- Email: [email protected]
Flash photolysis and pulse radiolysis studies on collagen Type I in acetic acid solution
Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45 www.elsevier.com/locate/jphotobiol
Flash photolysis and pulse radiolysis studies on collagen Type I in acetic acid solution Alina Sionkowska
*
Faculty of Chemistry, N. Copernicus University, Gagarin 7, 87-100 Torun, Poland Received 8 November 2005; received in revised form 28 December 2005; accepted 9 January 2006 Available online 28 February 2006
Abstract An investigation of the photochemical properties of collagen Type I in acetic acid solution was carried out using nanosecond laser irradiation. The transient spectra of collagen solution excited at 266 nm show two bands. One of them with maximum at 295 nm and the second one with maximum at 400 nm. The peak at 400 nm is assigned to tyrosyl radicals. The first peak of the transient absorption spectra at 295 nm is probably due to photoionisation producing collagen radical cation. The transient for collagen solution in acetic acid at 640 nm was not observed. It is evidence that there is no hydrated electron in the irradiated collagen solution. The reactions of hydrated electrons and OH radicals with collagen have been studied by pulse radiolysis. In the absorption spectra of products resulting from the reaction of collagen with e aq no characteristic maximum absorption in UV and visible light region has been observed. In the absorption spectra of products resulting from the reaction of the hydroxyl radicals with collagen two bands have been observed. The first one at 320 nm and the second one at 405 nm. Reaction of OH radicals with tyrosine residues in collagen chains gives rise to Tyr phenoxyl radicals (absorption at 400 nm). 2006 Elsevier B.V. All rights reserved. Keywords: Collagen; UV irradiation; Transient spectra; Photochemistry; Pulse radiolysis
1. Introduction Limited investigations of photo damage to human skin have revealed a down-regulation of collagen synthesis with a consequent overall loss of collagen [1]. However, all these studies are based on the assumption that the damage to the dermal collagen is through the normal degradative pathway of the remodelling process and scant attention has been paid to the possibility of direct damage to the collagen through free radicals or singlet oxygen formed during irradiation [2]. Structural proteins such as collagen and elastin undergo varied sequences of photochemical reactions when they are exposed to ultraviolet and longer wavelength radiation [2–4]. Because of the insolubility of structural proteins photochemists have been denied the use of *
Tel.: +48 56 6214302; fax: +48 566542477. E-mail address: [email protected].
1011-1344/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2006.01.007
many standard chemical analytical tools and methods. Even though the complexity of structural proteins limits the experimental techniques which can be employed, many insights of their photodegradation have been gained by studying model systems such as aromatic and aliphatic amino acids [5–8] or the photochemical behaviour of water soluble proteins [9]. Collagen is the most abundant biopolymer in animals where it provides the principal structural and mechanical support [10]. The main amino acids in collagen are: glycine, proline, hydroxyproline and alanine, with a small number of residues of phenylalanine and tyrosine. The ordered triple helical structure of collagen is stabilized by both interchain hydrogen bonds and by structural water molecules [11–15]. Collagen is readily available, non-toxic and has got the fibril architecture that is inherent in natural tissues. Therefore, collagen provides an excellent basis for biomaterials such as arterial prostheses and artificial skin [16,17]. There are 20 genetically distinct collagen types [11,12].
A. Sionkowska / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45
Type I collagen from young rat-tail tendon is soluble in diluted acetic acid solution. The same collagen Type I in bone is cross-linked and insoluble. Many studies have demonstrated modification of collagen induced by UV radiation: it has been shown that in solution collagen loses the ability to form natural fibrils after irradiation [18]. In addition, the fluorescence observed after UV irradiation is due to the presence of photo-products of phenylalanine and tyrosine residues in the collagen amino acid sequence [3,19–21]. Photocross-linking and photodegradation of collagen may also occur on exposure to UV radiation [3,22–24]. Destruction of Type I collagen under UVA (335–400 nm) and broadband solar simulating radiation (290–400 nm) has also been demonstrated [19]. The influence of UV radiation on collagen in films has also been investigated [25–30]. After UV irradiation of thin collagen films random-coil domains increased on the surface [27,29]. The material ejected from hydrated collagen gel by laser ablation was partially changed from the original collagen gel. In the ejected materials, low molecular weight polypeptides were found [30,31]. The aim of this work was to, study the photochemistry and radiation chemistry of collagen in acetic acid solutions. The main point of the work is to show what kind of radicals and short living species are involved in photochemical reaction in collagen. The paper deals with photoionisation and photodecomposition measurements of collagen in acetic acid solution at pH < 4 in the absence and presence of oxygen. The direct action of UV radiation is a novel approach to the photodegradation of collagen in acetic acid solution. Solutions of collagen were irradiated and the transient absorption spectra were recorded. Collagen absorbs mainly in the far UV so 266 nm laser irradiation from the Nd:YAG laser was employed. To better understand the reaction of hydrated electron and OH radicals with collagen the pulse radiolysis technique was employed. 2. Materials and methods 2.1. Sample preparation Collagen was obtained in our laboratory from tail tendons of young albino rats. Briefly, tendons were excised and washed in distilled water, and blended in a Waring blender in 0.4 M acetic acid, samples were then spun at 8000 rpm in a centrifuge and the soluble fraction were decanted. The concentration of collagen solution was determined using burette method. The preparation of collagen solution for flash photolysis and pulse radiolysis measurements was carried out with special care in order to avoid any structural changes. 2.2. Pulse radiolysis The pulse radiolysis experiments were carried out at the Free Radical Research Facility at Daresbury Laboratory,
39
UK, with a 12 MeV Radiation Dynamics Ltd. (UK) electron linear accelerator. We used single pulses of duration 0.22–2 ls and with a peak current of about 30 mA. The accelerator is normally operated at 10 Hz but the single pulse mode is achieved by modifying the pulses to the electron gun [32]. The detection system consists of a Xe arc lamp with a pulsing unit, high radiance Kratos monochromator and quartz optics. The sample cell, constructed by Spectrosil quartz, had an optical path length of 25 mm [33]. Optical transmissions at various wavelengths selected with the monochromator were observed as a function of time before and after the pulse using photoelectric detection. The output of the photomultiplier (EMI9558Q) was displayed on a Tektronix TDS 380 digitizing oscilloscope then transferred to a PC and fitted using in house software. Absorbed doses were determined from the transient ðSCNÞ2 formation in airsaturated 10 mM KSCN as described by Adams et al. [34], but using the updated Ge value of 2.59 · 104 m2 J1 obtained by Buxton and Stuart [35], G being the radiation chemical yield of ðSCNÞ2 and e its molar absorption coefficient at 475 nm. Saturation of such solutions with N2O results in a doubling of the ðSCNÞ2 yield. In water, three different primary radicals are produced by irradiation: solvated electrons (e aq ) and hydrogen atoms (H), which are reducing species and hydroxyl radicals (OH), which are oxidising species. For generating almost exclusively OH, aqueous solutions were saturated with N2O which converts e aq into hydroxyl radicals by dissociative electron attachment: ð1Þ N O þ e þ H O ! N þ OH þ OH 2
aq
2
2
2.3. Laser flash photolysis Laser flash photolysis experiments were also carried out at the FRRF facility at Daresbury Laboratory using a Nd:YAG laser. The system essentially consists of a Qswitched Nd:YAG (JK Lasers 2000 Series) laser capable of delivering up to 1 J of energy at 1064 nm in pulses of 12 ns duration as the excitation source [36]. The fundamental bean can be frequency doubled, tripled and quadrupled to deliver light of 532, 355 and 266 nm, respectively. The harmonies are separated using a beam splitter. The sample under investigation is contained in a quartz cell and changed after each laser pulse using a remote controlled flow system. The analysing beam generated by a xenon lamp, which can be pulsed to increase the analysing light up to 200 times in the UV region, passes through the sample at right angles to the laser beam. A shutter, which opens a few microseconds before the laser pulse and closes again after the event under examination is completed, and appropriate filters are placed between the analysing lamp and sample in order to minimise the photolysis of the sample by the monitoring light. The monitoring light is then dispersed in wavelength by a monochrometer and then onto
