Journal of Photochemistry and Photobiology B: Biology 141 (2014) 262–268
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Chemical changes in bovine serum albumin photoinduced by pterin Andrés H. Thomas a, Beatriz N. Zurbano a, Carolina Lorente a, Javier Santos b, Ernesto A. Roman b, M. Laura Dántola a,⇑ a Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, CCT La Plata-CONICET, Casilla de Correo 16, Sucursal 4, 1900 La Plata, Argentina b Instituto de Química y Físico-Químicas Biológicas (IQUIFIB), Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina
a r t i c l e
i n f o
Article history: Received 17 July 2014 Received in revised form 23 September 2014 Accepted 14 October 2014 Available online 19 October 2014
a b s t r a c t The exposure to UV-A radiation of bovine serum albumin (BSA) in aerated aqueous solution in the presence of pterin (Ptr), results in chemical and conformational modifications of the protein. Ptr belongs to a family of heterocyclic compounds that are well-known type I (electron-transfer) and type II (singlet oxygen) photosensitizers. The evolution of the photosensitized processes was followed by UV/vis spectrophotometry and fluorescence spectroscopy indicating that tryptophan (Trp) and tyrosine (Tyr) residues were affected. Additionally, conformational changes were evaluated by electrophoresis (SDS-PAGE) and size exclusion chromatography coupled with dynamic light scattering detection, showing that BSA undergoes dimerization, via the formation of Tyr radicals. The degradation of Trp residues takes place faster than the oligomerization of the protein. The photosensitized process is initiated by an electron transfer from BSA to the triplet excited stated of Ptr, being a purely dynamic mechanism. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Proteins, due to their relatively high abundance, their ability to bind chromophores, and the reactivity of particular amino acid residues, are one of the preferential targets of the photosensitized damaging effects of UV radiation on biological systems [1]. Currently, it is accepted that the photosensitization of proteins occurs mainly through the reactions of singlet oxygen (1O2) (type II mechanism) with tryptophan (Trp), tyrosine (Tyr), histidine, methionine and cysteine side-chains [2]. Especially, serum albumins are the most abundant plasma proteins, and their main biological function is the transport of a wide variety of molecules [3]. In addition, albumin is present in the human skin [4], where there is an autocrine synthesis and regulation [5]. It has been reported that in patients affected by vitiligo, epidermal albumin oxidation takes place [6]. Bovine serum albumin (BSA) has been commonly used as a model for the human protein due to its high homology with the human serum albumin (HSA) in the amino acidic sequence [7]. Pterins, heterocyclic compounds, are present in living systems mainly at three different redox states: fully oxidized (or aromatic) pterins, dihydro and tetrahydro derivatives. 5,6,7,8-Tetrahydrobiopterin (H4Bip) is an essential cofactor in the hydroxylation of the aromatic amino acids [8] and participates in the regulation of ⇑ Corresponding author at: C. C. 16, Sucursal 4, B1904DPI La Plata, Argentina. Tel.: +54 221 4257430x153; fax: +54 221 4254642. E-mail address: [email protected] (M. Laura Dántola). http://dx.doi.org/10.1016/j.jphotobiol.2014.10.007 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
melanin biosynthesis. Normal metabolism of H4Bip is altered in the skin in pathological conditions, such as vitiligo [9,10]. Vitiligo is a skin disorder characterized by the acquired loss of constitutional pigmentation, manifesting as white macules and patches [11], and the accumulation of reduced and oxidized pterins [12]. In diseased skin cells micromolar concentration of pterins have been determined, which is much higher than concentration found in healthy cells; e.g. in human keratinocytes and cells cultures from suction blister roofs the concentration of total biopterin was determined to be in the range 4–93 lM (41–950 pmol/mg of protein) depending on the cell type [13]. Under UV-A excitation (315–400 nm), unconjugated oxidized pterins can fluoresce, undergo photooxidation, and generate reactive oxygen species (ROS) [14]. In the presence of oxygen, pterin (Ptr) [14], the parent and unsubstituted compound of oxidized pterins, acts as a photosensitizer through both type I (electron transfer or hydrogen abstraction) [15] and type II (production of 1O2) [16] mechanisms. Moreover, Ptr photoinduces DNA damage and the oxidation of purine nucleotides [17–20]. Therefore, taking into account the accumulation of pterins in the human skin under pathological conditions described in the previous paragraph, the photochemistry and, especially their photosensitizing properties of these compounds are of particular interest for the study of skin disease. Two recent studies have suggested that degradation of proteins photosensitized by Ptr involves a type I mechanism [21,22]. The process is initiated by an electron transfer reaction from the protein (P) to the triplet excited state of Ptr (3Ptr*) (reaction 1),
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yielding the corresponding pair of radical ions (pterin radical anion (Ptr) and the protein radical cation (P+)). P+ leads to the irreversible damage and degradation of the protein (reaction 2), whereas Ptr, under aerobic conditions, transfers one electron to O2 regenerating Ptr and forming superoxide anion (O 2 ) (reaction 3), which, in turn, is converted into hydrogen peroxide (H2O2) by spontaneous disproportionation (reaction 4) [22]. Moreover, it was demonstrated that Ptr is able to photosensitize the oxidation of free Trp and Tyr in aqueous solution [23,24]. In particular, the study of the photosensitization of Tyr revealed that dimers of Tyr, or dityrosine are formed [24]. This amino acid plays a key role in polymerization and cross-linking of proteins via reactions initiated by Tyr radicals [25]. The accumulation of pterins at micromolar concentrations [9,12] in the skin of patients affected by vitiligo, in which the protection against UV radiation is lost, makes the investigation of the damage of proteins photoinduced by pterins important from a biomedical point of view. 3
Ptr þ P ! Ptr þ Pþ H2 O=O2
ð1Þ
Pþ ! Products
ð2Þ
Ptr þ O2 ! Ptr þ O 2
ð3Þ
2Hþ þ 2O 2 ! H 2 O2 þ O2
ð4Þ
The main aim of this work is to investigate the chemical changes that proteins undergo upon UV exposure in the presence of pterins. We have performed the experiments in aqueous solutions under UV-A irradiation, using BSA as a model protein and Ptr as a photosensitizer. The experiments were carried out at concentrations of Ptr of the same order of magnitude found in human skin affected by vitiligo [13]. Most of the experiments were performed at pH 6.0 ± 0.1, so that more than 95% of the Ptr (pKa = 7.9) [26] was in the acid form, the predominant form at physiological pH. The photochemical reactions were followed by UV/visible spectrophotometry, fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) coupled with dynamic light scattering detection. 2. Materials and methods
263
Tris–HCl (pH 7.0) and 50 mM NaCl. Twenty five milliliters of this solution was loaded in a SEPHADEX G-100 column and different fractions were collected. An SDS-PAGE was performed to evaluate which fractions contained the monomeric fraction of BSA. These tubes were used without change of buffer or concentration procedures. 2.3. Steady-state irradiation The continuous photolysis of the solutions containing Ptr and BSA were carried out irradiating in quartz cells (0.4 cm optical path length) at different temperatures using a temperature controller (LFI-3751 Wavelength Electronics). Two radiation sources were employed: (I) Rayonet RPR 3500 lamps (Southern N.E. Ultraviolet Co.) with emission centered at 350 nm (bandwidth (fwhm) 20 nm) and (II) a 450 W Xe lamp with a monochromator Horiba Jobin Yvon FL-1004 (single-grating spectrometer, 330 nm blaze grating). Photolysis experiments were performed in air equilibrated aqueous solutions. Aberchrome 540 (Aberchromics Ltd.) was used as an actinometer for the measurement of the incident photon flux density (q0;V n;p ) at the excitation wavelength, which is the amount of incident photons per time interval (q0n;p ) and divided by the volume (V) of the sample [27]. Aberchrome 540 is the anhydride form of the (E)-a-(2,5dimethyl-3-furylethylidene) (isopropylidene) succinic acid which, under irradiation in the spectral range 316–366 nm leads to a cyclized form. The reverse reaction to ring opening is induced by visible light. The method for the determination of q0;V n;p has been described in detail elsewhere [28]. The value of q0;V n;p measured for the radiation source I was 2.4 (±0.2) 105 Einstein L1 s1; the value of q0;V n;p measured for the radiation source II was 6.7 (±0.6) 106 Einstein L1 s1. Values of the absorbed photon flux density (qa;V n;p ) were calculated from q0;V n;p according to the Lambert–Beer law: A 0;V qa;V n;p ¼ qn;p ð1 10 Þ
