Journal of Photochemistry and Photobiology B: Biology 80 (2005) 115–121 www.elsevier.com/locate/jphotobiol
Photodegradation and photosensitization of mycosporine-like amino acids Kenia Whitehead *, John I. Hedges School of Oceanography, University of Washington, Seattle, WA 98195, USA Received 24 November 2004; received in revised form 16 March 2005; accepted 26 March 2005 Available online 11 May 2005
Abstract The photodegradation and photosensitization of several mycosporine-like amino acids (MAAs) were investigated. The photodegradation of the MAA, palythine, was tested with three photosensitizers: riboflavin, rose bengal and natural seawater. For comparison of degradation rates, the riboflavin-mediated photosensitization of six other MAAs was also examined. When riboflavin was used as a photosensitizer in distilled water, MAAs were undetectable after 1.5 h. Palythine showed little photodegradation when rose bengal was added as the photosensitizer (k = 0.12 · 103 m2 kJ1). Palythine dissolved in natural seawater containing high nitrate concentrations also showed slow photodegradation rate constants (k = 0.26 · 103 m2 kJ1) over a 24-h period of constant irradiation. Similar experiments in deep seawater with porphyra-334 and shinorine resulted in 75% of the initial MAA remaining after 4 h of irradiation and rates of 0.018 and 0.026 · 103 m2 kJ1, respectively. Experiments conducted in deep seawater with riboflavin additions resulted in photodegradation rate constants between 0.77 · 103 and 1.19 · 103 m2 kJ1 for shinorine and porphyra334, respectively. Photoproduct formation appeared to be minimal with the presence of a dehydration product of the cycloheximine ring structure indicated as well as the presence of amino acids. Evidence continues to build for the role of MAAs as potent and stable UV absorbers. This study further highlights the photostability of several MAAs in both distilled and seawater in the presence of photosensitizers. 2005 Elsevier B.V. All rights reserved. Keywords: Mycosporine-like amino acids; MAAs; Riboflavin; Photosensitization
1. Introduction The twenty-plus mycosporine-like amino acids (MAAs) now known to be synthesized by marine algae have absorption maxima in the UV-B (280–315 nm) and UV-A (315–400 nm) regions between 310 and 360 nm [1–4]. The basic MAA structure is an aminocyclohexenone or aminocycloheximine ring substituted with an imine or imino alcohol ([1]; Table 1). Differences in MAA absorption spectra reflect variations in the attached side groups to the core ring structure. Aquatic * Corresponding author. Present address: Institute for Systems Biology, 1441 N. 34th Street, Seattle, WA 98103, USA. Tel.: +1 206 732 1366; fax: +1 206 732 1299. E-mail address:
[email protected] (K. Whitehead).
1011-1344/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2005.03.008
organisms generally contain a variety of MAAs with each absorbing over a range of UV wavelengths to create a broad-band UV filter [5]. Chromatographic analyses have identified MAAs in all major divisions of algae, phytoplankton and cyanobacteria and a photoprotective role has been assigned to the MAAs [2,6]. Many studies have focused on the presence and potential roles of MAAs. However, fewer studies have looked at the photoreactivity and photosensitization of MAAs [7–9]. In an aqueous solution of the MAA, shinorine, no significant change in concentration was observed after 24 h of UV-A and UV-B irradiation [10]. MAAs extracted from a red alga, Gracilaria cornea, were irradiated at 75 C in distilled water and again no significant change in MAA absorption was observed although a slight increase in absorption at lower
116
K. Whitehead, J.I. Hedges / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 115–121
Table 1 Basic structure with R-groups, wavelength maximum (kmax, nm) and extinction coefficients (M cm1) of the MAAs ([1] and references therein) used in photodegradation experiments (table adapted from [20]) Base structure
R 6 5
HO HO
1 4
OMe 2 3
NH
MAA
R
kmax
e
Palythine Palythene Asterina-330 Palythinol Palythenic acid Shinorine Porphyra-334
N N–CH@CHCH3 (trans) N–CH2CH2 OH N–CH(CH3)CH2 OH N–C(CO2H)@CHCH3 N–CH(CO2H)CH2 OH N–CH(CO2H)CH(OH)CH3
320 360 330 332 337 334 334
36,200 50,000 43,800 43,500 29,200 44,668 42,300
COOH
2. Materials and methods 2.1. Photodegradation experiments Photodegradation experiments were conducted at the University of South Carolina (Columbia, SC, USA) in the laboratory of Dr. R. Benner. A Suntest XLS+ solar simulator with a 1500-W xenon arc lamp (Atlas Material Testing Technologies LLC, USA) was used for all incubation experiments (Fig. 1). The solar simulator was fitted with a UV filter to remove radiation at wavelengths below 290 nm thereby faithfully representing natural sunlight. All experiments were conducted at the same total intensity (400 W m2). Experiments varied in length from 2 to 60 h and were maintained at 18 C. Incubations were carried out in small quartz flasks (maximum volume of 60 ml). MAA standards were dissolved in either distilled water or deep seawater. The purity of the standards was assessed by absorption spectra and by LC/MS analysis both of which gave no indication of contaminants. For all experiments, the initial concentration of MAA standard was