40
A. Sionkowska / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45
a photodetector. The transmittance of light at this wavelength is detected by the photodetector before, during and after the laser pulse. The detector is also connected to an automatic back-off box which enables changes in transmittance to be observed by feeding back a signal equal and opposite to the detector anode current prior to the laser pulse, thus maintaining the anode current close to zero. The signal from the detector is captured and stored using a programmable digital oscilloscope. Back-off and energy meter readings are digitised using an Analogue to Digital Converter (ADC) attached to a DI-AN data acquisition system. Data acquisition and processing are obtained by repeating the above procedure at successive wavelengths [37]. Transient profiles taken at different wavelengths can be used to generate a difference spectrum at various times after the laser pulse. Thus, it is possible to determine whether more than one species contributes to the spectrum since if there are different species, and their lifetimes differ, their individual spectral contributions can be separated by recording spectra at different time delays. The transient profiles observed are usually either due to excited states or free radicals. First-order decay kinetics often indicate that the transient is an excited state, whereas free radicals usually decay by second-order kinetics. There are exceptions such as the first-order process when a radical decays intramolecularly, producing a different radical with a different absorption spectrum. In a deaerated solution with no quencher present a triplet state will decay radiationlessly via solvent induced deactivation (reaction (2)) and follow first-order kinetics, as described below (Eqs. (3)–(5)) 3
k1
M ! M 3
ð2Þ
d½ M t ¼ k 1 ½3 M t dt
ln½3 M t ¼ ln½3 M 0 k 1 t
ð4Þ 3
Where [ M*]0 is the initial concentration of M*. However, the change in absorbance (DAt) is proportional to the concentration of 3M* so DA can be substituted for 3M*. ln DAt ¼ ln DA0 k 1 t
ð5Þ
Where DA0 is the difference absorbance immediately after the laser pulse. Hence, a plot of lnDAt against t will give a straight line with gradient k1, and intercept lnDA0. The computer software uses an iterative least squares analysis to extract k1 and DA0 values from the integrated rate Eq. (6) which is a rearrangement of Eq. (5), with a constant DA1 added to account for any residual change in absorbance remaining after the decay of the species. DAt ¼ DA0 ek1 t þ DA1
i
Mixed first-order growth and decay kinetics can also be analysed in this way. The computer software in Daresbury can calculate k1 and k2 and DA1 and DA2 for a biexponential decay or growth and decay using Eq. (8). DAt ¼ DA1 ek1 t þ DA2 ek2 t
ð8Þ
A second-order decay arises in bimolecular reactions where both reactants are present in equal or approximately equal amounts. This occurs for triplet–triplet annihilation, radical–radical recombination, or when another solute (Q) quenches the initial excited state or radical. For triplet–triplet annihilation or radical–radical recombination both reactants are identical and thus, the rate constant is given by Eq. (9). d½3 M t ¼ k½3 M t2 dt
ð9Þ
Substituting DAt/el for [3M*] and integrating gives Eq. (10), so that a plot of 1/DAt against t will give a straight line with slope 2 k/el, where k is the second-order rate constant, and intercept 1/DA0. 1 1 2kt ¼ þ DAt DA0 el
ð10Þ
The software evaluates k/el from Eq. (11) (a rearrangement of Eq. (10)), again with the addition of the constant DA1. DA0 þ DA1 ð11Þ DAt ¼ 1 þ DA0 2kt=el
ð3Þ
Where k1 is the first-order rate constant for the decay of 3 * M , and [3M*]t is the concentration of 3M* after time t. Integration of Eq. (3) gives: 3
When more than one transient decaying with first-order kinetics contributes to the transient decay profile, the absorbance change is described by a sum of exponential decays, as in Eq. (7). X DAi eki t ð7Þ DAt ¼
ð6Þ
3. Results and discussion Model studies of the photodamage in proteins consider the photochemical reactions of free amino acids and of small peptides. Approximation of the results and mechanism to proteins is usually done. However, photodamage of proteins cannot be directly predicted from the behaviour of free amino acids or even isolated amino acids forming the peptide bonds. For this reason specific studies are required for each protein. Collagen absorbs mainly in the far UV but has an absorption tail out as far as 380 nm. The absorption spectra of collagen solution showed a wide peak in the region 250– 290 nm The main chromophores absorbing in the UV region are aromatic amino acids [3]. The absorbance at 266 nm was 0.3 (UV absorption spectra not shown). Laser irradiation with 266 nm from the Nd:YAG laser was employed. The transient absorption spectra obtained on subjecting an Ar saturated acetic acid solution of collagen to 266 nm laser irradiation are shown in Fig. 1. Two bands
A. Sionkowska / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45
not observe any transient at wavelength longer than 600 nm for tyrosine or for phenylalanine. The work published by Nikogosyan for photolysis of aromatic amino acids in aqueous solution [8] showed the minimum set of photochemical reactions. The initial step was: ð14Þ HA ! HAþ þ e
0.04
1
Absorbance
0.03
1- after the pulse of 35 μs 2- after the pulse of 310 μs
2
3- after the pulse of 878 μs
0.02
aq
HAþ ! A þ Hþ HAþ þ H2 O ! HA –OH þ Hþ
3 0.01
0 250
350
450
550
650
wavelength, nm Fig. 1. Transient absorption spectra of collagen in Argon saturated solution in acetic acid (pH 2.2): curve 1, after the pulse of 35 ls; curve 2, after the pulse of 310 ls; and curve 3, after the pulse of 878 ls.
were observed at the end of the pulse with two maxima at 295 nm and 400 nm. The species with kmax = 295 nm could be a tyrosine triplet. Tyrosine triplet usually gives a peak at 300 nm [8,9] however, the transient at 295 nm seems too long-lived for a triplet state. An obvious candidate for the second species is the tyrosyl radical (Tyrosine phenoxyl radicals) which is known to have an absorption band centred at 405 nm (absorption coefficient 2.8 · 103 M1 cm1) [38]. Tyrosine (TyrOH) is likely to deprotonate very rapidly on forming the neutral tyrosyl radical TyrO. The transient absorption spectra of collagen in air saturated acetic acid solution excited at 266 nm have shown two bands. One of them with maximum at 295 nm and the second one with maximum at 400 nm. This suggests that the bands are not due to the triplet state. The triplet state is usually quenched by oxygen. 3 TyrOH þ O ! TyrO þ Hþ þ O ð12Þ 2
3 3
2
TyrOH can absorb a second photon: TyrOH þ hm ! TyrO þ e þ Hþ aq
41
ð13Þ
To explain what kind of species are responsible for the transient the comparison with transient spectra of aromatic amino acids has been made. The photochemical properties of aromatic amino acids have been rather well investigated in the last three decades by Nikogosyan et al. [8,39]. They found that mechanism of photoionisation of the aromatic amino acids is monophotonic for kexc = 193 nm and mainly biphotonic for kexc = 248 nm. The comparison of quantum yield of decomposition of aromatic amino acids upon irradiation at 266 nm in O2 saturated solution have been made. It is the highest for tyrosine (19.7), whereas for tryptophan and phenylalanine is much lower (16.0 and 11.3, respectively) [8]. For free aromatic amino acids in water it was found that DA640 at the end of the pulse was the same in argon- and air-saturated solution [8]. It was assumed that this transient is due to the hydrated electron. In acidic condition, we did
ð15Þ ð16Þ
HA (aromatic amino acid) Because of the reactions of protons arising from the dissociation of acetic acid used as the solvent used in our experiment and protons arising from reactions (15) and (16) with hydrated electrons we did not observe any transient at 600–800 nm for aromatic amino acids (reaction 15 depends on the pKa). ð17Þ e þ H þ ! H aq
We did not observe any transient for collagen solution in acetic acid in 600–800 nm region. It is evidence that there is no hydrated electron in the irradiated collagen solution. In acidic collagen solution (pH < 7), hydrated electrons react rapidly with H+ ions as it has been shown in the reaction (17). The very active H radicals formed in this reaction may initiate several photochemical reactions and may give rise a solvent radical (acetic acid radical- R), which can react with the solute. _ þ RH _ ! COL H COL þ R
ð18Þ
ð19Þ Collagen-derived radicals had been found by Hawkins et al. using EPR spectroscopy [2]. They found signals in EPR spectra, which were assigned to the radical adduct of a large collagen-derived radical and also signals of a low molecular weight collagen-derived fragments. For collagen molecules the photochemical reactions maybe as follows: COL þ hm!1 COL 1 1
ð20Þ
COL ! fluorescence COL ! COLþ þ e
aq
ð21Þ ð22Þ
where COL represents collagen. In fact, fluorescence of collagen was observed in my previous work (excitation at 270 nm, emission at 305 nm) [3,21]. Hydrated electron was not detected by flash photolysis because of the reaction (17). Collagen radical cation may be a species absorbing at 295 nm probably due to photoionisation. However, due to the ground state absorption of proteins, the measurements of transient below 300 nm are not easy as well as the interpretation can be difficult.