ð5Þ
where A is the absorbance of the reactant at the excitation wavelength. 2.4. Analysis of irradiated solutions
2.1. General Pterin (Ptr, >99%) and bovine serum albumin (BSA, fatty acids free) were purchased from Schircks Laboratories and Sigma, respectively. Ptr was used without further purification. Sodium dodecyl sulfate (SDS, 99%), glycerol, 2-mercaptoethanol, bromophenol blue, glycine (Gly, >99% titration), ammonium persulphate (>98%) and N,N,N0 ,N0 -tetramethylethylene-diamine (TEMED, 99%) were provided by Sigma. Methanol was provided by Laboratorios Cicarelli. Acetic acid was provided by Anedra. Coomassie Brilliant Blue G was provided by Fluka. Acrylamide and N0 N0 -methylene-bis-acrylamide and trishydroxymethylaminomethane (Tris) were provided by Genbiotech. The pH measurements were performed using a pH-meter PHM220 (Radiometer Copenhagen) combined with a pH electrode pHC2011-8 (Radiometer Analytical). The pH of the aqueous solutions was adjusted by adding drops of HCl and NaOH solutions from a micropipette. The concentration of the acid and the base used for this purpose ranged from 0.1 M to 2 M. 2.2. BSA purification BSA was further purified to get rid of chemically altered protein and covalently bonded oligomeric species. Briefly, BSA was prepared in 40 mg/mL concentration in buffer containing 10 mM
2.4.1. UV/vis analysis Electronic absorption spectra were recorded on a Shimadzu UV1800 spectrophotometer. Measurements were made using quartz cells of 0.4 or 1 cm optical pathlength. The absorption spectra of the solutions were recorded at regular intervals of irradiation time. 2.4.2. Fluorescence spectroscopy Fluorescence measurements were performed on air-equilibrated aqueous solutions of Ptr and BSA using a Single-PhotonCounting equipment FL3 TCSPC-SP (Horiba Jobin Yvon). The equipment has been previously described in detail [29]. Briefly, in steady-state measurements the sample solution in a quartz cell was irradiated with a 450 W Xenon source through an excitation monochromator. The fluorescence, after passing through an emission monochromator, was registered at 90° with respect to the incident beam using a room-temperature R928P detector. The emission measurements were performed at 25 °C, using the temperature controller mentioned above. The total fluorescence intensities (IF) were calculated by integration of the fluorescence band centered at ca. 350 nm. The Trp fluorescence from BSA was corrected for inner filter effect. The fluorescence intensity was approximately corrected using the following equation [30]
IFcorr ¼ IFobs 10ðAexcþAemÞ=2
ð6Þ
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2.4.3. Electrophoretic analysis Protein damage was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples of protein solutions were boiled for 5 minutes in a 0.06 M Tris–HCl (pH 6.8) solution containing 2% SDS, 10% glycerol, 1% 2-mercaptoethanol (as reducing agent) and 0.02% bromophenol blue (as a tracking dye). Acrylamide (4%) stacking gel, 10% acrylamide resolving gel and running buffer containing 25 mM Tris, 192 mM Gly and 0.1% SDS, pH 8.3 were used. Electrophoresis was performed at 30 mA during 100 min. Gels were stained with 0.1% Coomassie Brilliant Blue G and destained with a solution of methanol and acetic acid during 2 h. The electrophoretic bands were quantified by scanning photodensitometry using the ImageJ 1.45s software. 2.4.4. Size exclusion chromatography (SEC) coupled with a dynamic light scattering Hydrodynamic behavior and oligomer formation of BSA was monitored by light scattering coupled to chromatography on a SEC–HPLC system equipped with a 280 nm UV detector coupled. Briefly, a Superdex S-200 HR 10/30 columns (Pharmacia Biotech, Sweden) coupled to a MiniDawin static light scattering detector (Wyatt) and an absorbance detector (Jasco UV 2075plus) was equilibrated at room temperature in buffer 20 mM Tris–HCl, 100 mM NaCl, pH 7.0. The scattered light was collected at three different angles and molar mass was obtained using ASTRA Software. In parallel, the scattered light at 90° was directed through an optical fiber to a Quasi Elastic Light Scattering module (Wyatt). Here, the time-dependent fluctuations in the intensity of the scattered light were registered and quantified via a second-order correlation function and the diffusion coefficient was obtained. This type of chromatographic resin lets us examine the presence of oligomers and soluble aggregates in the samples. The flow rate was 0.4 mL min1 and the injection volume was 100–200 lL. Samples were centrifuged at 14,000 rpm before loading onto the column. 2.5. Quantum yield determinations The quantum yield of BSA consumption (U-BSA) was determined under different experimental conditions. Values were obtained using Eq. (7).
UBSA ¼
ðd½BSA=dtÞ0 qa;V n;p
extinction coefficients of aromatic amino acids, Trp generally dominates the absorption, fluorescence and phosphorescence spectra of proteins that also contain either of the other two aromatic amino acids [31]. BSA contains Trp (Trp 214 and Trp 135) and Tyr residues which act as intrinsic fluorescence probes. When excitation wavelength of 280 nm is used, both Tyr and Trp residues are excited; in contrast, when wavelength of 295 nm is used, only the Trp residues are excited. The comparison of fluorescence spectra obtained by excitation at 280 and 295 nm showed that there are no changes in the shape and emission maximum of both spectra (data not shown), thus indicating that emission fluorescence comes mainly from Trp in BSA, under this experimental conditions. To determine if the photosensitization process modified the Trp residues of BSA, air-equilibrated aqueous solutions containing BSA and Ptr were irradiated in quartz cells during different periods of time at 37 °C, pH 6.0 ± 0.1 (350 nm, radiation source II, Materials and methods). Under these experimental conditions only Ptr was excited [22]. The corrected fluorescence spectra obtained by excitation at 280 nm of the irradiated samples were recorded between 295 and 400 nm. Although Ptr absorbs at the excitation wavelength, its emission is red shifted with respect to that of Trp, (maximum at 439 nm, [14]), so that, under our experimental conditions, the typical emission band of Trp could be adequately registered. The Trp fluorescence decreased as a function of irradiation time (Fig. 1), thus suggesting that Trp residues were damaged during irradiation. To confirm that the reaction observed was a photosensitized process, BSA aqueous solutions were exposed to UV-A radiation during different periods of time in the absence of Ptr. No changes in the total fluorescence intensity were detected, even after more than 60 min of irradiation, thus excluding the possibility that spurious effects of direct light absorption by the protein could affect its fluorescence (Fig. 1). In a recent work, we demonstrated that when air-equilibrated aqueous solutions containing BSA and Ptr were irradiated with UV-A radiation (350 nm, radiation source I, pH = 6.0 ± 0.1), the concentration of the BSA, estimated by means of SDS-PAGE, decreased as a function of irradiation time [22]. Therefore, irradiated air-equilibrated solutions containing BSA and Ptr were analyzed simultaneously by SDS-PAGE and fluorescence spectroscopy. The comparison of the two analyzed parameters showed that the rate of the decrease in total fluorescence intensity (related to Trp damage) was higher than the decrease of the BSA concentration (determined from the intensity of the BSA band) (Fig. 2).
