STUDY OF THE DENATURATION OF HUMAN SERUM ALBUMIN BY SODIUM DODECYL SULFATE USING THE INTRINSIC FLUORESCENCE OF ALBUMIN

Transcription

1 Journal of Applied Spectroscopy, Vol. 76, No. 4, 2009 STUDY OF THE DENATURATION OF HUMAN SERUM ALBUMIN BY SODIUM DODECYL SULFATE USING THE INTRINSIC FLUORESCENCE OF ALBUMIN I. M. Vlasova * and A. M. Saletsky UDC ; An analysis of the intrinsic tryptophan fluorescence of human serum albumin (HSA) confirms that the denaturation of HSA by sodium dodecyl sulfate takes place in two stages for different ph levels: the first is the disintegration of globules and the second is the complete unfolding of the amino acid chain of HSA. At ph levels below the isoelectric point (pi 4.7) of HSA, denaturation proceeds through both stages, but when the ph is above pi, denaturation ceases in the first stage. Key words: fluorescence, tryptophan, human serum albumin, denaturation, sodium dodecyl sulfate Introduction. Human serum albumin (HSA, 66.4 kdalton, isoelectric point pi 4.7) is the globular protein in human blood plasma [1]. The unique ability of HSA to bind a wide range of organic and inorganic ligands determines one of the basic functions of this protein the transport of physiological metabolites. Research on the denaturation of this protein is extremely important in connection with its physiological functions in blood, since this can provide information on the native conformation of the protein and, therefore, on the extent to which its physiological properties are maintained when various external factors act on it. Denaturation refers to a significant change in the secondary and tertiary structure of the protein, i.e., a breakup or disordering of the system of noncovalent interactions, which do not affect its covalent structure. Effective denaturing agents include the ionic detergents, of which the detergent sodium dodecyl sulfate (SDS) is widely used in biochemistry: H 3 C (CH 2 ) 11 OSO 3 Na + [2 7]. The denaturation of HSA by SDS has been studied [3 5] using Raman scattering spectroscopy, laser correlation spectroscopy, and fluorescence analysis using eosine as a probe. In this paper we investigate the mechanism by which HSA molecules are denatured by different concentrations of SDS at different levels of ph by studying the intrinsic tryptophan fluorescence of HSA, a method widely used to evaluate the conformation state of protein molecules [8 13]. The only amino acid residual of tryptophan (Trp-214) in HSA lies in domain II. The fluorescence properties of tryptophan in the HSA molecule are extremely sensitive to realignments of the protein globule. Changes in the spectral characteristics of the fluorescence of HSA tryptophan (intensity and position of the peak in the fluorescence spectrum and the polarization of the fluorescence) can be used to establish the conformational state of the protein globules in solutions with denaturing agents, such as SDS. Materials and Methods. Solutions of HSA were prepared by dilution of the protein to a concentration of 5 μm in two different buffer systems: 0.1 M CH 3 COOH KOH (ph ) and 0.1 M KH 2 PO M NaOH (ph ). Different concentrations of SDS ( mm) were added to solutions of HSA at different ph ( ). The fluorescence studies were made with a Perkin Elmer LS55 spectrofluorimeter and the spectral-fluorescence characteristics of the prepared samples were studied at room temperature. The tryptophan fluorescence of the HSA was recorded over nm with excitation by λ = 295 nm light. The degree r of anisotropy in the tryptophan fluorescence of the HSA was calculated from the values of I and I at the peak of the protein fluorescence spectrum, where I and I are the fluorescence intensities measured through a polarizer with its electric field axis directed parallel to or perpendicular to the polarization of the exciting light. The fluorescence spectra were processed using the Perkin Elmer FL Winlab program. To whom correspondence should be addressed. M. V. Lomonosov Moscow State University, Leninskie Gory, Moscow, , Russia; Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 76, No. 4, pp , July August Original article submitted March 26, /09/ Springer Science+Business Media, Inc.