A. Sionkowska / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45
To better understand the radical reactions in a collagen solution the reactions of hydrated electrons and OH radicals with collagen have been studied by pulse radiolysis. This method, do not usually yield information on the selectivity of damage at different sites within the molecule, unless it is possible to monitor specific absorptions from particular transients or products. For technical reasons, such an approach is usually only possible for aromatic and sulphur-containing residues. Most aliphatic sidechains and the carbon-centred radicals derived from hydrogen abstraction do not have readily detectable absorption. In pulse radiolysis, unlike laser flash photolysis where the solute is excited, the energy from the ionising radiation is absorbed by the most abundant species, which in dilute solutions will be the solvent. Upon absorption of the radiation the solvent-derived intermediates can interact with the solute thus forming solute transient intermediates. Hence, the choice of solvent is extremely important in determining the type of species formed. Solute excited states and radicals produced using pulse radiolysis can be formed via recombination, direct excitation, or energy transfer from excited solvent and sub-excitation electrons. The radiolysis of water occurs in two stages, firstly excited states, cations and electrons are produced (reaction (23)), then a variety of reactions occur, also generating the hydrogen atoms and the hydroxyl radical (reactions (24)–(27)) and the electron loses energy via excitation and ionisation of other molecules and becomes solvated (reaction (27)) H 2 O , H 2 O þ þ e þ H 2 O ð23Þ þ þ H O þ H O ! OH þ H O ð24Þ 2
2
In the absorption spectra of products resulting from the reaction of collagen with e aq no characteristic maximum absorption in UV and visible light has been observed. It is a good agreement with results published by Pietrucha et al. [40]. For model peptides it has been shown that the most reactive to the hydrated electron are: protonated imidazole ring of histidine, tryptophan residue, side –NH group of lysine and peptide bonds [41,42]. The reaction of the hydroxyl radicals with collagen leads to a transient spectrum which contains two bands. The first one at 320 nm and the second one at 405 nm (Fig. 2). The peak at 320 nm may come from radical of solvent (acetic acid radical). In Fig. 3, the transient absorption spectra resulting from the OH radicals that attack on 20 mM acetic acid (pH 3.3) saturated with N2O are shown for comparison. In the spectra one can see the transient suggesting the presence of free radicals. The maximum of the transient is located at about 320 nm. The acetic acid radicals were found by Metreveli et al. using ESR method for irradiated acetic acid solution [43]. These radicals (CH2COOH, R) may initiate many radical reactions in collagen. 0.12 0.1
Absorbance
42
ð26Þ
e ! e ðthermÞ ! eaq
ð27Þ
ð25Þ
2
These species can then rapidly react with each other so that further hydrogen atoms and hydrogen peroxide are generated: e þ Hþ ! H ð28Þ e aq þ eaq ! H2 þ 2OH OH þ OH ! H O
ð30Þ
2
2
ð31Þ
Many of the radicals formed will recombine to form water and the protons and hydroxide ions eventually neutralise one another. The solvated electron and hydrogen atom are extremely reactive reductants. The hydroxyl radical is a highly oxidising species. Predominantly, oxidising conditions are produced by saturating the solution with nitrous oxide (N2O) which reacts with the solvated electron to generate further oxidising hydroxyl radicals (reaction (32)). N O þ e þ H O ! N þ OH þ OH ð32Þ 2
aq
2
2
2
0.04
3
380
480
580
680
wavelength, nm Fig. 2. Transient absorption spectra resulting from the OH radicals attack on collagen solution in acetic acid (pH 3.3) saturated with N2O: curve 1, after the pulse of 6 ls; curve 2, after the pulse of 98 ls; and curve 3, after the pulse of 404 ls.
0.016
1 0.012
Absorbance
ð29Þ
0.06
0 280
aq
H þ H ! H2
0.08
0.02
3
H2 Oþ þ e ! H2 O H O ! OH þ H
1-after the pulse 6 μs 2-after the pulse 98 μs 3-after the pulse 404 μs
1
1- after the pulse 6 μs 2- after the pulse 34 μs 3- after the pulse 97 μs
2 0.008
0.004
0 280
3
330
380
430
480
wavelength, nm Fig. 3. Transient absorption spectra resulting from the OH radicals attack on 20 mM acetic acid (pH 3.3) saturated with N2O: curve 1, after the pulse of 6 ls; curve 2, after the pulse of 34 ls; and curve 3, after the pulse of 97 ls.
A. Sionkowska / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45
OH + CH3 COOH ! H2 O + CH2 COOH
ð33Þ
0.06
Reaction of OH radicals with tyrosine residues in collagen chains gives rise to Tyr phenoxyl radicals (absorption at 400 nm). This can occur via direct oxidation of the ring and subsequent deprotonation of the hydroxyl group. One of the major fates of this radical is self-dimerisation, with the formation of cross-linked product di-tyrosine. In the case of collagen the di-tyrosine cross-links were not found in collagen from rat tail tendon in acetic acid solution [23], but were found in collagen from calf skin irradiated in the presence of riboflavin as a photosenitizer in pH ca 7.4 [44]. In some cases, when oxidising conditions are required, milder oxidants may be needed, because the hydroxyl radical can react with the solute forming adducts as well as radical cations. Hydroxyl radicals can be converted into milder oxidants by the addition of halides, in our case we used KBr:
0.05
OH + Br ! Br + OH
Br + Br ! Br
ð35Þ
In Fig. 4, the transient spectra of collagen in the presence of KBr are presented. One band was observed at 380 nm in the transient spectra. Transient absorption spectra resulting from the OH radicals attack on 0.1 mM tyrosine and on 0.1 mM phenylalanine in 20 mM acetic acid (pH 3.3) saturated with N2O are presented for comparison in Figs. 5 and 6, respectively. The broad transient suggests that in the solution there are both, solvent and solute radicals. In general, it is assumed that the reactivity of biomolecules depends on the ionic conditions, which may change their charge and conformation. The unfolding of the protein molecule makes more group accessible for the reaction with photons. In the native state, the most of aromatic amino acids residues are situated in the hydrophobic core of the protein, so they are sterically hindered against the 0.25
1
Absorbance
0.2
0.15
0.1
2
1-after the pulse 1.9 μs 2-after the pulse 5.3 μs 3-after the pulse 19 μs
3
0.05
0 280
380
480
580
680
wavelength, nm Fig. 4. Transient absorption spectra resulting from the OH radicals attack on fish collagen solution in acetic acid (pH 3.3) saturated with N2O in the presence of KBr: curve 1, after the pulse of 1,9 ls; curve 2, after the pulse of 5.3 ls; and curve 3, after the pulse of 19 ls.
1-after the pulse 4.5 μs 2-after the pulse 16 μs
2
3-after the pulse 108 μs
0.04 0.03
3
0.02
0 280
330
380
430
480
wavelength, nm Fig. 5. Transient absorption spectra resulting from the OH radicals attack on 0.1 mM tyrosine in 20 mM acetic acid (pH 3.3) saturated with N2O: curve 1, after the pulse of 4.5 ls; curve 2, after the pulse of 16 ls; and curve 3, after the pulse of 108 ls. 0.06
ð34Þ
2
1
0.01
0.05
Absorbance
Absorbance
43
1-after the pulse 26 μs 2-after the pulse 132 μs 3-after the pulse 294 μs
1
0.04
2
0.03 0.02
3
0.01 0 280
300
320
340
360
380
400
420
wavelength, nm Fig. 6. Transient absorption spectra resulting from the OH radicals attack on 0.1 mM phenylalanine tyrosine in 20 mM acetic acid (pH 3.3) saturated with N2O: curve 1, after the pulse of 26 ls; curve 2, after the pulse of 132 ls; and curve 3, after the pulse of 294 ls.
attack of radicals. Schussler et al. [45] concluded on the basis on pulse radiolysis study on BSA that rate of radical reactions depends not only on amino acids composition of the protein, but on the ratio of the surface to the volume of the molecule. The greater is this ratio, the higher is the probability of radical reactions. In biological systems, radicals can be generated by multiple pathways. They can be formed by either the direct cleavage of bonds or by electron transfer reactions. Transfer of an electron to, or from a molecule results in the formation of radical anions or radical cations (for collagen the example is in reaction (22)). In most cases, these are relatively short-lived species which react rapidly with a range of targets to yield other radicals [41]. Radicals undergo a variety of reactions including hydrogen abstraction, electron transfer (oxidation or reduction of the molecule), addition, fragmentation, rearrangement, and dimerisation. These reactions are not only observed in vitro in the lab, all these reactions may occur in biological systems. In general, the nature of the radical formed on proteins depends on the nature and reactivity of the attacking agent. Free radical reactions in proteins play a major role in many oxidative processes in living body and may be implicated in a number of human diseases and ageing [9,41,42,2].
44
A. Sionkowska / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45
4. Conclusions In photochemical reactions, in collagen solution both free radicals and short-lived species are involved. The transient spectra of collagen solution (both argon and oxygen saturated) excited at 266 nm show two bands. One of them with maximum at 295 nm and the second one with maximum at 400 nm. The peak at 400 nm is due to the tyrosyl radicals. The first peak at the transient absorption spectra at 295 nm is probably due to the photoionisation of collagen. In the absorption spectra of products resulting from the reaction of collagen with e aq no characteristic maximum absorption in UV and visible light has been observed. The reaction of the hydroxyl radicals with collagen leads to a transient spectrum, which contains two bands. The first one at 320 nm and the second one at 405 nm. The peak at 320 nm may come from the acetic acid used as a solvent. Reaction of OH radicals with tyrosine residues in collagen chains gives rise to Tyr phenoxyl radicals (absorption at 400 nm). Acknowledgements Financial support from Scientific Research Committee (MNII-KBN, Grant No. 3T08E 3829), Poland, and European Commission (Transnational Access Grant) is gratefully acknowledged. Author thanks Dr. Ruth Edge and Dr. Suppiah Navaratnam for their help during the experiment. References [1] A.J. Bailey, Molecular mechanisms of ageing in connective tissues: review, Mechanisms of Ageing and Development 1222 (2001) 735– 755. [2] C.L. Hawkins, M.J. Davies, Oxidative damage to collagen and related substrates by metal/hydrogen peroxide systems: random attack or site-specific damage?, Biochimica et Biophysica Acta 1360 (1997) 84– 96. [3] A. Sionkowska, A. Kaminska, C.A. Miles, A.J. Bailey, The effect of UV radiation on the structure and properties of collagen, Polimery 6 (2001) 379–389. [4] G.J. Smith, New trends in photobiology: photodegradation of keratin and other structural proteins, Journal of Photochemistry and Photobiology Part B: Biology 27 (1995) 187–198. [5] D.N. Nikogosyan, H. Gorner, Photolysis (193 nm) of aliphatic amino acids in aqueous solution, Journal of Photochemistry and Photobiology Part B: Biology 30 (1995) 189–193. [6] H. Gorner, D.N. Nikogosyan, Indirect 248 nm 20 ns photolysis of aliphatic amino acids in aqueous solution, Journal of Photochemistry and Photobiology Part B: Biology 39 (1997) 84–89. [7] D.N. Nikogosyan, H. Gorner, Towards the laser photochemistry of the cornea: studies of the most common highly absorbing aliphatic amino acids in collagen, Journal of Photochemistry and Photobiology Part B: Biology 47 (1998) 63–67. [8] D.N. Nikogosyan, H. Gorner, Photolysis of aromatic amino acids in aqueous solution by nanosecond 248–193 nm laser light, Journal of Photochemistry and Photobiology Part B: Biology 13 (1992) 219– 234. [9] M.J. Davies, Singlet oxygen-mediated damage to proteins and its consequenced, Biochemical and Biophysical Research Communications 305 (2003) 761–770.