1.0
ð7Þ
whith (d[BSA]/dt)0: initial rate of BSA degradation, estimated as the Trp residues consumption (calculated using fluorescence analysis) a;V and qn;p : absorbed photon flux density by Ptr (Eq. (5)). 3. Results and discussion
0.8
Emission (10 7 counts)
where IFcorr and IFobs were the corrected and observed fluorescence intensities, respectively. Aexc and Aem were the absorbance of the system at excitation and emission wavelengths, respectively.
I F/ IF0
264
0.6
3.1. Effect on the tryptophan residues Taking into account the photosensitization of Trp by Ptr previously reported [23], and that Trp is particularly susceptible to a variety of oxidizing agents [2], it is straightforward to expect that Trp residues of BSA would be affected when this protein is exposed to UV-A radiation in the presence of Ptr. Therefore, the first aim of this work was to investigate the degradation of Trp in BSA, taking advantage of the fluorescence properties of this amino acid. Fluorescence of proteins comes mainly from the contributions of the three aromatic amino acids: Trp, Tyr and phenylalanine (Phe). However, as a result of the spectral distribution and relative
0.4
3 2 1 0
300 320 340 360 380
λ (nm)
0
10
20
30
40
50
60
time (min) Fig. 1. Evolution of the relative total fluorescence intensity (IF/I0F ) of Trp residues in an aqueous solution of BSA (15.0 lM) in the absence (s) and in the presence of Ptr (4.5 lM) (r) as a function of the irradiation time (emission temperature = 25 °C, kexc = 280 nm). Inset: corrected fluorescence spectra.
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3.2. New insights on the mechanism of photosensitization
3.3. Oligomerization of BSA In a recent work performed with free Tyr in aqueous solutions, we have demonstrated that the excitation of Ptr is followed by an electron transfer from the amino acid to the Ptr triplet excited state, leading to the formation of the corresponding ion radicals, Ptr and Tyr+ [24]. The latter undergoes oxygenation and dimerization to yield the so-called dimers of Tyr (denoted Tyr2), which are involved in cross-linking of proteins. Moreover, the dimerization of the peptide a-melanocyte-stimulating hormone (a-MSH) and the formation of Tyr2 were observed when the peptide was exposed to UV-A radiation in the presence of Ptr
Φ -BSA (10 −3)
10.0
1.1 ± 0.1
25.0
2.0 ± 0.2
40.0
2.5 ± 0.2
0
0.9
IF /IF
The mechanism of photodamage of BSA by Ptr involves an electron transfer process (vide supra) [22]. However, it is not clear how such a process takes place. BSA is able to bind Ptr, but the corresponding binding constant for the ground state complex formation is relatively low ((1.27 ± 0.03) 103 M1 at 25 °C) [22]. Therefore, the mechanism of photosensitization might be initiated by a purely dynamic process with the participation of free Ptr or by a static process in which the electron transfer would occur directly from the oxidizable amino acids, like Trp, to the protein-associated form of Ptr. To investigate this point, air-equilibrated aqueous solutions containing Ptr and BSA were irradiated at 350 nm (radiation source II, pH = 6.0 ± 0.1) for different periods of times at various temperatures. Higher temperature results in faster diffusion and hence larger amounts of molecular collision. Therefore, if a dynamic mechanism is responsible for the photosensitization of BSA by Ptr, we should observed more damage to Trp when the temperature increases. In contrast, if the electron transfer involves associated molecules, high temperature tends to disrupt the ground state complex, and, consequently, it should be observed a decrease in the photosensitized process with raising temperature. Under our experimental conditions, it was observed that the rate of Trp consumption, estimated from the evolution of its fluorescence intensity, was higher when the temperature increased (Fig. 3). This result confirmed that the photodamage to BSA by Ptr takes place via a purely dynamic mechanism.
T (°C) 1.0
0.8
0.7
0.6
0
3
6
9
18
21
[32]. To investigate if the photosensitization of BSA by Ptr leads to the generation of Tyr2, fluorescence experiments were carried out, taking advantage of its particular emission features. The absorption and emission spectra of Tyr2 are red shifted with respect to those of Tyr [33]. Therefore solutions containing Ptr and BSA were exposed to UVA radiation (350 nm, radiation source II, pH = 6.0 ± 0.1) and the emission spectra under excitation at 310 nm were registered at various irradiation times. For a given irradiation time, the spectrum of the solution before irradiation, that means the fluorescence of Ptr and Trp, was subtracted from the spectrum registered. Results show that an emission band with a maximum coinciding with that expected for the Tyr2 [34] was present in the irradiated solutions and that the intensity increased as a function of irradiation time (Fig. 4), which is strong evidence in favor of the photodimerization of Tyr residues of BSA induced by Ptr.
10
60
[BSA]/[BSA]0
40
IF /IF0
Emission (10 counts)
0.6
30
5
20
5
0.4 0.2 0.0
0
0
5
10
10
20
20
30
30
50
40
50
time (min) Fig. 2. Evolution of the relative total fluorescence intensity (IF/IF0) and the relative BSA concentration ([BSA]/[BSA]0), estimated by means of SDS-PAGE, of an aqueous solution of BSA (5.6 lM) in the presence of Ptr (50.0 lM) as a function of the irradiation time. Inset: electrophoretic bands (picture) of BSA as a function of irradiation time. The irradiation time (min) appears above each band. Emission temperature = 25 °C, kexc = 280 nm.
0 IF (10 5 counts)
IF /IF0 - [BSA]/[BSA]0
15
Fig. 3. Evolution of the relative total fluorescence intensity (IF/I0F ) of an aqueous solution of BSA (3.0 lM) and Ptr (22.5 lM) as a function of the irradiation time (min) at various irradiation temperatures. Emission temperature: 25 °C, kexc = 280 nm. Table: quantum yield of BSA consumption (U-BSA) at each temperature.
1.0 0.8
12
time (min)
-5
-10
340
360
8 6 4 2 0
0
380
10
20
30 40 50 time (min)
400
420
60
440
460
λ (nm) Fig. 4. Corrected fluorescence spectra (kexc = 310 nm) of an aqueous solution of BSA (15.0 lM) and Ptr (4.5 lM) irradiated at 350 nm at 37 °C. The irradiation time (min) appears above each spectrum. For each time, the spectrum of the solution before irradiation was subtracted. Inset: increase of the fluorescence intensity at 402 nm as a function of irradiation time.
A.H. Thomas et al. / Journal of Photochemistry and Photobiology B: Biology 141 (2014) 262–268
0
10
20
30
50
BSA photoproducts
BSA
1.0
relative concentration
266
BSA monomer, [Ptr] = 0 μM BSA dimer, [Ptr] = 0 μM BSA monomer, [Ptr] = 75 μM BSA dimer, [Ptr] = 75 μM
0.8
0.2
Fig. 5. SDS-PAGE analysis of BSA (5 lM) solutions irradiated in the presence of Ptr (50 lM) at 350 nm at room temperature. The irradiation time (min) appears above each band.
Taking into account the fluorescence results previously shown, and in order to confirm that the photosensitization of BSA by Ptr produces dimers or oligomers, aqueous solutions containing purified BSA and Ptr were exposed to UV-A radiation (350 nm, radiation source I) during different periods of time and the treated samples were analyzed by SDS-PAGE. In this case, a larger sample volume was used to improve the detection of products. The electrophoretic patterns showed that simultaneously with the decrease of BSA optical density, photoproducts with molecular weight higher than BSA were formed (Fig. 5).
Absorbance (relative scale)
BSA monomer
(a)
1.0
0.8
0.6 BSA photoproduct
0.4
0.2
(b)
1.0
2.0 0.8 1.5 0.6 1.0
5
0.4
Molar Mass ( 10 Da)
Normalized light scattering (90 degrees)
0.0
0.0
0
50
100
150
200
250
time (min) Fig. 7. Evolution of the relative concentration of BSA monomer and BSA dimer in the absence and in the presence of Ptr as a function of irradiation time. [BSA] = 150 lM, [Ptr] = 75 lM, irradiation source I (350 nm), pH = 6.0 ± 0.1.
