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Journal of the Chilean Chemical Society

versão On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.52 n.2 Concepción jun. 2007

http://dx.doi.org/10.4067/S0717-97072007000200017 

 

J. Chil. Chem. Soc, 52, Nº 2 (2007) págs.: 1196-1197

 

BINDING OF ROSE BENGAL TO BOVINE SERUM ALBUMIN

 

E. ABUIN,* A. ASPÉE, E. LISSI and L. LEÓN

Universidad de Santiago de Chile, Facultad de Química y Biología, Casilla 40- Correo 33, Santiago, Chile

Dirección para correspondencia


ABSTRACT

The association of Rose Bengal (RB) with bovine serum albumin (BSA) was investigated by absorbance spectroscopy. The binding constant was determined from the effect observed in the absorbance of RB at 548 nm upon addition of the protein according with the Benesi-Hildebrand treatment. Results were obtained in phosphate buffer at pH = 7.0. The effect of the salinity of the buffer and the sensitivity of the binding constant to the presence of urea were also studied. The results obtained allow to conclude that the binding of RB to BSA is dominated by hydrophobic effects.

Keywords : Rose Bengal; bovine serum albumin


INTRODUCTION

Rose Bengal is one of the compounds most frequently employed as sensitizers for the generation of singlet oxygen,1-5 which is known to produce oxidative damage to proteins and enzymes.5-7 On the other hand, serum albumins are the most abundant of the proteins in blood plasma, accounting for ca. 60% of the total protein,8,9 and is the major macromolecule contributing to the osmotic pressure of blood.10 They have the property to bind a wide variety of endogenous and exogenous compounds such as fatty acids, lysolecithin, bilirubin, warfarin, tryptophan, steroids, anaesthetics, surfactants11-16 and several dyes.16,17 The abundance of serum albumins in blood plasma makes it the potentially major targets for photooxidation and damage. It has been reported that protein-bound chromophores are capable of inducing protein and cell components oxidation, and the photodynamic effect of Rose Bengal has been shown to be related to the damage of unsaturated fatty acids or histidine, and on beef heart submitochondrial particles.18 Binding studies of sensitizers with proteins, particularly with the most abundant BSA, are useful for the understanding of the proteins oxidation reaction mechanism. In spite of this, investigations on the interaction of BSA with RB, widely used as sensitizer, have not been conducted.

In this work, we report the results of a study on the binding of RB to BSA and discuss on the effects of buffer salinity and the addition of urea upon the binding constant.

EXPERIMENTAL

Bovine serum albumin (BSA), and Rose Bengal (RB), were purchased from Sigma Chemical Co, and used without treatment. Urea was a sample from Scharlam. Na2HPO4 and NaH2PO4 used for the preparation of buffers were Merck products. Water employed was obtained from a Modulab Type II water purification system. Experiments were performed in buffer phosphate at pH 7.0 at room temperature.

The absorption spectra and the absorbances at 548 nm were recorded on a Hewlett Packard 8453 spectrophotometer.

Experiments were carried out as follows. A concentrated solution of RB in ethanol was prepared and an aliquot of it was added to the buffer solution to obtain an absorbance of 0.75 at 548 nm (corresponding to a concentration equal to 8,3 µM; this concentration is similar to that employed in studies where RB is used as sensitizer.1 The spectrum of this sample was recorded and afterwards aliquots of an appropriate BSA solution were successively added to cover a range of BSA concentrations up to 4,4 µM, registering the absorbance spectra after each addition.

RESULTS AND DISCUSSION

Addition of BSA to a solution of RB (8,3 µM) leads to a change in the RB absorption spectra as shown in Figure 1 where a clear isosbestic point can be observed. We assigned this behaviour to the presence of two species: RB free (band at 548 nm) and RB bound to the protein (band at 562 nm). This interpretation was in line with the RB absorption spectra obtained in n-butanol and n-butanol / water mixtures (data not shown) which shows a decrease in the band at 562 nm with the water content, concomitant with an increase in the absorbance at lower wavelengths (ca. at 550 nm).


The binding of RB to the protein was quantified using the Benesi-Hildebrand treatment,19 (Eq. 1), by plotting the reciprocal of the difference in the absorbance at 548 nm elicited by BSA addition vs the reciprocal of the BSA concentration.

In Eq. (1), ∆ε is the difference of the absorption coefficient between the free and bound states and Kb is the binding constant defined by Eq. (2).

where the subscripts b and free correspond to the analytical concentrations of RB associated with the protein and free, respectively.

The plot obtained according with the Benesi-Hildebrand treatment is shown in Figure 2.


From the ratio between the intercept and the slope of the line of Figure 2, a value of Kb equal to (374 000 ± 7 800 ) M-1 was obtained in 10 mM phosphate buffer. This value is within the order of the binding constants of exogenous ligands that are typically in the range 104 – 108 M-1,20 and implies that 50 % of the dye is bound to the protein when the BSA concentration is 2.7 μM. This concentration is two orders of magnitude smaller than that present in blood plasma.