[10] F.H. Silver, Connective tissue structure, in: Biological Materials: Structure Mechanical Properties and Modelling of Soft Tissues, University Press, New York, USA, 1987, p. 7. [11] A.J. Bailey, R.G. Paul, Collagen – is not so simple protein, Journal of the Society of Leather Technologists and Chemists 82 (1998) 104–108. [12] R. Van der Rest, M. Garrone, Collagen family of proteins, FASEB Journal 5 (1991) 2814–2823. [13] D.O. Ellis, S. McGavin, The structure of collagen – on X-ray study, Journal of Ultrastructure Research 32 (1970) 191–211. [14] J. Prockop, A. Fertala, The collagen fibril: the almost crystalline structure, Journal of Structural Biology 122 (1998) 111–118. [15] J. Bella, M. Eaton, B. Brodsky, H.M. Berman, Crystal and molecular structure of a collagen-like peptide at 1.9 A resolution, Science 266 (1994) 75–81. [16] A. Huc, Collagen biomaterials characteristics and applications, Journal of the American Leather Chemists Association 80 (1985) 195–212. [17] K. Matsumoto, T. Nakamura, Y. Shimizu, H. Ueda, T. Sekine, Y. Yamamoto, T. Yiyotami, Y. Takomoto, A novel surgical material made from collagen with high mechanical strength: a collagen sandwich membrane, OSAIO Journal 45 (4) (1999) 288–292. [18] D.R. Cooper, R.J. Davidson, The effect of ultraviolet irradiation on soluble collagen, Biochemistry Journal 97 (1965) 139–147. [19] M. Menter, J.D. Williamson, K. Carlyle, C.L. Moore, I. Willis, Photochemistry of Type I acid soluble calf skin collagen: dependence on excitation wavelength, Photochemistry and Photobiology 62 (1995) 402–408. [20] D.V. Crabtree, E. Fujimori, Fluorescent labeled collagen: age related differences and fluorescence changes during the lag phase of fibril formation, Biopolymers 19 (1980) 1081–1091. [21] A. Sionkowska, A. Kamin´ska, Changes induced by ultraviolet light in fluorescence of collagen in the presence of b-carotene, Journal of Photochemistry and Photobiology A-Chemistry 120 (1999) 207–210. [22] A. Torikai, H. Shibata, Effect of ultraviolet radiation on photodegradation of collagen, Journal of Applied Polymer Science 73 (3) (1999) 1257–1266. [23] C.A. Miles, A. Sionkowska, S.L. Hulin, T.J. Sims, N.C. Avery, A.J. Bailey, Identification of an intermediate state in the helix-coil degradation of collagen by ultraviolet light, Journal of Biological Chemistry 275 (42) (2000) 33014–33020. [24] Y. Kato, S. Uchida, S. Kawakishi, Oxidative degradation of collagen and its model peptide by ultraviolet irradiation, Journal of Agricultural Food Chemistry 40 (1992) 373–379. [25] A. Sionkowska, T.J. Wess, Mechanical properties of UV-irradiated collagen Type I from rat tail tendon, International Journal of Biological Macromolecules 34 (2004) 9–12. [26] K. Vizarova, D. Bakos, M. Rehakova, V. Macho, Modification of layered atelocollagen by ultraviolet irradiation and chemical crosslinking: structure stability and mechanical properties, Biomaterials 15 (1994) 1082–1086. [27] A. Sionkowska, Modification of collagen films by ultraviolet irradiation, Polymer Degradation and Stability 68 (2000) 147–151. [28] M.S. Kital, Y.M. Sobou, A.P. Sviridov, A.I. Omelchenko, Manifestation of photochemical reactions in bone tissue on exposure to the ultraviolet radiation of an excimer laser, Biophysics 41 (1996) 1151– 1157. [29] Y. Tsuboi, N. Kimoto, M. Kabeshita, A. Itaya, Pulsed laser deposition of collagen and keratin, Journal of Photochemistry and Photobiology A-Chemistry 145 (2001) 209–214. [30] K. Tsunoda, D. Kumaki, T. Takahashi, H. Yajiama, T. Ishii, H. Itoh, Characterization of materials ejected by excimer laser ablation of hydrated collagen gel, Applied Surface Science 197–198 (2002) 782– 785. [31] S. Lazare, V. Tokarev, A. Sionkowska, M. Wisniewski, Surface foaming of collagen, chitosan and other biopolymer films by KrF excimer laser ablation in the photomechanical regime, Applied Physics A: Materials Science and Processing 81 (2005) 465–470.
A. Sionkowska / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45 [32] D.J. Holder, D. Allan, E.J. Land, S. Navaratnam, in: Proceedings of European Particle Accelerator Conference, Paris, 2002, pp. 2804– 2806.. [33] J. Butler, B.W. Hodgson, B.M. Hoey, E.J. Land, J.S. Lea, E.J. Lindley, F.A.P. Rushton, A.J. Swallow, Experimental studies of some moderately fast processes initiated by radiation, Radiation Physics and Chemistry 34 (1989) 633–646. [34] G.E. Adams, J.W. Boag, B.D. Michael, J. Currant, The pulse radiolysis of aqueous solutions of thiocyanate ion, in: J.H. Baxendale, M. Ebert, J.P. Keene, A.J. Swallow (Eds.), Pulse Radiolysis, Academic Press, London, 1965, pp. 117–129. [35] G.V. Buxton, C.R. Stuart, Re-evaluation of thiocyanate dosimeter for pulse radiolysis, Journal of the Chemical Society-Faraday Transactions 91 (1995) 279–281. [36] S. Navaratnam, G.O. Phillips, Application of Laser Flash Photolysis to Study Photoreceptor Pigments, in: F. Lenci, F. Ghetti, G. Colombetti, D.P. Hader, Pill-Soon Song (Eds.), Biophysics of photoreceptors and photomovements in microorganisms, Plenum, New York, 1991, pp. 139–148. [37] S. Navaratnam, in: H.H. Tonnesen (Ed.), Photostability of Drugs and Drug Formulations, CRC Press, Boca Raton, 2004, pp. 255– 284. [38] Y.P. Tsentalovich, O.B. Morozova, Laser flash photolysis and time resolved CIDNP study of photoreaction of 2,2 0 -dipyridyl
[39]
[40]
[41] [42]
[43]
[44]
[45]
45
with N-acetyl tyrosine in aqueous solutions, Journal of Photochemistry and Photobiology A-Chemistry 131 (2000) 33–40. D.N. Nikogosyan, H. Gorner, Laser-induced photodecomposition of aminoacids and peptides: extrapolation to corneal collagen, IEEE Journal of Selected Topics in Quantum Electronics 5 (1999) 1107– 1115. K. Pietrucha, L. Gora, C.J. Doillon, Reaction of a hydrated electron with gentamycin and collagen – a pulse radiolysis study, Radiation Physics and Chemistry 47 (1996) 93–97. M.J. Davies, The oxidative environment and protein damage – review, Biochimica et Biophysica Acta 1703 (2005) 93–109. M.J. Davies, Photo-oxidation of proteins and its role in cataractogenesis, Journal of Photochemistry and Photobiology B: Chemistry 63 (2001) 114–125. N. Metreveli, L. Namicheishvili, K. Jariashvili, E. Chikvaidze, G. Mrevlishvili, Microcalorimetric and electron spin resonance study of the influence of ultraviolet irradiation on collagen, Bulletin of the Georgian Academy of Science 165 (3) (2002) 552–555. Y. Kato, K. Uchida, S. Kawakishi, Aggregation of collagen exposed to UVA in the presence of riboflavin: a plausible role of tyrosine modification, Photochemistry and Photobiology 59 (3) (1994) 343–349. H. Schussler, S. Navaratnam, L. Distel, Pulse radiolysis study on histones and serum albumin under different ionic condition, Radiation Physics and Chemistry 61 (2001) 123–128.