2 Results and Discussion. The protein macromolecules are capable of undergoing structural readjustments when acted on by various factors and agents. Two types of dynamic readjustments of this sort are possible: a transition of the protein from its native state to a denatured functionally inactive state (a denaturation transition) and a transition between two functionally active forms (a functional transition). We first examine the functional transition of HSA in the absence of SDS. The modifications in the intramolecular dynamics of HSA in solutions as the ph of the solutions vary within the range studied here ( ) when SDS is not present are functional transitions, i.e., they take place within the confines of the native structure of the protein. The dependence of the peak intensity (I fl max ) in the spectrum of the tryptophan fluorescence of HSA was determined as a function of the ph of solutions without SDS (Fig. 1a, inset). As the ph is raised from 3.5 to 6.0, I fl max rises; this indicates a change in the surroundings of the amino acid group of the tryptophan in the HSA molecules owing to small conformational readjustments. The dependence of the wavelength of the peak in the spectrum of the tryptophan fluorescence (λ fl max ) as a function of the ph of solutions without SDS was also obtained (Fig. 1b, inset). It is evident that besides an increase in the intensity of the tryptophan fluorescence of HSA with increasing ph there is a red shift in the fluorescence spectrum. A change in the ph (reduction from 6.0 to 3.5) leads to changes in the ionization state of the protein groups which trigger conformational changes in the protein within the native structure; this is the so-called N F transition. This conformational functional transition in HSA changes the surroundings of the protein chromophore the amino acid group of the tryptophan and, therefore, modifies the tryptophan fluorescence of the HSA. This N F transition of HSA as the ph is reduced from 6.0 to 3.5 is related to protonation of the carboxyl groups of the protein and is typical of all serum albumins [1]. The F-form of HSA, which exists for low ph solutions, is characterized by a looser structure with disordered hydrophobic regions. We now examine the denaturation transitions of HSA produced by different concentrations of SDS at different ph levels. For characterizing the conformational changes in the protein during denaturation, it is possible to use the parameters of the intrinsic protein (tryptophan) fluorescence: the intensity and position of the spectral peak of the steady state fluorescence, as well as the degree of anisotropy in the polarized fluorescence. Spectra of the tryptophan steady-state fluorescence of HSA in solutions containing different concentrations of SDS for different ph levels were obtained. Figure 1 shows the dependence of I fl max on the SDS concentration for different values of the ph. In the solutions with SDS the tryptophan fluorescence of HSA is quenched; this explained by denaturation of the protein, so that as the protein globule unfolds the chromophore tryptophan group becomes more accessible to water molecules which quench its fluorescence emission. Figure 1a shows that the stronger quenching of the intrinsic tryptophan fluorescence of HSA observed in solutions with SDS takes place at lower ph levels in solutions with equal concentrations of SDS. This behavior is indicative of an electrostatic mechanism for the interaction of the HSA with the SDS. SDS molecules in solution dissociate into positively charged sodium ions and dodecyl sulfate anions. It is these which interact with the HSA. On the whole, HSA molecules are positively charged for ph < pi of HSA. Thus, at low ph, the overall positively charged HSA macromolecules bond rapidly to the dodecyl sulfate anions, so that quenching of the tryptophan fluorescence of the HSA in solutions with SDS is strong compared to solutions which do not contain SDS. As the ph is raised, the overall positive charge of the HSA macromolecules decreases, and when ph > pi, the HSA molecules acquire an overall negative charge. Thus, for high ph (>pi), there