4.0
3.0
-2
-1
Irradiance (W m nm )
wavelengths was detected [11]. The photophysical parameters of the MAAs, porphyra-334 and shinorine (kmax = 334 nm) were studied and both compounds were determined to be highly photostable with 96–98% of the absorbed energy released into the medium as heat [12,13]. The cis–trans isomers, palythene and usujirene, showed photoisomerization during exposure to monochromatic radiation with little loss of total MAA [14]. This high degree of photostability of MAAs supports their role as sunscreen compounds. While pure solutions of MAAs have shown a high degree of photostability, there has been mounting evidence that they are susceptible to photosensitization. Previous experiments showed the photooxidation of MAAs in the presence of other organic compounds to occur in a temperature- and wavelength-dependent manner [15,16]. Photochemical reaction in freshwater of the fungalderived mycosporine, mycosporine glutamine in the presence of a photosensitizer produced 2-hydroxy glutaric acid in addition to the aminocyclohexenone ring [15]. Hence, MAAs are photostable and their photodegradation does not appear to occur via a direct mechanism, but appears to require the presence of a photosensitizing agent for indirect photodegradation [13]. In light of these previous experiments, we conducted several photodegradation incubations to examine the photosensitization of the MAA, palythine (kmax = 320 nm). Palythine was chosen as it represents the basic cycloheximine unit common to most MAAs and we possessed adequate amounts of purified standard material. Palythine was photosensitized by the addition of riboflavin or rose bengal to the distilled water medium. Several other MAAs were also tested using riboflavin as the photosensitizer and their photodegradation rate constants are presented and compared. Experiments were also conducted in a seawater medium to measure photodegradation rate constants with naturally occurring photosensitizers (e.g., nitrate and other components) present in deep seawater [17,18]. The potential production of MAA-derived photoproducts was also monitored.
2.0
1.0
0.0 250
300
350
400
450
500
550
600
650
700
750
Wavelength (nm)
Fig. 1. Spectral irradiance of the xenon lamp used in the solar simulator. A filter was used to cutoff all radiation below 290 nm.
K. Whitehead, J.I. Hedges / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 115–121
0.1 lM. Riboflavin or rose bengal were added as photosensitizers at a molar ratio of 10:1 photosensitizer to MAA (initial concentration of 1 lM). Riboflavinmediated experiments were conducted in distilled water with seven different MAA standards: palythine, palythinol, shinorine, palythenic acid, asterina-330, palythene and porphyra-334 (Table 2). In the interest of saving standard material, the natural seawater experiments were conducted with only three MAAs: palythine, shinorine and porphyra-334, while only palythine was photosensitized with rose bengal in distilled water. The deep seawater was collected near the BermudaAtlantic time series (BATS) station (3216.7 0 N, 6858.3 0 W) from a depth of 1365 m on July 9, 2001 by Dr. Benner. The seawater was ultrafiltered so that dissolved organic compounds greater than 30 kDa was removed. Deep seawater was used because the various inorganic and organic components are more representative of naturally occurring photosensitizers in marine systems. In particular, the high nitrate concentration (16.7 lM) provides a photochemical source for reactive oxygen species (mainly hydroxyl radicals) via direct photochemical reactions [19,20]. All samples were stored in combusted glass vials with acid washed teflon tops. Absorption spectra were measured on a Shimadzu UV-1601 UV–Vis spectrophotometer using 1-cm quartz cells. 2.2. LC–MS analysis of MAAs Frozen samples were brought back to the University of Washington (Seattle, Washington) for LC/MS analysis. Duplicate samples were analyzed without any pretreatment or pre-concentration and 20 ll amounts were injected directly onto the column. Separation and quanTable 2 Experimentally determined photodegradation rate constants, k (m2 kJ1), in the presence of molecular oxygen for each experiment and photosensitized with riboflavin, rose bengal or seawater MAA
Photosensitizer
k · 103 (m2 kJ1)
Palythine Palythine Palythine Palythinol Shinorine Palythenic acid Asterina-330 Palythene Porphyra-334 Palythine Shinorine Porphyra-334 Shinorine Porphyra-334
None Rose bengal Riboflavin Riboflavin Riboflavin Riboflavin Riboflavin Riboflavin Riboflavin Seawater Seawater Seawater Seawater + Riboflavin Seawater + Riboflavin
N.D. 0.12 3.17 4.18 7.39 5.52 7.00 36.8 4.98 0.26 .018 .026 .77 1.19
The calculated rates are statistically significant (r2 = 0.87–0.99; p < 0.01). N.D., no degradation detected.