Flash photolysis and pulse radiolysis studies on collagen Type I in acetic acid solution Alina Sionkowska
*
Faculty of Chemistry, N. Copernicus University, Gagarin 7, 87-100 Torun, Poland Received 8 November 2005; received in revised form 28 December 2005; accepted 9 January 2006 Available online 28 February 2006
Abstract An investigation of the photochemical properties of collagen Type I in acetic acid solution was carried out using nanosecond laser irradiation. The transient spectra of collagen solution excited at 266 nm show two bands. One of them with maximum at 295 nm and the second one with maximum at 400 nm. The peak at 400 nm is assigned to tyrosyl radicals. The first peak of the transient absorption spectra at 295 nm is probably due to photoionisation producing collagen radical cation. The transient for collagen solution in acetic acid at 640 nm was not observed. It is evidence that there is no hydrated electron in the irradiated collagen solution. The reactions of hydrated electrons and OH radicals with collagen have been studied by pulse radiolysis. In the absorption spectra of products resulting from the reaction of collagen with e aq no characteristic maximum absorption in UV and visible light region has been observed. In the absorption spectra of products resulting from the reaction of the hydroxyl radicals with collagen two bands have been observed. The first one at 320 nm and the second one at 405 nm. Reaction of OH radicals with tyrosine residues in collagen chains gives rise to Tyr phenoxyl radicals (absorption at 400 nm). 2006 Elsevier B.V. All rights reserved. Keywords: Collagen; UV irradiation; Transient spectra; Photochemistry; Pulse radiolysis
1. Introduction Limited investigations of photo damage to human skin have revealed a down-regulation of collagen synthesis with a consequent overall loss of collagen [1]. However, all these studies are based on the assumption that the damage to the dermal collagen is through the normal degradative pathway of the remodelling process and scant attention has been paid to the possibility of direct damage to the collagen through free radicals or singlet oxygen formed during irradiation [2]. Structural proteins such as collagen and elastin undergo varied sequences of photochemical reactions when they are exposed to ultraviolet and longer wavelength radiation [2–4]. Because of the insolubility of structural proteins photochemists have been denied the use of *
Tel.: +48 56 6214302; fax: +48 566542477. E-mail address: [email protected].
1011-1344/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2006.01.007
many standard chemical analytical tools and methods. Even though the complexity of structural proteins limits the experimental techniques which can be employed, many insights of their photodegradation have been gained by studying model systems such as aromatic and aliphatic amino acids [5–8] or the photochemical behaviour of water soluble proteins [9]. Collagen is the most abundant biopolymer in animals where it provides the principal structural and mechanical support [10]. The main amino acids in collagen are: glycine, proline, hydroxyproline and alanine, with a small number of residues of phenylalanine and tyrosine. The ordered triple helical structure of collagen is stabilized by both interchain hydrogen bonds and by structural water molecules [11–15]. Collagen is readily available, non-toxic and has got the fibril architecture that is inherent in natural tissues. Therefore, collagen provides an excellent basis for biomaterials such as arterial prostheses and artificial skin [16,17]. There are 20 genetically distinct collagen types [11,12].
A. Sionkowska / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45
Type I collagen from young rat-tail tendon is soluble in diluted acetic acid solution. The same collagen Type I in bone is cross-linked and insoluble. Many studies have demonstrated modification of collagen induced by UV radiation: it has been shown that in solution collagen loses the ability to form natural fibrils after irradiation [18]. In addition, the fluorescence observed after UV irradiation is due to the presence of photo-products of phenylalanine and tyrosine residues in the collagen amino acid sequence [3,19–21]. Photocross-linking and photodegradation of collagen may also occur on exposure to UV radiation [3,22–24]. Destruction of Type I collagen under UVA (335–400 nm) and broadband solar simulating radiation (290–400 nm) has also been demonstrated [19]. The influence of UV radiation on collagen in films has also been investigated [25–30]. After UV irradiation of thin collagen films random-coil domains increased on the surface [27,29]. The material ejected from hydrated collagen gel by laser ablation was partially changed from the original collagen gel. In the ejected materials, low molecular weight polypeptides were found [30,31]. The aim of this work was to, study the photochemistry and radiation chemistry of collagen in acetic acid solutions. The main point of the work is to show what kind of radicals and short living species are involved in photochemical reaction in collagen. The paper deals with photoionisation and photodecomposition measurements of collagen in acetic acid solution at pH < 4 in the absence and presence of oxygen. The direct action of UV radiation is a novel approach to the photodegradation of collagen in acetic acid solution. Solutions of collagen were irradiated and the transient absorption spectra were recorded. Collagen absorbs mainly in the far UV so 266 nm laser irradiation from the Nd:YAG laser was employed. To better understand the reaction of hydrated electron and OH radicals with collagen the pulse radiolysis technique was employed. 2. Materials and methods 2.1. Sample preparation Collagen was obtained in our laboratory from tail tendons of young albino rats. Briefly, tendons were excised and washed in distilled water, and blended in a Waring blender in 0.4 M acetic acid, samples were then spun at 8000 rpm in a centrifuge and the soluble fraction were decanted. The concentration of collagen solution was determined using burette method. The preparation of collagen solution for flash photolysis and pulse radiolysis measurements was carried out with special care in order to avoid any structural changes. 2.2. Pulse radiolysis The pulse radiolysis experiments were carried out at the Free Radical Research Facility at Daresbury Laboratory,
39
UK, with a 12 MeV Radiation Dynamics Ltd. (UK) electron linear accelerator. We used single pulses of duration 0.22–2 ls and with a peak current of about 30 mA. The accelerator is normally operated at 10 Hz but the single pulse mode is achieved by modifying the pulses to the electron gun [32]. The detection system consists of a Xe arc lamp with a pulsing unit, high radiance Kratos monochromator and quartz optics. The sample cell, constructed by Spectrosil quartz, had an optical path length of 25 mm [33]. Optical transmissions at various wavelengths selected with the monochromator were observed as a function of time before and after the pulse using photoelectric detection. The output of the photomultiplier (EMI9558Q) was displayed on a Tektronix TDS 380 digitizing oscilloscope then transferred to a PC and fitted using in house software. Absorbed doses were determined from the transient ðSCNÞ2 formation in airsaturated 10 mM KSCN as described by Adams et al. [34], but using the updated Ge value of 2.59 · 104 m2 J1 obtained by Buxton and Stuart [35], G being the radiation chemical yield of ðSCNÞ2 and e its molar absorption coefficient at 475 nm. Saturation of such solutions with N2O results in a doubling of the ðSCNÞ2 yield. In water, three different primary radicals are produced by irradiation: solvated electrons (e aq ) and hydrogen atoms (H), which are reducing species and hydroxyl radicals (OH), which are oxidising species. For generating almost exclusively OH, aqueous solutions were saturated with N2O which converts e aq into hydroxyl radicals by dissociative electron attachment: ð1Þ N O þ e þ H O ! N þ OH þ OH 2
aq
2
2
2.3. Laser flash photolysis Laser flash photolysis experiments were also carried out at the FRRF facility at Daresbury Laboratory using a Nd:YAG laser. The system essentially consists of a Qswitched Nd:YAG (JK Lasers 2000 Series) laser capable of delivering up to 1 J of energy at 1064 nm in pulses of 12 ns duration as the excitation source [36]. The fundamental bean can be frequency doubled, tripled and quadrupled to deliver light of 532, 355 and 266 nm, respectively. The harmonies are separated using a beam splitter. The sample under investigation is contained in a quartz cell and changed after each laser pulse using a remote controlled flow system. The analysing beam generated by a xenon lamp, which can be pulsed to increase the analysing light up to 200 times in the UV region, passes through the sample at right angles to the laser beam. A shutter, which opens a few microseconds before the laser pulse and closes again after the event under examination is completed, and appropriate filters are placed between the analysing lamp and sample in order to minimise the photolysis of the sample by the monitoring light. The monitoring light is then dispersed in wavelength by a monochrometer and then onto
40
A. Sionkowska / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45
