditions. The results showed that BSA and HSA fluorescence was quenched by HU through dynamic quenching mechanism. HU interacted with SAs through hydrophobic forces. The remarkable change of amide I peak position in the BSA and HSA infrared spectrum after interaction with HU indicated that secondary structure of BSA and HSA has been changed. Since, the pharmaceutical firms need standardized screens for protein binding in the first step of new drug design, this kind of study of interaction between BSA and HSA with HU would be useful in pharmaceutical industry, life sciences and clinical medicine.
Keerti M. Naik thanks UGC, New Delhi for the award of Research Fellowship in Science for Meritorious Students (RFSMS).
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