is weaker bonding of the dodecyl sulfate anions to the overall negatively charged HSA molecules, which still do retain some positively charged segments, so that weak quenching of the tryptophan fluorescence of HSA is observed in solutions with SDS. The dependences of I fl max on [SDS] for different ph levels (Fig. 1a) obtained from the experimental data can explain the two-stage mechanism for denaturation of HSA in the presence of SDS. Here the behavior of the variation in I fl max with [SDS] differs, depending on whether the ph of the solution is higher or lower than pi for HSA. It is clear (Fig. 1a) that for ph < pi of HSA (ph ), denaturation of HSA in the presence of SDS is a stepwise, two-stage process. For concentrations [SDS] < 1 mm, the tryptophan fluorescence of the HSA is quenched, while for [SDS] = 1 2 mm I fl max is essentially constant. The first transition stage of denaturation of the protein takes place at these concentrations of SDS (up to 2 mm): the protein globules of the HSA are loosened, but complete unfolding has not yet taken place. As [SDS] is increased to 3 mm, further quenching of the tryptophan fluorescence of the HSA is observed. This indicates that the protein molecules change from a transitional, loose state to the second denaturation 537

3 Fig. 1. The intensity (a) and wavelength (b) of the peak in the tryptophan fluorescence spectrum (λ exc = 295 nm) of HSA (5 μm) as functions of the concentration of sodium dodecyl sulfate for ph 3.5 (1), 4.0 (2), 4.5 (3), 5.0 (4), 5.5 (5), and 6.0 (6). The insets show I fl (a) and λ fl (b) at the spectral maximum of the tryptophan fluorescence (λ exc = 295 nm) of HSA (5 μm) as functions of the ph of solutions without SDS. stage a completely unfolded state. When the SDS concentration is raised beyond 3 mm (to 7 mm), stronger quenching of the tryptophan fluorescence of HSA does not occur; this indicates complete denaturation of the HSA molecules. Further increases in [SDS] change nothing in this system, as is confirmed by data from [3 5]. The two-stage quenching of the intrinsic fluorescence of the HSA as [SDS] is increased for ph < pi of HSA is explained by a two-stage mechanism for its denaturation and subsequent conformational readjustments of the protein globule which lead to exposure of the tryptophan and increased access to the tryptophan by water molecules which quench its fluorescence. When ph > pi of HSA (ph ), denaturation of HSA by SDS proceeds slowly and (Fig. 2) the twostage process for denaturation of HSA in the presence of SDS only reaches the first stage within the range of [SDS] that was studied. When [SDS] is raised to 1 2 mm, the tryptophan fluorescence of HSA is quenched, i.e., denaturation loosening of the protein globules (the first state of denaturation) is observed, which leads to increased accessibility of the tryptophan of the HSA to water molecules which quench it. Higher concentrations of SDS (>2 mm) at these ph levels do not lead to the next, second stage of denaturation. The mechanism for denaturation of HSA by SDS was also studied by analyzing the position of λ fl max. Figure 1b shows the variation in λ fl max with [SDS] for different ph levels. When an initial concentration of SDS (

4 Fig. 2. The degree r of anisotropy in the tryptophan fluorescence (λ exc = 295 nm) of HSA (5 μm) as a function of [SDS] for ph 3.5 (1), 4.0 (2), 4.5 (3), 5.0 (4), 5.5 (5), 6.0 (6). mm) is added to the solution, there is a sharp blue shift in λ fl max (by 8 12 nm, depending on ph). Further addition of SDS (to mm) still leads to a small blue shift in the tryptophan fluorescence spectra of HSA. The blue shift in λ fl max in solutions with SDS is explained by a change in the nearest surroundings of the tryptophan chromophore group of the HSA owing to the denaturation readjustment of the protein globules and to the influence of the dodecyl sulfate anions that bond to the protein. The polarized tryptophan fluorescence of HSA was also studied. The degree (r) of anisotropy of the tryptophan fluorescence of HSA was determined as a function of [SDS] for different values of the ph (Fig. 2). r was calculated