a photodetector. The transmittance of light at this wavelength is detected by the photodetector before, during and after the laser pulse. The detector is also connected to an automatic back-off box which enables changes in transmittance to be observed by feeding back a signal equal and opposite to the detector anode current prior to the laser pulse, thus maintaining the anode current close to zero. The signal from the detector is captured and stored using a programmable digital oscilloscope. Back-off and energy meter readings are digitised using an Analogue to Digital Converter (ADC) attached to a DI-AN data acquisition system. Data acquisition and processing are obtained by repeating the above procedure at successive wavelengths [37]. Transient profiles taken at different wavelengths can be used to generate a difference spectrum at various times after the laser pulse. Thus, it is possible to determine whether more than one species contributes to the spectrum since if there are different species, and their lifetimes differ, their individual spectral contributions can be separated by recording spectra at different time delays. The transient profiles observed are usually either due to excited states or free radicals. First-order decay kinetics often indicate that the transient is an excited state, whereas free radicals usually decay by second-order kinetics. There are exceptions such as the first-order process when a radical decays intramolecularly, producing a different radical with a different absorption spectrum. In a deaerated solution with no quencher present a triplet state will decay radiationlessly via solvent induced deactivation (reaction (2)) and follow first-order kinetics, as described below (Eqs. (3)–(5)) 3
k1
M ! M 3
ð2Þ
d½ M t ¼ k 1 ½3 M t dt
ln½3 M t ¼ ln½3 M 0 k 1 t
ð4Þ 3
Where [ M*]0 is the initial concentration of M*. However, the change in absorbance (DAt) is proportional to the concentration of 3M* so DA can be substituted for 3M*. ln DAt ¼ ln DA0 k 1 t
ð5Þ
Where DA0 is the difference absorbance immediately after the laser pulse. Hence, a plot of lnDAt against t will give a straight line with gradient k1, and intercept lnDA0. The computer software uses an iterative least squares analysis to extract k1 and DA0 values from the integrated rate Eq. (6) which is a rearrangement of Eq. (5), with a constant DA1 added to account for any residual change in absorbance remaining after the decay of the species. DAt ¼ DA0 ek1 t þ DA1
i
Mixed first-order growth and decay kinetics can also be analysed in this way. The computer software in Daresbury can calculate k1 and k2 and DA1 and DA2 for a biexponential decay or growth and decay using Eq. (8). DAt ¼ DA1 ek1 t þ DA2 ek2 t
ð8Þ
A second-order decay arises in bimolecular reactions where both reactants are present in equal or approximately equal amounts. This occurs for triplet–triplet annihilation, radical–radical recombination, or when another solute (Q) quenches the initial excited state or radical. For triplet–triplet annihilation or radical–radical recombination both reactants are identical and thus, the rate constant is given by Eq. (9). d½3 M t ¼ k½3 M t2 dt
ð9Þ
Substituting DAt/el for [3M*] and integrating gives Eq. (10), so that a plot of 1/DAt against t will give a straight line with slope 2 k/el, where k is the second-order rate constant, and intercept 1/DA0. 1 1 2kt ¼ þ DAt DA0 el
ð10Þ
The software evaluates k/el from Eq. (11) (a rearrangement of Eq. (10)), again with the addition of the constant DA1. DA0 þ DA1 ð11Þ DAt ¼ 1 þ DA0 2kt=el
ð3Þ
Where k1 is the first-order rate constant for the decay of 3 * M , and [3M*]t is the concentration of 3M* after time t. Integration of Eq. (3) gives: 3
When more than one transient decaying with first-order kinetics contributes to the transient decay profile, the absorbance change is described by a sum of exponential decays, as in Eq. (7). X DAi eki t ð7Þ DAt ¼
ð6Þ
3. Results and discussion Model studies of the photodamage in proteins consider the photochemical reactions of free amino acids and of small peptides. Approximation of the results and mechanism to proteins is usually done. However, photodamage of proteins cannot be directly predicted from the behaviour of free amino acids or even isolated amino acids forming the peptide bonds. For this reason specific studies are required for each protein. Collagen absorbs mainly in the far UV but has an absorption tail out as far as 380 nm. The absorption spectra of collagen solution showed a wide peak in the region 250– 290 nm The main chromophores absorbing in the UV region are aromatic amino acids [3]. The absorbance at 266 nm was 0.3 (UV absorption spectra not shown). Laser irradiation with 266 nm from the Nd:YAG laser was employed. The transient absorption spectra obtained on subjecting an Ar saturated acetic acid solution of collagen to 266 nm laser irradiation are shown in Fig. 1. Two bands
A. Sionkowska / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45
not observe any transient at wavelength longer than 600 nm for tyrosine or for phenylalanine. The work published by Nikogosyan for photolysis of aromatic amino acids in aqueous solution [8] showed the minimum set of photochemical reactions. The initial step was: ð14Þ HA ! HAþ þ e
0.04
1
Absorbance
0.03
1- after the pulse of 35 μs 2- after the pulse of 310 μs
2
3- after the pulse of 878 μs
0.02
aq
HAþ ! A þ Hþ HAþ þ H2 O ! HA –OH þ Hþ
3 0.01
0 250
350
450
550
650
wavelength, nm Fig. 1. Transient absorption spectra of collagen in Argon saturated solution in acetic acid (pH 2.2): curve 1, after the pulse of 35 ls; curve 2, after the pulse of 310 ls; and curve 3, after the pulse of 878 ls.
were observed at the end of the pulse with two maxima at 295 nm and 400 nm. The species with kmax = 295 nm could be a tyrosine triplet. Tyrosine triplet usually gives a peak at 300 nm [8,9] however, the transient at 295 nm seems too long-lived for a triplet state. An obvious candidate for the second species is the tyrosyl radical (Tyrosine phenoxyl radicals) which is known to have an absorption band centred at 405 nm (absorption coefficient 2.8 · 103 M1 cm1) [38]. Tyrosine (TyrOH) is likely to deprotonate very rapidly on forming the neutral tyrosyl radical TyrO. The transient absorption spectra of collagen in air saturated acetic acid solution excited at 266 nm have shown two bands. One of them with maximum at 295 nm and the second one with maximum at 400 nm. This suggests that the bands are not due to the triplet state. The triplet state is usually quenched by oxygen. 3 TyrOH þ O ! TyrO þ Hþ þ O ð12Þ 2
3 3
2
TyrOH can absorb a second photon: TyrOH þ hm ! TyrO þ e þ Hþ aq
41
ð13Þ
To explain what kind of species are responsible for the transient the comparison with transient spectra of aromatic amino acids has been made. The photochemical properties of aromatic amino acids have been rather well investigated in the last three decades by Nikogosyan et al. [8,39]. They found that mechanism of photoionisation of the aromatic amino acids is monophotonic for kexc = 193 nm and mainly biphotonic for kexc = 248 nm. The comparison of quantum yield of decomposition of aromatic amino acids upon irradiation at 266 nm in O2 saturated solution have been made. It is the highest for tyrosine (19.7), whereas for tryptophan and phenylalanine is much lower (16.0 and 11.3, respectively) [8]. For free aromatic amino acids in water it was found that DA640 at the end of the pulse was the same in argon- and air-saturated solution [8]. It was assumed that this transient is due to the hydrated electron. In acidic condition, we did
ð15Þ ð16Þ
HA (aromatic amino acid) Because of the reactions of protons arising from the dissociation of acetic acid used as the solvent used in our experiment and protons arising from reactions (15) and (16) with hydrated electrons we did not observe any transient at 600–800 nm for aromatic amino acids (reaction 15 depends on the pKa). ð17Þ e þ H þ ! H aq
We did not observe any transient for collagen solution in acetic acid in 600–800 nm region. It is evidence that there is no hydrated electron in the irradiated collagen solution. In acidic collagen solution (pH < 7), hydrated electrons react rapidly with H+ ions as it has been shown in the reaction (17). The very active H radicals formed in this reaction may initiate several photochemical reactions and may give rise a solvent radical (acetic acid radical- R), which can react with the solute. _ þ RH _ ! COL H COL þ R
ð18Þ
ð19Þ Collagen-derived radicals had been found by Hawkins et al. using EPR spectroscopy [2]. They found signals in EPR spectra, which were assigned to the radical adduct of a large collagen-derived radical and also signals of a low molecular weight collagen-derived fragments. For collagen molecules the photochemical reactions maybe as follows: COL þ hm!1 COL 1 1
ð20Þ
COL ! fluorescence COL ! COLþ þ e
aq
ð21Þ ð22Þ
where COL represents collagen. In fact, fluorescence of collagen was observed in my previous work (excitation at 270 nm, emission at 305 nm) [3,21]. Hydrated electron was not detected by flash photolysis because of the reaction (17). Collagen radical cation may be a species absorbing at 295 nm probably due to photoionisation. However, due to the ground state absorption of proteins, the measurements of transient below 300 nm are not easy as well as the interpretation can be difficult.