To further investigate this point the irradiated samples were analyzed by size-exclusion chromatography coupled with light scattering and absorbance detectors. In Fig. 6a it can be seen that the peak corresponding to BSA decreased as a function of irradiation time, while new absorbance product appeared at lower retention time (tR), indicating that the molecular weight of this photoproduct is higher than that of the BSA. In order to determine the molecular weight of the photoproducts, the scattering profiles obtained for different irradiation times were analyzed. As shown in Fig. 6b, at time zero, around tR 31 min there is a peak of scattered light corresponding to an specie of 62 (+/3) kDa corresponding to the monomeric BSA, being the main specie in solution. When the irradiation time increased, besides the monomeric species, another peaks started to contribute to the scattered light (tR 28 min), this peak corresponding to an specie of 132 (+/11) kDa, indicating that this contribution is mainly due to an increase in the dimeric species. As it can be seen, as the dimeric peak increased, the monomeric peak decreased suggesting that oligomer formation is due to an interaction of monomers. As a control, aqueous solutions of BSA were exposed to UV-A radiation in the absence of Ptr and analyzed by light scattering. In Fig. 7 it can be observed that the loss of monomer and, appearance of dimer is much greater in BSA solutions when Ptr is present if we compare with BSA solution irradiated in the absence of photosensitizer. The relative concentration of BSA monomer and BSA dimer were calculated as [BSA monomer]/([BSA monomer]+[BSA dimer]) and [BSA dimer]/([BSA monomer]+[BSA dimer]), respectively. These results confirm that the dimerization due to a photosensitization process.
0.5
0.2
4. Conclusion
0.0
0.0 20
22
24
26
28 30 tR (min)
32
34
Fig. 6. Chromatographic analysis of a solution of BSA (150 lM) irradiated in the presence of Ptr (75.0 lM) (irradiation: source I, pH = 6.0 ± 0.1) (a) chromatograms obtained using an absorbance detector (k = 280 nm), irradiation times: 0, 1 h, 3 h and 4 h. (b) Scattered light at 0 (dotted line) and 240 min (solid line) irradiation time. Black and white circles are the molecular mass of the main peak for the 0 and 240 min irradiation time, respectively. Black triangles are the molecular weights of the photoproducts.
The photosensitization of bovine serum albumin (BSA) by pterin (Ptr), the parent compound of oxidized pterins, in aqueous solution under UV-A irradiation was investigated. The photoinduced oxidation of BSA is a purely dynamic process and results in the oxidation of the protein in at least two different and specific sites: tryptophan (Trp) and tyrosine (Tyr) residues. The Trp degradation results in a fast decrease of the fluorescence intensity, which is observed before structural changes in SDS-PAGE analysis. In addition, Tyr residues contribute to dimerization of the protein, since dimers of Tyr residues were detected clearly by fluorescence. The
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size exclusion chromatography analysis indicated unequivocally that Ptr photoinduces cross-linking of BSA, most likely due to dimerization between Tyr residues. Taking into account that oxidized pterins are present in human skin under pathological conditions in which the protection against UV radiation fails due to the lack of melanin and that albumin is one of the most abundant proteins present in skin, the results shown in this work, using BSA and Ptr as model compounds, are relevant from a biomedical point of view and can have further ramification. In particular, the photosensitized reactions described in our studies might significantly contribute to the protein damage reported in various skin diseases. 5. Abbreviations A Aem Aexc qa;V n;p BSA IFcorr Tyr2 Gly HSA H2O2 q0;V n;p q0n;p TEMED IFobs Phe P P+ Ptr Ptr
U-BSA ROS tR 1 O2 SEC SDS SDSPAGE O2 H4Bip IF 3 Ptr* Tris Trp Tyr V a-MSH
absorbance absorbance of the system at emission wavelength absorbance of the system at excitation wavelength absorbed photon flux density bovine serum albumin corrected fluorescence intensity dimers of Tyr glycine human serum albumin hydrogen peroxide incident photon flux densitity incident photons per time interval N,N,N0 ,N0 -tetramethylethylene-diamine observed fluorescence intensity phenylalanine protein protein radical cation pterin pterin radical anion quantum yield of BSA consumption reactive oxygen species retention time singlet oxygen size exclusion chromatography sodium dodecyl sulfate sodium dodecyl sulfate polyacrylamide gel electrophoresis superoxide anion 5,6,7,8-tetrahydrobiopterin total fluorescence intensities triplet excited state of Ptr trishydroxymethylaminomethane tryptophan tyrosine volume a-melanocyte-stimulating hormone
Acknowledgements The present work was partially supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET; Grant PIP 11220090100425), Agencia de Promoción Científica y Tecnológica (ANPCyT; Grants PICT 2012-0508), and Universidad Nacional de La Plata (UNLP; Grant X586). B. N. Z. thanks CONICET for doctoral research fellowship. A. H. T., C. L., E. A. R, J. S. and M. L. D are research members of CONICET. The authors thank Dr. Catalá Ángel for his crucial contributions in SDS-PAGE analysis.
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Raman Spectrosc. 35 (2004) 125–130. [7] T. Peters, All about Albumin Proteins, first ed., Academic press, New York, 1996 (Chapter 3). [8] I. Ziegler, Production of pteridines during hematopoiesis and T-lymphocyte proliferation: potential participation in the control of cytokine signal transmission, Med. Res. Rev. 10 (1990) 95–114. [9] K.U. Schallreuter, J.M. Wood, M.R. Pittelkow, M. Gütlich, K.R. Lemke, ; W. Rödl, N.N. Swanson, K. Hitzemann, I. Ziegler, Regulation of melanin biosynthesis in the human epidermis by tetrahydrobiopterin, Science 263 (1994) 1444–1446. [10] C.A. Nichol, G.K. Smith, D.S. Duch, Biosynthesis and metabolism of tetrahydrobiopterin and molybdopterin, Annu. Rev. Biochem. 54 (1985) 729–764. [11] S.J. Glassman, Vitiligo, reactive oxygen species and T-cells, Clin. Sci. 120 (2011) 99–120. [12] H. Rokos, W.D. Beazley, K.U. Schallreuter, Oxidative stress in vitiligo: photooxidation of pterins produces H2O2 and pterin-6-carboxylic acid, Biochem. Biophys. Res. Commun. 292 (2002) 805–811. [13] K.U. Schallreuter, J. Moore, J.M. Wood, W.D. Beazley, E.M.J. Peters, L.K. Marles, S.C. Behrens-Williams, R. Dummer, N. Blau, B.J. Thöny, Epidermal H2O2 accumulation alters tetrahydrobiopterin (6BH4) recycling in vitiligo: identification of a general mechanism in regulation of all 6BH4-dependent processes?, J Invest. Dermatol. 116 (2001) 167–174. [14] C. Lorente, A.H. Thomas, Photophysics and photochemistry of pterins in aqueous solution, Acc. Chem. Res. 39 (2006) 395–402. [15] C. Lorente, G. Petroselli, M.L. Dántola, E. Oliveros, A.H. Thomas, Electron transfer initiated reactions photoinduced by pterins, Pteridines 22 (2011) 111–119. [16] E. Oliveros, M.L. Dántola, M. Vignoni, A.H. Thomas, C. Lorente, Production and quenching of reactive oxygen species by pterin derivatives, an intriguing class of biomolecules, Pure Appl. Chem. 83 (2011) 801–811. [17] K. Ito, S. Kawanishi, Photoinduced hydroxylation of deoxyguanosine in DNA by pterins: sequence specificity and mechanism, Biochemistry 36 (1997) 1774– 1781. [18] T. Offer, B.N. Ames, S.W. Bailey, E.A. Sabens, M. Nozawa, J.E. Ayling, 5Methyltetrahydrofolate inhibits photosensitization reactions and strand breaks in DNA, FASEBJ 21 (2007) 2101–2107. [19] G. Petroselli, M.L. Dántola, F.M. Cabrerizo, A.L. Capparelli, C. Lorente, E. Oliveros, A.H. Thomas, Oxidation of 20 -deoxyguanosine50 -monophosphate photoinduced by pterin: type I versus type II mechanism, J. Am. Chem. Soc. 130 (2008) 3001–3011. [20] G. Petroselli, R. Erra-Balsells, F.M. Cabrerizo, C. Lorente, A.L. Capparelli, A.M. Braun, E. Oliveros, A.H. Thomas, Photosensitization of 20 -deoxyadenosine-50 monophosphate by pterin, Org. Biomol. Chem. 5 (2007) 2792–2799. [21] M.L. Dántola, A.D. Gojanovich, A.H. Thomas, Inactivation of tyrosinase photoinduced by pterin, Biochem. Biophys. Res. Commun. 