117
tification of the MAAs was carried out on a Thermo Finnegan model LCQ LC/MS (San Jose, California) using a modified version of the LC/MS method we have previously described [21]. Calibration curves for MAA quantification were established using MAA standards with concentrations determined spectrophotometrically using the known wavelength maxima and extinction coefficients for each MAA. The purity of the standards was confirmed by spectrophotometry and mass spectral examination. LC separation was carried out in a Develosil RP-aqueous column (spheri-5, 150 · 2-mm i.d.) at a flow rate of 250 ll min1 over a gradient between water and MeOH, both modified with 0.05% formic acid. The gradient started at 100% (water) for 2 min, decreased to 75% A from 2 to 5 min, decreased to 60% from 5–7 min, decreased to 35% from 7 to 12 min, and decreased again to 25% from 12 to 14 min. The column was then re-equilibrated for 10 min in 100% water prior to the next injection. Absorption spectra were obtained by a diode array detector (DAD) scanning between 250 and 600 nm at 1 nm increments. Flow was directed from the DAD into the electrospray source of the MS without splitting. The electrospray source was operated at a capillary voltage at 4.8 kV, N2 gas flow at 70 units, auxiliary N2 gas flow at 10 units, and a capillary temperature at 335 C. For each spectrum, three ÔmicroscansÕ were collected with a maximum ion collection time of 50 ms. MAAs were detected by selective ion monitoring (SIM) which was oscillated with full mass spectral scans from 150 to 1000 m/z. The ions monitored by SIM corresponded to the [M + H]+ for the various MAAs used in the experiments and are indicated in Table 1. The SIM detection limit for the MAAs in seawater was 0.1 ng with an average precision of ±2% of the measured value. Palythine was used to optimize the ion optics and trap conditions. The mass spectrometer was operated at unit mass resolution and calibrated using a standard solution prescribed by Thermo Finnigan (caffeine, MRFA and Ultramark). All solvents used were of environmental analytical grade from Burdick and Jackson.
3. Results and discussion 3.1. Photosensitization Initial experiments conducted here were consistent with previous studies indicating that appreciable MAA photodegradation requires light, oxygen and a strong photosensitizer [15]. Experiments carried out in an anoxic system with palythine dissolved in distilled water showed no measurable photodegradation (data not shown). A loss of 4% in the absorption of palythine (kmax = 320 nm) was observed after 24 h of irradiation at 400 W m2 in oxygenated water (Fig. 2). There is also a slight increase in absorption at lower wavelengths
118
K. Whitehead, J.I. Hedges / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 115–121
indicating the possible photoproduction of compounds absorbing at lower wavelengths (see below). Addition of rose bengal as a source of singlet oxygen resulted in the degradation of palythine (Fig. 3) with a loss of 50% after 2 h of exposure (total dose = 3000 kJ m2). Whereas the addition of the photosensitizer, riboflavin, resulted in the complete degradation of all palythine within 1 h (total dose = 1440 kJ m2; Fig. 3). The rapid, riboflavin-mediated photodegradation was investigated in several other MAAs for comparison of their relative photosensitivity. Addition of riboflavin at a 10:1 molar ratio resulted the complete photodegradation of all MAAs tested within 1.5 h (dose after 77 min = 1848 kJ m2; Fig. 4). However, the dose at which the individual MAAs fell below mass spectral
0.024
t=0h t = 24 h
Absorption (A.U.)
0.018
SIM detection varied. Palythene had the fastest degradation and fell below detection after only 15 min of irradiation (dose = 240 kJ m2). Dark controls retained 82–100% of the initial concentration (Fig. 4; triangles). Photodegradation experiments with palythine, shinorine and porphyra-334 conducted in deep seawater demonstrated substantial loss (Fig. 5). Palythine was almost completely degraded over the 24 h incubation with little loss in the dark control (Fig. 5A). Shinorine and porphyra-334 were not photodegraded as rapidly, with 80% of the initial dissolved porphyra-334 remaining after 24 h (Fig. 5B). This experiment was repeated in deep seawater with riboflavin added at a 10:1 molar ratio of riboflavin to porphyra-334. Porphyra-334 was then completely photodegraded by the end of the incubation time (Fig. 5B). For all deep seawater experiments, at least 90% of the initially added MAA concentration remained after the experiment in the dark control. 3.2. Experimental rate constants Dosage-based experimental rate constants (k, m2 kJ1) were calculated for all experiments. Generally, photooxidation rate constants are assumed to be pseudo first-order [22]. Construction of VanÕt Hoff plots (not shown) gave reaction orders between 1.00 and 1.42 illustrating the validity of the pseudo first-order approach. The first-order rate constants were estimated using a 2parameter decay function:
0.012
0.006
0 280
310
340
370
400
Wavelength (nm)
Fig. 2. Absorption spectra of palythine irradiated at 400 W m2 for initial (t = 0 h; thick line) and final (t = 24 h; thin line) time points.