A. Sionkowska / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45
To better understand the radical reactions in a collagen solution the reactions of hydrated electrons and OH radicals with collagen have been studied by pulse radiolysis. This method, do not usually yield information on the selectivity of damage at different sites within the molecule, unless it is possible to monitor specific absorptions from particular transients or products. For technical reasons, such an approach is usually only possible for aromatic and sulphur-containing residues. Most aliphatic sidechains and the carbon-centred radicals derived from hydrogen abstraction do not have readily detectable absorption. In pulse radiolysis, unlike laser flash photolysis where the solute is excited, the energy from the ionising radiation is absorbed by the most abundant species, which in dilute solutions will be the solvent. Upon absorption of the radiation the solvent-derived intermediates can interact with the solute thus forming solute transient intermediates. Hence, the choice of solvent is extremely important in determining the type of species formed. Solute excited states and radicals produced using pulse radiolysis can be formed via recombination, direct excitation, or energy transfer from excited solvent and sub-excitation electrons. The radiolysis of water occurs in two stages, firstly excited states, cations and electrons are produced (reaction (23)), then a variety of reactions occur, also generating the hydrogen atoms and the hydroxyl radical (reactions (24)–(27)) and the electron loses energy via excitation and ionisation of other molecules and becomes solvated (reaction (27)) H 2 O , H 2 O þ þ e þ H 2 O ð23Þ þ þ H O þ H O ! OH þ H O ð24Þ 2
2
In the absorption spectra of products resulting from the reaction of collagen with e aq no characteristic maximum absorption in UV and visible light has been observed. It is a good agreement with results published by Pietrucha et al. [40]. For model peptides it has been shown that the most reactive to the hydrated electron are: protonated imidazole ring of histidine, tryptophan residue, side –NH group of lysine and peptide bonds [41,42]. The reaction of the hydroxyl radicals with collagen leads to a transient spectrum which contains two bands. The first one at 320 nm and the second one at 405 nm (Fig. 2). The peak at 320 nm may come from radical of solvent (acetic acid radical). In Fig. 3, the transient absorption spectra resulting from the OH radicals that attack on 20 mM acetic acid (pH 3.3) saturated with N2O are shown for comparison. In the spectra one can see the transient suggesting the presence of free radicals. The maximum of the transient is located at about 320 nm. The acetic acid radicals were found by Metreveli et al. using ESR method for irradiated acetic acid solution [43]. These radicals (CH2COOH, R) may initiate many radical reactions in collagen. 0.12 0.1
Absorbance
42
ð26Þ
e ! e ðthermÞ ! eaq
ð27Þ
ð25Þ
2
These species can then rapidly react with each other so that further hydrogen atoms and hydrogen peroxide are generated: e þ Hþ ! H ð28Þ e aq þ eaq ! H2 þ 2OH OH þ OH ! H O
ð30Þ
2
2
ð31Þ
Many of the radicals formed will recombine to form water and the protons and hydroxide ions eventually neutralise one another. The solvated electron and hydrogen atom are extremely reactive reductants. The hydroxyl radical is a highly oxidising species. Predominantly, oxidising conditions are produced by saturating the solution with nitrous oxide (N2O) which reacts with the solvated electron to generate further oxidising hydroxyl radicals (reaction (32)). N O þ e þ H O ! N þ OH þ OH ð32Þ 2
aq
2
2
2
0.04
3
380
480
580
680
wavelength, nm Fig. 2. Transient absorption spectra resulting from the OH radicals attack on collagen solution in acetic acid (pH 3.3) saturated with N2O: curve 1, after the pulse of 6 ls; curve 2, after the pulse of 98 ls; and curve 3, after the pulse of 404 ls.
0.016
1 0.012
Absorbance
ð29Þ
0.06
0 280
aq
H þ H ! H2
0.08
0.02
3
H2 Oþ þ e ! H2 O H O ! OH þ H
1-after the pulse 6 μs 2-after the pulse 98 μs 3-after the pulse 404 μs
1
1- after the pulse 6 μs 2- after the pulse 34 μs 3- after the pulse 97 μs
2 0.008
0.004
0 280
3
330
380
430
480
wavelength, nm Fig. 3. Transient absorption spectra resulting from the OH radicals attack on 20 mM acetic acid (pH 3.3) saturated with N2O: curve 1, after the pulse of 6 ls; curve 2, after the pulse of 34 ls; and curve 3, after the pulse of 97 ls.
A. Sionkowska / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45
OH + CH3 COOH ! H2 O + CH2 COOH
ð33Þ
0.06
Reaction of OH radicals with tyrosine residues in collagen chains gives rise to Tyr phenoxyl radicals (absorption at 400 nm). This can occur via direct oxidation of the ring and subsequent deprotonation of the hydroxyl group. One of the major fates of this radical is self-dimerisation, with the formation of cross-linked product di-tyrosine. In the case of collagen the di-tyrosine cross-links were not found in collagen from rat tail tendon in acetic acid solution [23], but were found in collagen from calf skin irradiated in the presence of riboflavin as a photosenitizer in pH ca 7.4 [44]. In some cases, when oxidising conditions are required, milder oxidants may be needed, because the hydroxyl radical can react with the solute forming adducts as well as radical cations. Hydroxyl radicals can be converted into milder oxidants by the addition of halides, in our case we used KBr:
0.05
OH + Br ! Br + OH
Br + Br ! Br
ð35Þ
In Fig. 4, the transient spectra of collagen in the presence of KBr are presented. One band was observed at 380 nm in the transient spectra. Transient absorption spectra resulting from the OH radicals attack on 0.1 mM tyrosine and on 0.1 mM phenylalanine in 20 mM acetic acid (pH 3.3) saturated with N2O are presented for comparison in Figs. 5 and 6, respectively. The broad transient suggests that in the solution there are both, solvent and solute radicals. In general, it is assumed that the reactivity of biomolecules depends on the ionic conditions, which may change their charge and conformation. The unfolding of the protein molecule makes more group accessible for the reaction with photons. In the native state, the most of aromatic amino acids residues are situated in the hydrophobic core of the protein, so they are sterically hindered against the 0.25
1
Absorbance
0.2
0.15
0.1
2
1-after the pulse 1.9 μs 2-after the pulse 5.3 μs 3-after the pulse 19 μs
3
0.05
0 280
380
480
580
680
wavelength, nm Fig. 4. Transient absorption spectra resulting from the OH radicals attack on fish collagen solution in acetic acid (pH 3.3) saturated with N2O in the presence of KBr: curve 1, after the pulse of 1,9 ls; curve 2, after the pulse of 5.3 ls; and curve 3, after the pulse of 19 ls.
1-after the pulse 4.5 μs 2-after the pulse 16 μs
2
3-after the pulse 108 μs
0.04 0.03
3
0.02
0 280
330
380
430
480
wavelength, nm Fig. 5. Transient absorption spectra resulting from the OH radicals attack on 0.1 mM tyrosine in 20 mM acetic acid (pH 3.3) saturated with N2O: curve 1, after the pulse of 4.5 ls; curve 2, after the pulse of 16 ls; and curve 3, after the pulse of 108 ls. 0.06
ð34Þ
2
1
0.01
0.05
Absorbance
Absorbance
43
1-after the pulse 26 μs 2-after the pulse 132 μs 3-after the pulse 294 μs
1
0.04
2
0.03 0.02
3
0.01 0 280
300
320
340
360
380
400
420
wavelength, nm Fig. 6. Transient absorption spectra resulting from the OH radicals attack on 0.1 mM phenylalanine tyrosine in 20 mM acetic acid (pH 3.3) saturated with N2O: curve 1, after the pulse of 26 ls; curve 2, after the pulse of 132 ls; and curve 3, after the pulse of 294 ls.
attack of radicals. Schussler et al. [45] concluded on the basis on pulse radiolysis study on BSA that rate of radical reactions depends not only on amino acids composition of the protein, but on the ratio of the surface to the volume of the molecule. The greater is this ratio, the higher is the probability of radical reactions. In biological systems, radicals can be generated by multiple pathways. They can be formed by either the direct cleavage of bonds or by electron transfer reactions. Transfer of an electron to, or from a molecule results in the formation of radical anions or radical cations (for collagen the example is in reaction (22)). In most cases, these are relatively short-lived species which react rapidly with a range of targets to yield other radicals [41]. Radicals undergo a variety of reactions including hydrogen abstraction, electron transfer (oxidation or reduction of the molecule), addition, fragmentation, rearrangement, and dimerisation. These reactions are not only observed in vitro in the lab, all these reactions may occur in biological systems. In general, the nature of the radical formed on proteins depends on the nature and reactivity of the attacking agent. Free radical reactions in proteins play a major role in many oxidative processes in living body and may be implicated in a number of human diseases and ageing [9,41,42,2].
44
A. Sionkowska / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45
4. Conclusions In photochemical reactions, in collagen solution both free radicals and short-lived species are involved. The transient spectra of collagen solution (both argon and oxygen saturated) excited at 266 nm show two bands. One of them with maximum at 295 nm and the second one with maximum at 400 nm. The peak at 400 nm is due to the tyrosyl radicals. The first peak at the transient absorption spectra at 295 nm is probably due to the photoionisation of collagen. In the absorption spectra of products resulting from the reaction of collagen with e aq no characteristic maximum absorption in UV and visible light has been observed. The reaction of the hydroxyl radicals with collagen leads to a transient spectrum, which contains two bands. The first one at 320 nm and the second one at 405 nm. The peak at 320 nm may come from the acetic acid used as a solvent. Reaction of OH radicals with tyrosine residues in collagen chains gives rise to Tyr phenoxyl radicals (absorption at 400 nm). Acknowledgements Financial support from Scientific Research Committee (MNII-KBN, Grant No. 3T08E 3829), Poland, and European Commission (Transnational Access Grant) is gratefully acknowledged. Author thanks Dr. Ruth Edge and Dr. Suppiah Navaratnam for their help during the experiment. References [1] A.J. Bailey, Molecular mechanisms of ageing in connective tissues: review, Mechanisms of Ageing and Development 1222 (2001) 735– 755. [2] C.L. Hawkins, M.J. Davies, Oxidative damage to collagen and related substrates by metal/hydrogen peroxide systems: random attack or site-specific damage?, Biochimica et Biophysica Acta 1360 (1997) 84– 96. [3] A. Sionkowska, A. Kaminska, C.A. Miles, A.J. Bailey, The effect of UV radiation on the structure and properties of collagen, Polimery 6 (2001) 379–389. [4] G.J. Smith, New trends in photobiology: photodegradation of keratin and other structural proteins, Journal of Photochemistry and Photobiology Part B: Biology 27 (1995) 187–198. [5] D.N. Nikogosyan, H. Gorner, Photolysis (193 nm) of aliphatic amino acids in aqueous solution, Journal of Photochemistry and Photobiology Part B: Biology 30 (1995) 189–193. [6] H. Gorner, D.N. Nikogosyan, Indirect 248 nm 20 ns photolysis of aliphatic amino acids in aqueous solution, Journal of Photochemistry and Photobiology Part B: Biology 39 (1997) 84–89. [7] D.N. Nikogosyan, H. Gorner, Towards the laser photochemistry of the cornea: studies of the most common highly absorbing aliphatic amino acids in collagen, Journal of Photochemistry and Photobiology Part B: Biology 47 (1998) 63–67. [8] D.N. Nikogosyan, H. Gorner, Photolysis of aromatic amino acids in aqueous solution by nanosecond 248–193 nm laser light, Journal of Photochemistry and Photobiology Part B: Biology 13 (1992) 219– 234. [9] M.J. Davies, Singlet oxygen-mediated damage to proteins and its consequenced, Biochemical and Biophysical Research Communications 305 (2003) 761–770.