A data treatment based on Eqs. (1) and (2) implies a two pseudophase model. This assumption is only valid for the association to proteins when the occupation number n (i.e., the average number of RB molecules bound per BSA) is considerably smaller than one.21 Under the experimental conditions employed in this work, n can be calculated from the derived Kb value, rendering values lower than 0.11 over all the range of BSA concentrations employed. Furthermore, an alternative data treatment based on an extrapolated value for the absorbance of the bound RB (obtained by extrapolating the absorption at infinite BSA concentration) allows an evaluation of the free and bound dye at each BSA concentration. A plot of [RB]bound/[BSA] vs [RB]free is almost lineal (data not shown) rendering support to the data treatment based on Eqs. (1) and (2).

The factors affecting the interactions between additives and proteins may include electrostatic and/or hydrophobic interactions. In order to investigate on the relevance of each one of these factors in the association of RB with BSA, we have measured the sensitivity of the binding constant to the buffer salinity and to the presence of urea.

In the presence of 30 mM phosphate buffer the effect of BSA addition on the absorption spectra of RB was similar to that shown in Fig.1. A value of Kb equal to (335 000 ± 5 200) M-1 was obtained from the Benesi-Hildebrand treatment, indicating that the salinity of the medium does no appreciably affect the binding, pointing to a minor importance of electrostatic interactions.

Figure 3 shows the effect of BSA addition on the absorption spectrum of RB in the presence of 8 M urea.


It is seen that the band at 562 nm does not appear in the presence of the denaturant. This result indicate that in the expanded protein,15 RB is bound to domains that are much less hydrophobic that in the native form. Furthermore, the binding constant was strongly decreased in the presence of urea, giving a value equal to (12 300 ± 2 100) M-1. All these data would imply that the binding of RB to BSA is mainly dominated by hydrophobic effects and that, in presence of urea, the binding domains are less hydrophobic and, hence, of lower affinity.

ACKNOWLEDGMENTS

Thanks are given to Dicyt (USACH) and Fondecyt (Grants # 1050058, and 1050137) for financial support.

 

REFERENCES

1. A. Wright, W.A. Bubb, C. Hawkins, M. Davies. Photochem. Photobiol. 76, 35 (2002).        [ Links ]

2. A. Wright, C.L. Hawkins, M.J. Davies. Redox Rep. 5, 159 (2000).        [ Links ]

3. A. Wright, C.L. Hawkins, M.J. Davies. Free Rad. Biol. Med. 34, 367 (2003).        [ Links ]

4. P.E. Morgan, R.T. Dean, M. Davies. Free Rad. Biol. Med. 36, 484 (2004).        [ Links ]

5. M.A. Biasutti, A. Posadaz, N.A. García. J. Peptide Research. 62, 11 (2003).        [ Links ]

6. M.J. Davies. Biochem. Biophys. Res. Commun. 305, 761 (2003).        [ Links ]

7. M.J. Davies. J. Photochem. Photobiol. B.: Biology. 63, 114 (2001).        [ Links ]

8. J.R. Brown, P. Shockley P. Lipid – Protein Interactions, Vol. 1, Wiley, New York, 1982.        [ Links ]

9. D.C. Carter, J.X. Ho. Adv. Protein Chem. 45, 153 (1994).        [ Links ]

10. E.L. Gelamo, M. Tabak. Spectrochim. Acta A. 56, 2255 (2000).        [ Links ]

11. E.L. Gelamo, C.H.T.P. Silva, M. Tabak. Biochim. Biophys. Acta. 1594, 84 (2002)        [ Links ]

12. H. Gharibi, S. Javadian, M. Hashemianzadeh. Colloid and Surfaces A: Physicochem. Eng. Aspects. 232, 77 (2004).        [ Links ]

13. A.A. Rafati, A.- K. Bordbar, H. Gharibi, M.-K. Amini, M.-A. Safarpur. Bull. Chem. Soc. Jpn.. 77, 1111 (2004).        [ Links ]

14. A. A. Rafati, H. Gharibi, H. Iloukhani. Phys. Chem. Lipids. 41, 509 (2003)        [ Links ]

15. X. Díaz, E. Abuin, E. Lissi. Photochem. Photobiol. A: Chemistry, 155, 157 (2003).        [ Links ]

16. Yan-Jun Hu, Yi Liu, Ru-Ming Zaho, Jia-Xin Dong, Song-Sheng Qu. J. Photochem. Photobiol. 179, 324 (2006).        [ Links ]

17. Zhao Hongwei, Ge Min, Zhang Zhaoxia, Wang Wenfeng, Wu Guoshong. Spectrochim. Acta A. 65, 811 (2006).        [ Links ]

18. C. Giulivi, M. Sarcansky, E. Rosenfeld, A. Boveris. Photochem. Photobiol. 52, 745 (1990).        [ Links ]

19. E. Otto, Chemomet. Intell. Lab. 39, 85 (1997).        [ Links ]

20. D.C. Carter, J.X. Ho, Adv. Protein Chem. 45, 153 (1994).        [ Links ]

21. L. Sepúlveda, E. Lissi, F. Quina. Adv. Colloid Interface Sci, 25, 1 (1986).        [ Links ]

 

e-mail: eabuin@lauca.usach.cl

 

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