from the intensities I and I at the peak of the HSA fluorescence spectra. Changes in the polarization of the fluorescence are known to have two causes: first, rotational diffusion of fluorophores and second, nonradiative energy transfer between fluorophores. Given the choice of experimental conditions (highly dilute solutions of the protein [5 μm] were studied and the tryptophan fluorescence was excited at the far long wavelength edge [295 nm]), there is no contribution from the second (nonradiative energy transfer). Thus, for the given experimental conditions only rotational diffusion of the fluorophore (the tryptophan group of the HSA molecule) has a significant effect on the polarization of the fluorescence of the HSA tryptophan. This polarization of the fluorescence of the tryptophan group of the HSA molecule is, in general, caused both by rotation of the entire protein molecule (Brownian diffusional motion) and by rotation of domain II of the HSA, which contains the tryptophan group, and the rotation of the chromophore (tryptophan) itself relative to its nearest surroundings owing to dipole-orientational relaxation of the chromophore after excitation. The measurements of the polarized steady state fluorescence of HSA make it possible to analyze the rotation of the entire protein molecule, while the contribution from the rotation of the domain containing the tryptophan and the rotation of the tryptophan group with respect to the nearby surroundings are considered to be negligible. Figure 2 shows that r increases in the region up to 1 mm SDS for all values of the ph; this is indicative of the first stage of denaturation of the HSA loosening of the globules, which reduces the rotational diffusion coefficient for the HSA molecules and leads to an increase in r. Subsequent increases (above 1 mm) in [SDS] for ph > pi of HSA do not change r. This shows that denaturation has come to a halt in the first stage. The way r varies with [SDS] is different for ph < pi of HSA (Fig. 2): for [SDS] = 1 2 mm, r is essentially constant and for [SDS] = 2 4 mm r increases further, which indicates a decrease in the rotational diffusion coefficient of the HSA molecules, which in turn is explained by the increase, reported in [4], in the linear-longitudinal dimensions of the HSA molecules as the amino acid chain unfolds. Thus, the HSA molecules enter the second stage of denaturation the stage of complete unfolding. As [SDS] is raised above 4 mm (to 7 mm), r is essentially constant, which indicates that the HSA molecules have been completely denatured. Based on the two stage increase in r as 539

5 [SDS] is raised, we may conclude that for ph < pi of HSA, denaturation of HSA in the presence of SDS is a stepwise, two-stage process. As opposed to the behavior of I fl max and r, which reflects the multistage denaturation of HSA by SDS, λfl max gives evidence only of a denaturation process with no signs of its occurring in stages. This is explained by the fact that the variation in λ fl max is sensitive only to the change in the nearest surroundings of the tryptophan, while the variation in I fl max and r bears traces both of local readjustments in the nearest surroundings of the tryptophan surroundings and of more distant peripheral changes in the HSA globules. Tryptophan-214 (Trp(214)) is known to lie in the hydrophobic pocket of HSA, which consists of Lys(212) Ala(213) Trp(214) Ala(215) Val(216) Ala(217) Arg(218), and is located on the surface of subdomain 4 in domain II. This hydrophobic pocket, which is responsible for the bonding of organic anions, contains five nonpolar amino acid groups which form a region bounded by polar cation (for the ph levels used in the present work) groups of lateral chains of lysine-212 and arginine-218, positioned at the entrance to the hydrophobic pocket. These two amino acid groups (Lys(212) and Arg(218)), located at the entrance to the hydrophobic pocket, within which Trp(214) is found, are the first targets for the dodecyl sulfate anions. From this point of view, because of the interaction of Lys(212) and Arg(218) with dodecyl sulfate anions, when SDS is added in