Normalized concentration
1
riboflavin rose bengal
0.8
0.6
0.4
0.2
0 0
500
1000
1500
2000
2500
3000
-2
Dose (kJ m )
Fig. 3. The photodegradation of palythine photosensitized with riboflavin (circles; n = 12; r2 = 0.92; p < 0.01) and with rose bengal (diamonds; n = 12; r2 = 0.89; p < 0.01). Palythine concentrations are presented as values normalized to the initial concentration. Dark controls remained within 5% of initial concentrations.
k ¼ 1=DI lnð½MAA=½MAA0 Þ;
ð1Þ
where [MAA] is the MAA concentration after irradiation at a given dose, [MAA]0 is the initial concentration prior to irradiation, k is the experimentally determined photodegradation rate constant (m2 kJ1) and DI is the total dose (kJ m2). The calculated photodegradation rates and the range of r2 values are given in Table 2. Photosensitization and subsequent photodegradation of palythine by rose Bengal was slow relative to that observed with the addition of riboflavin; 0.12 versus 3.17 m2 kJ1, respectively. The resistance of palythine to degradation by singlet oxygen is in agreement with recent reports of antioxidant activity in another MAA, mycosporine-glycine [23–25]. This may also indicate that MAAs are more effectively degraded by type I photooxidation rather than the singlet oxygen type II mechanism. Riboflavin has been shown to be a source of both singlet oxygen and the superoxide radical anion and may be acting in a similar manner to promote the photosensitive oxidation of MAA. Photodegradation rate constants varied between 3.17 and 7.39 · 103 m2 kJ1 for MAAs photosensitized with riboflavin (Table 2) with the exception of palythene with k = 36.8 · 103 m2 kJ1.
Normalized concentration
Normalized concentration
Normalized concentration
K. Whitehead, J.I. Hedges / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 115–121
porphyra-334
palythinol
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0
200
400
600
800
1000 1200 1400
0.0
0
200
shinorine 1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2 0
200
400
600
400
800
1000 1200 1400
0.0
0
200
400
asterina-330 1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2 0
200
400
600
800
1000 1200 1400
800
600
800
1000 1200 1400
palythene
1.0
0.0
600
palythenic acid
1.0
0.0
119
1000 1200 1400
-2
Dose (kJ m )
0.0
0
200
400
600
800
1000 1200 1400
-2
Dose (kJ m )
Fig. 4. The decrease in MAA concentration over dose for six MAAs tested in distilled water and photosensitized with riboflavin (circles). MAA concentrations are presented as values normalized to the initial concentration. The dark control for each MAA is indicated by a triangle. Photodegradation rate constants and r2 are presented in Table 1.
The order of magnitude increase in the photodegradation of palythene may be attributed to the extra double bond in the R-group (Table 1) which would be attractive to electrophiles such as hydroxyl or peroxy radicals. Palythenic acid also contains a double bond in the R-group (Table 1), however, the adjacent carbonyl carbon would contribute to decreasing the nucleophilicity of the double bond and reducing its attractiveness to electrophiles. The differences in the photodegradation rates appear to be related to the chemistry of the R-group and its relation to the photosensitization reaction with the simplest MAA, palythine, having the slowest degradation rate. Experiments conducted in seawater with palythine, shinorine and porphyra showed slow photodegradation ranging from 0.018 to 0.26 · 103 m2 kJ1. The addition of riboflavin to the seawater system with shinorine and porphyra gave an increase in photodegradation (0.77– 1.19 · 103 m2 kJ1) relative to the non-photosensitized deep seawater, but were below the rate constants observed in distilled water with riboflavin present (Table 2). The decrease in riboflavin-sensitized photodegradation in deep seawater may be due to the abundance of
other substrates (e.g., components within dissolved organic matter pool) available to quench the excited triplet state of the photosensitizer. Overall, rapid photodegradation of MAAs requires a strong photosensitizer such as riboflavin, while more natural photosensitizer sources such as the components present in seawater will photodegrade MAAs more slowly over a period of days under high irradiance conditions. 3.3. MAA-derived photoproducts Besides MAA quantification, the LC/MS data were also examined for the presence of MAA photodegradation products whose potential presence was suggested by a slight increase in absorbance at lower wavelengths (