[10] F.H. Silver, Connective tissue structure, in: Biological Materials: Structure Mechanical Properties and Modelling of Soft Tissues, University Press, New York, USA, 1987, p. 7. [11] A.J. Bailey, R.G. Paul, Collagen – is not so simple protein, Journal of the Society of Leather Technologists and Chemists 82 (1998) 104–108. [12] R. Van der Rest, M. Garrone, Collagen family of proteins, FASEB Journal 5 (1991) 2814–2823. [13] D.O. Ellis, S. McGavin, The structure of collagen – on X-ray study, Journal of Ultrastructure Research 32 (1970) 191–211. [14] J. Prockop, A. Fertala, The collagen fibril: the almost crystalline structure, Journal of Structural Biology 122 (1998) 111–118. [15] J. Bella, M. Eaton, B. Brodsky, H.M. Berman, Crystal and molecular structure of a collagen-like peptide at 1.9 A resolution, Science 266 (1994) 75–81. [16] A. Huc, Collagen biomaterials characteristics and applications, Journal of the American Leather Chemists Association 80 (1985) 195–212. [17] K. Matsumoto, T. Nakamura, Y. Shimizu, H. Ueda, T. Sekine, Y. Yamamoto, T. Yiyotami, Y. Takomoto, A novel surgical material made from collagen with high mechanical strength: a collagen sandwich membrane, OSAIO Journal 45 (4) (1999) 288–292. [18] D.R. Cooper, R.J. Davidson, The effect of ultraviolet irradiation on soluble collagen, Biochemistry Journal 97 (1965) 139–147. [19] M. Menter, J.D. Williamson, K. Carlyle, C.L. Moore, I. Willis, Photochemistry of Type I acid soluble calf skin collagen: dependence on excitation wavelength, Photochemistry and Photobiology 62 (1995) 402–408. [20] D.V. Crabtree, E. Fujimori, Fluorescent labeled collagen: age related differences and fluorescence changes during the lag phase of fibril formation, Biopolymers 19 (1980) 1081–1091. [21] A. Sionkowska, A. Kamin´ska, Changes induced by ultraviolet light in fluorescence of collagen in the presence of b-carotene, Journal of Photochemistry and Photobiology A-Chemistry 120 (1999) 207–210. [22] A. Torikai, H. Shibata, Effect of ultraviolet radiation on photodegradation of collagen, Journal of Applied Polymer Science 73 (3) (1999) 1257–1266. [23] C.A. Miles, A. Sionkowska, S.L. Hulin, T.J. Sims, N.C. Avery, A.J. Bailey, Identification of an intermediate state in the helix-coil degradation of collagen by ultraviolet light, Journal of Biological Chemistry 275 (42) (2000) 33014–33020. [24] Y. Kato, S. Uchida, S. Kawakishi, Oxidative degradation of collagen and its model peptide by ultraviolet irradiation, Journal of Agricultural Food Chemistry 40 (1992) 373–379. [25] A. Sionkowska, T.J. Wess, Mechanical properties of UV-irradiated collagen Type I from rat tail tendon, International Journal of Biological Macromolecules 34 (2004) 9–12. [26] K. Vizarova, D. Bakos, M. Rehakova, V. Macho, Modification of layered atelocollagen by ultraviolet irradiation and chemical crosslinking: structure stability and mechanical properties, Biomaterials 15 (1994) 1082–1086. [27] A. Sionkowska, Modification of collagen films by ultraviolet irradiation, Polymer Degradation and Stability 68 (2000) 147–151. [28] M.S. Kital, Y.M. Sobou, A.P. Sviridov, A.I. Omelchenko, Manifestation of photochemical reactions in bone tissue on exposure to the ultraviolet radiation of an excimer laser, Biophysics 41 (1996) 1151– 1157. [29] Y. Tsuboi, N. Kimoto, M. Kabeshita, A. Itaya, Pulsed laser deposition of collagen and keratin, Journal of Photochemistry and Photobiology A-Chemistry 145 (2001) 209–214. [30] K. Tsunoda, D. Kumaki, T. Takahashi, H. Yajiama, T. Ishii, H. Itoh, Characterization of materials ejected by excimer laser ablation of hydrated collagen gel, Applied Surface Science 197–198 (2002) 782– 785. [31] S. Lazare, V. Tokarev, A. Sionkowska, M. Wisniewski, Surface foaming of collagen, chitosan and other biopolymer films by KrF excimer laser ablation in the photomechanical regime, Applied Physics A: Materials Science and Processing 81 (2005) 465–470.
A. Sionkowska / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 38–45 [32] D.J. Holder, D. Allan, E.J. Land, S. Navaratnam, in: Proceedings of European Particle Accelerator Conference, Paris, 2002, pp. 2804– 2806.. [33] J. Butler, B.W. Hodgson, B.M. Hoey, E.J. Land, J.S. Lea, E.J. Lindley, F.A.P. Rushton, A.J. Swallow, Experimental studies of some moderately fast processes initiated by radiation, Radiation Physics and Chemistry 34 (1989) 633–646. [34] G.E. Adams, J.W. Boag, B.D. Michael, J. Currant, The pulse radiolysis of aqueous solutions of thiocyanate ion, in: J.H. Baxendale, M. Ebert, J.P. Keene, A.J. Swallow (Eds.), Pulse Radiolysis, Academic Press, London, 1965, pp. 117–129. [35] G.V. Buxton, C.R. Stuart, Re-evaluation of thiocyanate dosimeter for pulse radiolysis, Journal of the Chemical Society-Faraday Transactions 91 (1995) 279–281. [36] S. Navaratnam, G.O. Phillips, Application of Laser Flash Photolysis to Study Photoreceptor Pigments, in: F. Lenci, F. Ghetti, G. Colombetti, D.P. Hader, Pill-Soon Song (Eds.), Biophysics of photoreceptors and photomovements in microorganisms, Plenum, New York, 1991, pp. 139–148. [37] S. Navaratnam, in: H.H. Tonnesen (Ed.), Photostability of Drugs and Drug Formulations, CRC Press, Boca Raton, 2004, pp. 255– 284. [38] Y.P. Tsentalovich, O.B. Morozova, Laser flash photolysis and time resolved CIDNP study of photoreaction of 2,2 0 -dipyridyl
[39]
[40]
[41] [42]
[43]
[44]
[45]
45
with N-acetyl tyrosine in aqueous solutions, Journal of Photochemistry and Photobiology A-Chemistry 131 (2000) 33–40. D.N. Nikogosyan, H. Gorner, Laser-induced photodecomposition of aminoacids and peptides: extrapolation to corneal collagen, IEEE Journal of Selected Topics in Quantum Electronics 5 (1999) 1107– 1115. K. Pietrucha, L. Gora, C.J. Doillon, Reaction of a hydrated electron with gentamycin and collagen – a pulse radiolysis study, Radiation Physics and Chemistry 47 (1996) 93–97. M.J. Davies, The oxidative environment and protein damage – review, Biochimica et Biophysica Acta 1703 (2005) 93–109. M.J. Davies, Photo-oxidation of proteins and its role in cataractogenesis, Journal of Photochemistry and Photobiology B: Chemistry 63 (2001) 114–125. N. Metreveli, L. Namicheishvili, K. Jariashvili, E. Chikvaidze, G. Mrevlishvili, Microcalorimetric and electron spin resonance study of the influence of ultraviolet irradiation on collagen, Bulletin of the Georgian Academy of Science 165 (3) (2002) 552–555. Y. Kato, K. Uchida, S. Kawakishi, Aggregation of collagen exposed to UVA in the presence of riboflavin: a plausible role of tyrosine modification, Photochemistry and Photobiology 59 (3) (1994) 343–349. H. Schussler, S. Navaratnam, L. Distel, Pulse radiolysis study on histones and serum albumin under different ionic condition, Radiation Physics and Chemistry 61 (2001) 123–128.