concentrations up to 1 mm major changes occur in the nearest surroundings of the Trp(214), which lead, on one hand, to a blue shift in λ fl max and, on the other, to partial opening of the hydrophobic pocket with Trp(214) and partial exposure of the Trp(214) (because of the loosening of the globule) to water molecules which quench its fluorescence; this produces the first large reduction in I fl max (the first stage of denaturation) and an increase in r. As [SDS] is raised (>1 2 mm), depending on the ph, the bonding of new dodecyl sulfate anions leads to: (1) complete denaturation (the second stage of denaturation) of the HSA for ph < pi of the protein, involving complete exposure of the Trp(214) to water molecules that quench it, which causes a second large increase in I fl max, i.e., the unfolding of the globules leading to reduced rotational diffusion of the HSA molecules and, therefore, to an increase in r (here the nearest surroundings of Trp(214) change little, so that the blue shift in λ fl max is very small); (2) the absence of a second HSA denaturation stage for ph > pi of the protein here only a slight change in the surroundings of the Trp(214) takes place which shows up as a small blue shift in λ fl max. Conclusion. The intrinsic tryptophan fluorescence of human serum albumin (both the native protein and denatured by sodium dodecyl sulfate) has been used to study the functional transitions of HSA within the confines of the native structure as the ph is varied in the absence of SDS and the denaturing transitions of HSA produced by SDS at different ph levels. The tryptophan fluorescence of HSA gives an indication of a functional transition (N F transition) of HSA within the confines of its native structure (in the absence of SDS) when the ph is varied from 6.0 to 3.5 that leads to a reduction in I fl max and a blue shift in λfl max. An analysis of the intrinsic tryptophan fluorescence of HSA (λ fl max, Ifl max, and r) has been used to study the denaturing transitions of HSA acted on by SDS at different ph levels. The two-stage changes in I fl max and r as the concentration of SDS is increased indicate that the denaturation of the protein occurs in two stages: the first stage involves loosening of the protein globules and the second, complete unfolding of the amino acid chain of the HSA. When ph > pi of HSA, denaturation passes through two stages, and when ph < pi of HSA, denaturation is slow and ceases in the first stage. The variation in the tryptophan fluorescence of HSA shows that denaturation by SDS is more efficient for ph of the HSA, and that as the ph is increased, the efficiency of denaturation of HSA by SDS falls off. Acknowledgement. This work was supported by the Russian Foundation for Basic Research (grant No ). REFERENCES 1. Yu. A. Gryzunov and G. E. Dobretsov, Human Serum Albumin in Clinical Medicine [in Russian], IRIUS, Moscow (1994). 2. L. A. Osterman, Protein and Nucleic Acid Research Techniques [in Russian], MTsNMO, Moscow (2002). 3. I. M. Vlasova, D. V. Polyansky, and A. M. Saletsky, Laser Phys. Lett., 4, No. 12, (2007). 4. A. N. Baranov, I. M. Vlasova, V. E. Mikrin, and A. M. Saletskii, Zh. Prikl. Spektr., 71, (2004). 5. I. M. Vlasova, A. Yu. Zemlyanskii, and A. M. Saletskii, Zh. Prikl. Spektr., 73, (2004). 540

6 6. P. Dutta, P. Sen, A. Halder, S. Mukherjee, S. Sen, and K. Bhattacharyya, Chem. Phys. Lett., 377, (2003). 7. E. L. Gelamo, C. H. T. P. Silva, H. Imasato, and M. Tabak, Biochim. Biophys. Acta, 1594, (2002). 8. L. V. Levshin and A. M. Saletskii, Optical Methods for Studying Molecular Systems. Part 1. Molecular Spectroscopy [in Russian], Izd-vo MGU, Moscow (1994). 9. Yu. A. Vladimirov, Protein Photochemistry and Luminescence [in Russian], Nauka, Moscow (1965). 10. E. A. Permyakov, The Technique of Intrinsic Protein Luminescence [in Russian], Nauka, Moscow (2003). 11. A. P. Demchenko, Luminescence and Dynamics of Protein Structure [in Russian], Naukova dumka, Kiev (1988) 12. X. Diaz, E. Abuin, and E. Lissi, J. Photochem. Photobiol. A: Chem., 155, (2003). 13. A. Brahma, C. Mandal, and D. Bhattacharyya, Biochim. Biophys. Acta, 1751, (2005). 541