424 (2012) 568– 572. [22] A.H. Thomas, C. Lorente, K. Roitman, M.M. Morales, M.L. Dántola, Photosensitization of bovine serum albumin by pterin: a mechanistic study, J. Photochem. Photobiol. B. 120 (2013) 52–58. [23] A.H. Thomas, M.P. Serrano, V. Rahal, P. Vicendo, C. Claparols, E. Oliveros, C. Lorente, Tryptophan oxidation photosensitizided by pterin, Free Radic. Biol. Med. 63 (2013) 467–475. [24] C. Castaño, M.L. Dántola, E. Oliveros, A.H. Thomas, C. Lorente, Oxidation of tyrosine photoinduced by pterin in aqueous solution, Photochem. Photobiol. 89 (2013) 1448–1455. [25] D.A. Malencik, S.R. Anderson, Dityrosine formation in calmodulin: crosslinking and polymerization catalyzed by Arthromyces peroxidase, Biochemistry 35 (1996) 4375–4386. [26] V.D. Monópoli, A.H. Thomas, A.L. Capparelli, Kinetics and equilibrium study of nickel(II) complexation by pterin and 6-carboxypterin, Int. J. Chem. Kinet. 32 (2000) 231–237. [27] S.E. Braslavsky, Glossary of terms used in photochemistry, (IUPAC recommendations) third ed., Pure Appl. 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[30] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, third Ed., Springer, New York, 2006. [31] D.B. Wetlaufer, Ultraviolet spectra of proteins and amino acids, Adv. Protein Chem. 17 (1962) 303–390. [32] C. Castaño, C. Lorente, N. Martins-Froment, E. Oliveros, A.H. Thomas, Degradation of a-melanocyte-stimulating hormone photosensitized by pterin, Org. Biomol. Chem. 12 (2014) 3877–3886.
[33] R. Amadò, R. Aeschbach, H. Neukom, Dityrosine: in vitro production and characterization, Methods Enzymol. 107 (1984) 377–388. [34] G.S. Harms, S.W. Pauls, J.F. Hedstrom, C.K. Johnson, Fluorescence and rotational dynamics of dityrosine, J. Fluoresc. 7 (1997) 283–292.
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Chemical changes in bovine serum albumin photoinduced by pterin Andrés H. Thomas a, Beatriz N. Zurbano a, Carolina Lorente a, Javier Santos b, Ernesto A. Roman b, M. Laura Dántola a,⇑ a Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, CCT La Plata-CONICET, Casilla de Correo 16, Sucursal 4, 1900 La Plata, Argentina b Instituto de Química y Físico-Químicas Biológicas (IQUIFIB), Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina
a r t i c l e
i n f o
Article history: Received 17 July 2014 Received in revised form 23 September 2014 Accepted 14 October 2014 Available online 19 October 2014
a b s t r a c t The exposure to UV-A radiation of bovine serum albumin (BSA) in aerated aqueous solution in the presence of pterin (Ptr), results in chemical and conformational modifications of the protein. Ptr belongs to a family of heterocyclic compounds that are well-known type I (electron-transfer) and type II (singlet oxygen) photosensitizers. The evolution of the photosensitized processes was followed by UV/vis spectrophotometry and fluorescence spectroscopy indicating that tryptophan (Trp) and tyrosine (Tyr) residues were affected. Additionally, conformational changes were evaluated by electrophoresis (SDS-PAGE) and size exclusion chromatography coupled with dynamic light scattering detection, showing that BSA undergoes dimerization, via the formation of Tyr radicals. The degradation of Trp residues takes place faster than the oligomerization of the protein. The photosensitized process is initiated by an electron transfer from BSA to the triplet excited stated of Ptr, being a purely dynamic mechanism. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Proteins, due to their relatively high abundance, their ability to bind chromophores, and the reactivity of particular amino acid residues, are one of the preferential targets of the photosensitized damaging effects of UV radiation on biological systems [1]. Currently, it is accepted that the photosensitization of proteins occurs mainly through the reactions of singlet oxygen (1O2) (type II mechanism) with tryptophan (Trp), tyrosine (Tyr), histidine, methionine and cysteine side-chains [2]. Especially, serum albumins are the most abundant plasma proteins, and their main biological function is the transport of a wide variety of molecules [3]. In addition, albumin is present in the human skin [4], where there is an autocrine synthesis and regulation [5]. It has been reported that in patients affected by vitiligo, epidermal albumin oxidation takes place [6]. Bovine serum albumin (BSA) has been commonly used as a model for the human protein due to its high homology with the human serum albumin (HSA) in the amino acidic sequence [7]. Pterins, heterocyclic compounds, are present in living systems mainly at three different redox states: fully oxidized (or aromatic) pterins, dihydro and tetrahydro derivatives. 5,6,7,8-Tetrahydrobiopterin (H4Bip) is an essential cofactor in the hydroxylation of the aromatic amino acids [8] and participates in the regulation of ⇑ Corresponding author at: C. C. 16, Sucursal 4, B1904DPI La Plata, Argentina. Tel.: +54 221 4257430x153; fax: +54 221 4254642. E-mail address: [email protected] (M. Laura Dántola). http://dx.doi.org/10.1016/j.jphotobiol.2014.10.007 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
melanin biosynthesis. Normal metabolism of H4Bip is altered in the skin in pathological conditions, such as vitiligo [9,10]. Vitiligo is a skin disorder characterized by the acquired loss of constitutional pigmentation, manifesting as white macules and patches [11], and the accumulation of reduced and oxidized pterins [12]. In diseased skin cells micromolar concentration of pterins have been determined, which is much higher than concentration found in healthy cells; e.g. in human keratinocytes and cells cultures from suction blister roofs the concentration of total biopterin was determined to be in the range 4–93 lM (41–950 pmol/mg of protein) depending on the cell type [13]. Under UV-A excitation (315–400 nm), unconjugated oxidized pterins can fluoresce, undergo photooxidation, and generate reactive oxygen species (ROS) [14]. In the presence of oxygen, pterin (Ptr) [14], the parent and unsubstituted compound of oxidized pterins, acts as a photosensitizer through both type I (electron transfer or hydrogen abstraction) [15] and type II (production of 1O2) [16] mechanisms. Moreover, Ptr photoinduces DNA damage and the oxidation of purine nucleotides [17–20]. Therefore, taking into account the accumulation of pterins in the human skin under pathological conditions described in the previous paragraph, the photochemistry and, especially their photosensitizing properties of these compounds are of particular interest for the study of skin disease. Two recent studies have suggested that degradation of proteins photosensitized by Ptr involves a type I mechanism [21,22]. The process is initiated by an electron transfer reaction from the protein (P) to the triplet excited state of Ptr (3Ptr*) (reaction 1),
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yielding the corresponding pair of radical ions (pterin radical anion (Ptr) and the protein radical cation (P+)). P+ leads to the irreversible damage and degradation of the protein (reaction 2), whereas Ptr, under aerobic conditions, transfers one electron to O2 regenerating Ptr and forming superoxide anion (O 2 ) (reaction 3), which, in turn, is converted into hydrogen peroxide (H2O2) by spontaneous disproportionation (reaction 4) [22]. Moreover, it was demonstrated that Ptr is able to photosensitize the oxidation of free Trp and Tyr in aqueous solution [23,24]. In particular, the study of the photosensitization of Tyr revealed that dimers of Tyr, or dityrosine are formed [24]. This amino acid plays a key role in polymerization and cross-linking of proteins via reactions initiated by Tyr radicals [25]. The accumulation of pterins at micromolar concentrations [9,12] in the skin of patients affected by vitiligo, in which the protection against UV radiation is lost, makes the investigation of the damage of proteins photoinduced by pterins important from a biomedical point of view. 3
Ptr þ P ! Ptr þ Pþ H2 O=O2
ð1Þ
Pþ ! Products
ð2Þ
Ptr þ O2 ! Ptr þ O 2
ð3Þ
2Hþ þ 2O 2 ! H 2 O2 þ O2
ð4Þ
The main aim of this work is to investigate the chemical changes that proteins undergo upon UV exposure in the presence of pterins. We have performed the experiments in aqueous solutions under UV-A irradiation, using BSA as a model protein and Ptr as a photosensitizer. The experiments were carried out at concentrations of Ptr of the same order of magnitude found in human skin affected by vitiligo [13]. Most of the experiments were performed at pH 6.0 ± 0.1, so that more than 95% of the Ptr (pKa = 7.9) [26] was in the acid form, the predominant form at physiological pH. The photochemical reactions were followed by UV/visible spectrophotometry, fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) coupled with dynamic light scattering detection. 2. Materials and methods
263
Tris–HCl (pH 7.0) and 50 mM NaCl. Twenty five milliliters of this solution was loaded in a SEPHADEX G-100 column and different fractions were collected. An SDS-PAGE was performed to evaluate which fractions contained the monomeric fraction of BSA. These tubes were used without change of buffer or concentration procedures. 2.3. Steady-state irradiation The continuous photolysis of the solutions containing Ptr and BSA were carried out irradiating in quartz cells (0.4 cm optical path length) at different temperatures using a temperature controller (LFI-3751 Wavelength Electronics). Two radiation sources were employed: (I) Rayonet RPR 3500 lamps (Southern N.E. Ultraviolet Co.) with emission centered at 350 nm (bandwidth (fwhm) 20 nm) and (II) a 450 W Xe lamp with a monochromator Horiba Jobin Yvon FL-1004 (single-grating spectrometer, 330 nm blaze grating). Photolysis experiments were performed in air equilibrated aqueous solutions. Aberchrome 540 (Aberchromics Ltd.) was used as an actinometer for the measurement of the incident photon flux density (q0;V n;p ) at the excitation wavelength, which is the amount of incident photons per time interval (q0n;p ) and divided by the volume (V) of the sample [27]. Aberchrome 540 is the anhydride form of the (E)-a-(2,5dimethyl-3-furylethylidene) (isopropylidene) succinic acid which, under irradiation in the spectral range 316–366 nm leads to a cyclized form. The reverse reaction to ring opening is induced by visible light. The method for the determination of q0;V n;p has been described in detail elsewhere [28]. The value of q0;V n;p measured for the radiation source I was 2.4 (±0.2) 105 Einstein L1 s1; the value of q0;V n;p measured for the radiation source II was 6.7 (±0.6) 106 Einstein L1 s1. Values of the absorbed photon flux density (qa;V n;p ) were calculated from q0;V n;p according to the Lambert–Beer law: A 0;V qa;V n;p ¼ qn;p ð1 10 Þ
ð5Þ
where A is the absorbance of the reactant at the excitation wavelength. 2.4. Analysis of irradiated solutions
2.1. General Pterin (Ptr, >99%) and bovine serum albumin (BSA, fatty acids free) were purchased from Schircks Laboratories and Sigma, respectively. Ptr was used without further purification. Sodium dodecyl sulfate (SDS, 99%), glycerol, 2-mercaptoethanol, bromophenol blue, glycine (Gly, >99% titration), ammonium persulphate (>98%) and N,N,N0 ,N0 -tetramethylethylene-diamine (TEMED, 99%) were provided by Sigma. Methanol was provided by Laboratorios Cicarelli. Acetic acid was provided by Anedra. Coomassie Brilliant Blue G was provided by Fluka. Acrylamide and N0 N0 -methylene-bis-acrylamide and trishydroxymethylaminomethane (Tris) were provided by Genbiotech. The pH measurements were performed using a pH-meter PHM220 (Radiometer Copenhagen) combined with a pH electrode pHC2011-8 (Radiometer Analytical). The pH of the aqueous solutions was adjusted by adding drops of HCl and NaOH solutions from a micropipette. The concentration of the acid and the base used for this purpose ranged from 0.1 M to 2 M. 2.2. BSA purification BSA was further purified to get rid of chemically altered protein and covalently bonded oligomeric species. Briefly, BSA was prepared in 40 mg/mL concentration in buffer containing 10 mM
2.4.1. UV/vis analysis Electronic absorption spectra were recorded on a Shimadzu UV1800 spectrophotometer. Measurements were made using quartz cells of 0.4 or 1 cm optical pathlength. The absorption spectra of the solutions were recorded at regular intervals of irradiation time. 2.4.2. Fluorescence spectroscopy Fluorescence measurements were performed on air-equilibrated aqueous solutions of Ptr and BSA using a Single-PhotonCounting equipment FL3 TCSPC-SP (Horiba Jobin Yvon). The equipment has been previously described in detail [29]. Briefly, in steady-state measurements the sample solution in a quartz cell was irradiated with a 450 W Xenon source through an excitation monochromator. The fluorescence, after passing through an emission monochromator, was registered at 90° with respect to the incident beam using a room-temperature R928P detector. The emission measurements were performed at 25 °C, using the temperature controller mentioned above. The total fluorescence intensities (IF) were calculated by integration of the fluorescence band centered at ca. 350 nm. The Trp fluorescence from BSA was corrected for inner filter effect. The fluorescence intensity was approximately corrected using the following equation [30]
IFcorr ¼ IFobs 10ðAexcþAemÞ=2
ð6Þ
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2.4.3. Electrophoretic analysis Protein damage was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples of protein solutions were boiled for 5 minutes in a 0.06 M Tris–HCl (pH 6.8) solution containing 2% SDS, 10% glycerol, 1% 2-mercaptoethanol (as reducing agent) and 0.02% bromophenol blue (as a tracking dye). Acrylamide (4%) stacking gel, 10% acrylamide resolving gel and running buffer containing 25 mM Tris, 192 mM Gly and 0.1% SDS, pH 8.3 were used. Electrophoresis was performed at 30 mA during 100 min. Gels were stained with 0.1% Coomassie Brilliant Blue G and destained with a solution of methanol and acetic acid during 2 h. The electrophoretic bands were quantified by scanning photodensitometry using the ImageJ 1.45s software. 2.4.4. Size exclusion chromatography (SEC) coupled with a dynamic light scattering Hydrodynamic behavior and oligomer formation of BSA was monitored by light scattering coupled to chromatography on a SEC–HPLC system equipped with a 280 nm UV detector coupled. Briefly, a Superdex S-200 HR 10/30 columns (Pharmacia Biotech, Sweden) coupled to a MiniDawin static light scattering detector (Wyatt) and an absorbance detector (Jasco UV 2075plus) was equilibrated at room temperature in buffer 20 mM Tris–HCl, 100 mM NaCl, pH 7.0. The scattered light was collected at three different angles and molar mass was obtained using ASTRA Software. In parallel, the scattered light at 90° was directed through an optical fiber to a Quasi Elastic Light Scattering module (Wyatt). Here, the time-dependent fluctuations in the intensity of the scattered light were registered and quantified via a second-order correlation function and the diffusion coefficient was obtained. This type of chromatographic resin lets us examine the presence of oligomers and soluble aggregates in the samples. The flow rate was 0.4 mL min1 and the injection volume was 100–200 lL. Samples were centrifuged at 14,000 rpm before loading onto the column. 2.5. Quantum yield determinations The quantum yield of BSA consumption (U-BSA) was determined under different experimental conditions. Values were obtained using Eq. (7).
UBSA ¼
ðd½BSA=dtÞ0 qa;V n;p
extinction coefficients of aromatic amino acids, Trp generally dominates the absorption, fluorescence and phosphorescence spectra of proteins that also contain either of the other two aromatic amino acids [31]. BSA contains Trp (Trp 214 and Trp 135) and Tyr residues which act as intrinsic fluorescence probes. When excitation wavelength of 280 nm is used, both Tyr and Trp residues are excited; in contrast, when wavelength of 295 nm is used, only the Trp residues are excited. The comparison of fluorescence spectra obtained by excitation at 280 and 295 nm showed that there are no changes in the shape and emission maximum of both spectra (data not shown), thus indicating that emission fluorescence comes mainly from Trp in BSA, under this experimental conditions. To determine if the photosensitization process modified the Trp residues of BSA, air-equilibrated aqueous solutions containing BSA and Ptr were irradiated in quartz cells during different periods of time at 37 °C, pH 6.0 ± 0.1 (350 nm, radiation source II, Materials and methods). Under these experimental conditions only Ptr was excited [22]. The corrected fluorescence spectra obtained by excitation at 280 nm of the irradiated samples were recorded between 295 and 400 nm. Although Ptr absorbs at the excitation wavelength, its emission is red shifted with respect to that of Trp, (maximum at 439 nm, [14]), so that, under our experimental conditions, the typical emission band of Trp could be adequately registered. The Trp fluorescence decreased as a function of irradiation time (Fig. 1), thus suggesting that Trp residues were damaged during irradiation. To confirm that the reaction observed was a photosensitized process, BSA aqueous solutions were exposed to UV-A radiation during different periods of time in the absence of Ptr. No changes in the total fluorescence intensity were detected, even after more than 60 min of irradiation, thus excluding the possibility that spurious effects of direct light absorption by the protein could affect its fluorescence (Fig. 1). In a recent work, we demonstrated that when air-equilibrated aqueous solutions containing BSA and Ptr were irradiated with UV-A radiation (350 nm, radiation source I, pH = 6.0 ± 0.1), the concentration of the BSA, estimated by means of SDS-PAGE, decreased as a function of irradiation time [22]. Therefore, irradiated air-equilibrated solutions containing BSA and Ptr were analyzed simultaneously by SDS-PAGE and fluorescence spectroscopy. The comparison of the two analyzed parameters showed that the rate of the decrease in total fluorescence intensity (related to Trp damage) was higher than the decrease of the BSA concentration (determined from the intensity of the BSA band) (Fig. 2).
1.0
ð7Þ
whith (d[BSA]/dt)0: initial rate of BSA degradation, estimated as the Trp residues consumption (calculated using fluorescence analysis) a;V and qn;p : absorbed photon flux density by Ptr (Eq. (5)). 3. Results and discussion
0.8
Emission (10 7 counts)
where IFcorr and IFobs were the corrected and observed fluorescence intensities, respectively. Aexc and Aem were the absorbance of the system at excitation and emission wavelengths, respectively.
I F/ IF0
264
0.6
3.1. Effect on the tryptophan residues Taking into account the photosensitization of Trp by Ptr previously reported [23], and that Trp is particularly susceptible to a variety of oxidizing agents [2], it is straightforward to expect that Trp residues of BSA would be affected when this protein is exposed to UV-A radiation in the presence of Ptr. Therefore, the first aim of this work was to investigate the degradation of Trp in BSA, taking advantage of the fluorescence properties of this amino acid. Fluorescence of proteins comes mainly from the contributions of the three aromatic amino acids: Trp, Tyr and phenylalanine (Phe). However, as a result of the spectral distribution and relative
0.4
3 2 1 0
300 320 340 360 380
λ (nm)
0
10
20
30
40
50
60
time (min) Fig. 1. Evolution of the relative total fluorescence intensity (IF/I0F ) of Trp residues in an aqueous solution of BSA (15.0 lM) in the absence (s) and in the presence of Ptr (4.5 lM) (r) as a function of the irradiation time (emission temperature = 25 °C, kexc = 280 nm). Inset: corrected fluorescence spectra.
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3.2. New insights on the mechanism of photosensitization
3.3. Oligomerization of BSA In a recent work performed with free Tyr in aqueous solutions, we have demonstrated that the excitation of Ptr is followed by an electron transfer from the amino acid to the Ptr triplet excited state, leading to the formation of the corresponding ion radicals, Ptr and Tyr+ [24]. The latter undergoes oxygenation and dimerization to yield the so-called dimers of Tyr (denoted Tyr2), which are involved in cross-linking of proteins. Moreover, the dimerization of the peptide a-melanocyte-stimulating hormone (a-MSH) and the formation of Tyr2 were observed when the peptide was exposed to UV-A radiation in the presence of Ptr
Φ -BSA (10 −3)
10.0
1.1 ± 0.1
25.0
2.0 ± 0.2
40.0
2.5 ± 0.2
0
0.9
IF /IF
The mechanism of photodamage of BSA by Ptr involves an electron transfer process (vide supra) [22]. However, it is not clear how such a process takes place. BSA is able to bind Ptr, but the corresponding binding constant for the ground state complex formation is relatively low ((1.27 ± 0.03) 103 M1 at 25 °C) [22]. Therefore, the mechanism of photosensitization might be initiated by a purely dynamic process with the participation of free Ptr or by a static process in which the electron transfer would occur directly from the oxidizable amino acids, like Trp, to the protein-associated form of Ptr. To investigate this point, air-equilibrated aqueous solutions containing Ptr and BSA were irradiated at 350 nm (radiation source II, pH = 6.0 ± 0.1) for different periods of times at various temperatures. Higher temperature results in faster diffusion and hence larger amounts of molecular collision. Therefore, if a dynamic mechanism is responsible for the photosensitization of BSA by Ptr, we should observed more damage to Trp when the temperature increases. In contrast, if the electron transfer involves associated molecules, high temperature tends to disrupt the ground state complex, and, consequently, it should be observed a decrease in the photosensitized process with raising temperature. Under our experimental conditions, it was observed that the rate of Trp consumption, estimated from the evolution of its fluorescence intensity, was higher when the temperature increased (Fig. 3). This result confirmed that the photodamage to BSA by Ptr takes place via a purely dynamic mechanism.
T (°C) 1.0
0.8
0.7
0.6
0
3
6
9
18
21
[32]. To investigate if the photosensitization of BSA by Ptr leads to the generation of Tyr2, fluorescence experiments were carried out, taking advantage of its particular emission features. The absorption and emission spectra of Tyr2 are red shifted with respect to those of Tyr [33]. Therefore solutions containing Ptr and BSA were exposed to UVA radiation (350 nm, radiation source II, pH = 6.0 ± 0.1) and the emission spectra under excitation at 310 nm were registered at various irradiation times. For a given irradiation time, the spectrum of the solution before irradiation, that means the fluorescence of Ptr and Trp, was subtracted from the spectrum registered. Results show that an emission band with a maximum coinciding with that expected for the Tyr2 [34] was present in the irradiated solutions and that the intensity increased as a function of irradiation time (Fig. 4), which is strong evidence in favor of the photodimerization of Tyr residues of BSA induced by Ptr.
10
60
[BSA]/[BSA]0
40
IF /IF0
Emission (10 counts)
0.6
30
5
20
5
0.4 0.2 0.0
0
0
5
10
10
20
20
30
30
50
40
50
time (min) Fig. 2. Evolution of the relative total fluorescence intensity (IF/IF0) and the relative BSA concentration ([BSA]/[BSA]0), estimated by means of SDS-PAGE, of an aqueous solution of BSA (5.6 lM) in the presence of Ptr (50.0 lM) as a function of the irradiation time. Inset: electrophoretic bands (picture) of BSA as a function of irradiation time. The irradiation time (min) appears above each band. Emission temperature = 25 °C, kexc = 280 nm.
0 IF (10 5 counts)
IF /IF0 - [BSA]/[BSA]0
15
Fig. 3. Evolution of the relative total fluorescence intensity (IF/I0F ) of an aqueous solution of BSA (3.0 lM) and Ptr (22.5 lM) as a function of the irradiation time (min) at various irradiation temperatures. Emission temperature: 25 °C, kexc = 280 nm. Table: quantum yield of BSA consumption (U-BSA) at each temperature.
1.0 0.8
12
time (min)
-5
-10
340
360
8 6 4 2 0
0
380
10
20
30 40 50 time (min)
400
420
60
440
460
λ (nm) Fig. 4. Corrected fluorescence spectra (kexc = 310 nm) of an aqueous solution of BSA (15.0 lM) and Ptr (4.5 lM) irradiated at 350 nm at 37 °C. The irradiation time (min) appears above each spectrum. For each time, the spectrum of the solution before irradiation was subtracted. Inset: increase of the fluorescence intensity at 402 nm as a function of irradiation time.
A.H. Thomas et al. / Journal of Photochemistry and Photobiology B: Biology 141 (2014) 262–268
0
10
20
30
50
BSA photoproducts
BSA
1.0
relative concentration
266
BSA monomer, [Ptr] = 0 μM BSA dimer, [Ptr] = 0 μM BSA monomer, [Ptr] = 75 μM BSA dimer, [Ptr] = 75 μM
0.8
0.2
Fig. 5. SDS-PAGE analysis of BSA (5 lM) solutions irradiated in the presence of Ptr (50 lM) at 350 nm at room temperature. The irradiation time (min) appears above each band.
Taking into account the fluorescence results previously shown, and in order to confirm that the photosensitization of BSA by Ptr produces dimers or oligomers, aqueous solutions containing purified BSA and Ptr were exposed to UV-A radiation (350 nm, radiation source I) during different periods of time and the treated samples were analyzed by SDS-PAGE. In this case, a larger sample volume was used to improve the detection of products. The electrophoretic patterns showed that simultaneously with the decrease of BSA optical density, photoproducts with molecular weight higher than BSA were formed (Fig. 5).
Absorbance (relative scale)
BSA monomer
(a)
1.0
0.8
0.6 BSA photoproduct
0.4
0.2
(b)
1.0
2.0 0.8 1.5 0.6 1.0
5
0.4
Molar Mass ( 10 Da)
Normalized light scattering (90 degrees)
0.0
0.0
0
50
100
150
200
250
time (min) Fig. 7. Evolution of the relative concentration of BSA monomer and BSA dimer in the absence and in the presence of Ptr as a function of irradiation time. [BSA] = 150 lM, [Ptr] = 75 lM, irradiation source I (350 nm), pH = 6.0 ± 0.1.
To further investigate this point the irradiated samples were analyzed by size-exclusion chromatography coupled with light scattering and absorbance detectors. In Fig. 6a it can be seen that the peak corresponding to BSA decreased as a function of irradiation time, while new absorbance product appeared at lower retention time (tR), indicating that the molecular weight of this photoproduct is higher than that of the BSA. In order to determine the molecular weight of the photoproducts, the scattering profiles obtained for different irradiation times were analyzed. As shown in Fig. 6b, at time zero, around tR 31 min there is a peak of scattered light corresponding to an specie of 62 (+/3) kDa corresponding to the monomeric BSA, being the main specie in solution. When the irradiation time increased, besides the monomeric species, another peaks started to contribute to the scattered light (tR 28 min), this peak corresponding to an specie of 132 (+/11) kDa, indicating that this contribution is mainly due to an increase in the dimeric species. As it can be seen, as the dimeric peak increased, the monomeric peak decreased suggesting that oligomer formation is due to an interaction of monomers. As a control, aqueous solutions of BSA were exposed to UV-A radiation in the absence of Ptr and analyzed by light scattering. In Fig. 7 it can be observed that the loss of monomer and, appearance of dimer is much greater in BSA solutions when Ptr is present if we compare with BSA solution irradiated in the absence of photosensitizer. The relative concentration of BSA monomer and BSA dimer were calculated as [BSA monomer]/([BSA monomer]+[BSA dimer]) and [BSA dimer]/([BSA monomer]+[BSA dimer]), respectively. These results confirm that the dimerization due to a photosensitization process.
0.5
0.2
4. Conclusion
0.0
0.0 20
22
24
26
28 30 tR (min)
32
34
Fig. 6. Chromatographic analysis of a solution of BSA (150 lM) irradiated in the presence of Ptr (75.0 lM) (irradiation: source I, pH = 6.0 ± 0.1) (a) chromatograms obtained using an absorbance detector (k = 280 nm), irradiation times: 0, 1 h, 3 h and 4 h. (b) Scattered light at 0 (dotted line) and 240 min (solid line) irradiation time. Black and white circles are the molecular mass of the main peak for the 0 and 240 min irradiation time, respectively. Black triangles are the molecular weights of the photoproducts.
The photosensitization of bovine serum albumin (BSA) by pterin (Ptr), the parent compound of oxidized pterins, in aqueous solution under UV-A irradiation was investigated. The photoinduced oxidation of BSA is a purely dynamic process and results in the oxidation of the protein in at least two different and specific sites: tryptophan (Trp) and tyrosine (Tyr) residues. The Trp degradation results in a fast decrease of the fluorescence intensity, which is observed before structural changes in SDS-PAGE analysis. In addition, Tyr residues contribute to dimerization of the protein, since dimers of Tyr residues were detected clearly by fluorescence. The
A.H. Thomas et al. / Journal of Photochemistry and Photobiology B: Biology 141 (2014) 262–268
size exclusion chromatography analysis indicated unequivocally that Ptr photoinduces cross-linking of BSA, most likely due to dimerization between Tyr residues. Taking into account that oxidized pterins are present in human skin under pathological conditions in which the protection against UV radiation fails due to the lack of melanin and that albumin is one of the most abundant proteins present in skin, the results shown in this work, using BSA and Ptr as model compounds, are relevant from a biomedical point of view and can have further ramification. In particular, the photosensitized reactions described in our studies might significantly contribute to the protein damage reported in various skin diseases. 5. Abbreviations A Aem Aexc qa;V n;p BSA IFcorr Tyr2 Gly HSA H2O2 q0;V n;p q0n;p TEMED IFobs Phe P P+ Ptr Ptr
U-BSA ROS tR 1 O2 SEC SDS SDSPAGE O2 H4Bip IF 3 Ptr* Tris Trp Tyr V a-MSH
absorbance absorbance of the system at emission wavelength absorbance of the system at excitation wavelength absorbed photon flux density bovine serum albumin corrected fluorescence intensity dimers of Tyr glycine human serum albumin hydrogen peroxide incident photon flux densitity incident photons per time interval N,N,N0 ,N0 -tetramethylethylene-diamine observed fluorescence intensity phenylalanine protein protein radical cation pterin pterin radical anion quantum yield of BSA consumption reactive oxygen species retention time singlet oxygen size exclusion chromatography sodium dodecyl sulfate sodium dodecyl sulfate polyacrylamide gel electrophoresis superoxide anion 5,6,7,8-tetrahydrobiopterin total fluorescence intensities triplet excited state of Ptr trishydroxymethylaminomethane tryptophan tyrosine volume a-melanocyte-stimulating hormone
Acknowledgements The present work was partially supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET; Grant PIP 11220090100425), Agencia de Promoción Científica y Tecnológica (ANPCyT; Grants PICT 2012-0508), and Universidad Nacional de La Plata (UNLP; Grant X586). B. N. Z. thanks CONICET for doctoral research fellowship. A. H. T., C. L., E. A. R, J. S. and M. L. D are research members of CONICET. The authors thank Dr. Catalá Ángel for his crucial contributions in SDS-PAGE analysis.
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