Photocatalytic decomposition of pollen allergenic extracts of Cupresus Arizonica and Platanus Hybrida

Photocatalytic decomposition of pollen allergenic extracts of Cupresus Arizonica and Platanus Hybrida E. Jimenez-Relinquea, M. Sapiñaa, R. Nevshupaa, E. Romanb, M. Castellotea,* a

Spanish National Research Council, Institute of Construction Science “Eduardo Torroja“ (IETcc-

CSIC), C/Serrano Galvache, 4, 28033 Madrid, Spain b

Spanish National Research Council, Institute of Materials Science of Madrid (ICMM-CSIC),

C/Sor Juana Inés de la Cruz, 3, 28049 Madrid, Spain *The author to whom correspondence should be addressed. E-mail: [email protected]

Abstract The effect of TiO2 photocatalytic treatment on allergen proteins of pollen was evaluated. The allergenic activity and chemical composition of pollen allergenic extracts (PAE) of two species: Cupressus Arizonica and Platanus Hybrida acerifolia were characterized using three experimental techniques: scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and Fluorescence spectrophotometry with the fluorescein diacetate probe (FDA). SEM and XPS experiments have shown that the total amount of organic matter of PAE decreased during the photocatalytic treatment. Furthermore, FDA fluorescence probe provided an indication that the enzymatic activity was completely inhibited after photocatalytic treatment. This result demonstrated evidences of the efficiency of the photocatalysis process in deactivation of pollen allergens.

Keywords: Photocatalysis, pollen allergen, Cupressus Arizonica, Platanus Hybrida, XPS, FDA

1. Introduction Humans health can be considerably influenced by allergies that can be induced either directly by airborne pollen or in combination with the common pollutants in urban air, indirectly by increasing the risk of atopic sensitization and aggravating the symptoms in sensitized subjects [1, 2]. The cost for the global economy related to respiratory allergic diseases has been estimated to be as high as €3 billion per year [3, 4]. Furthermore, these diseases cause around 250.000 avoidable asthma deaths in the world. Though certain reductions in morbidity and mortality associated with exposures to pollen in indoor environments, e.g., buildings, vehicles cabins etc., can be achieved by air filtering, this widely used 1   

technique can be only modestly effective in reducing adverse allergy and asthma outcomes and it is not very effective in reducing acute health symptoms in subjects with allergies and asthma [5]. Inadequate maintenance of the filtering system may even become a source of pollutants [6]. Evidently, air filtering cannot mitigate the problems associated with airborne pollen in outdoor environment. A good alternative to conventional air filtering can be air cleaning by heterogeneous photocatalysis (HPhC), which can be used for elimination of biological and chemical pollutants in both indoor and outdoor applications [7-9]. Our recent exploratory study [10] has shown that photocatalysis can also reduce the number of pollen particles in aqueous solutions and on mortars specimens. Here the question arises: can allergic activity of pollen be deactivated by HPhC? Allergic activity is associated with enzymatic proteins such as pectate lyase, inhibitor of invertase, etc. Depending on the species, these proteins are predominantly localized in endoplasmic reticulum, Golgi cisternae and vesicles [11] situating in the central cytoplasmic part or in the polysaccharide matrix of internal stratum of a sporoderm – intine [12]. Normally, intine is protected by a hard outer stratum – exine – formed by sporopollenin [13] that is extremely resistant to chemical degradations. However, during hydration in water or humid air, initially tough exine is disrupted and shed that bares intine (See Supplementary materials). The latter consists of various layers composed mainly of pectine, cellulose, callose and mucilage and, to a smaller extent, of proteins, pigments and aromatic compounds [13, 14]. Considering that chemical resistance of intine is not too high, it is reasonable to expect that it can be prone to photocatalytic degradation. However, this question has not been studied so far. Therefore, the present work is aimed at studying whether HPhC is effective for degradation of pollen allergenic extracts (PAE) mainly composed of the pollen core such as intine, mucilage and allergenic proteins for two species: Cupressus Arizonica (Cypress Arizona) and Platanus Hybrida (acerifolia) (Sycamore/London Plane). These species were selected considering their important negative impact on human health in causing polyposis in Mediterranean countries and their extensive use in medicine for diagnosis and immunotherapy purposes.

2.

Materials and methods

2.1

Materials and procedure

Lyophilized pollen allergenic extracts (PAE) of Cupressus Arizonica and Platanus Hybrida

(acerifolia), hereinafter referred to as Cup. and Pla., correspondingly, in a form of cake hermetically sealed in vials were purchased from Greerlab. Immediately before the experiments 2.5 ml of extra pure (18 MΩ-cm) water was added to a vial to obtain a liquid extract. PAE contained disintegrated pollen grains without exine.   2   

Aeroxide® TiO2 P25 powder supplied by Evonik was used as a photocatalyst (PhC).Two kinds of photocatalytic experiments have been carried out in this study. In the first one, supported photocatalyst was used. For this purpose, a drop of 30 µL of TiO2 P25 suspension (6 g/100 ml) was extended and dried over the surface of a gilded glass support or aluminium sample carriers for Xray photoelectron spectroscopy (XPS) and Energy-Dispersive X-ray Spectroscopy (EDS) measurements, respectively. Then PAE (30 µl) was placed on the top of the PhC layer and dried. In the second one, for fluorescein diacetate probe (FDA) analysis, the photocatalyst (0.0033 g) was added to the PAE solution (1 ml) to form suspension. In this study, four test configurations, listed in Table 1, have been performed. This allowed to study not only the effect of photocatalysis, by contrasting cases III with IV, but also discrimination of the photocatalytic reactions from photolysis (I vs. II) and chemical reactivity of PhC (I vs. III). LEDs with sharp emission in the UV region at 365 nm and power of 5 W were used for illumination during 24 h in all cases. The experiments were carried out at ambient temperature and pressure and the relative humidity of air in the range 30 – 40%. Table 1. Nomenclature of the experimental configurations

2.2

Designation

I

II

III

IV

Configuration

PAE

PAE + UV

PAE/PhC

PAE/PhC + UV

Characterization techniques

2.2.1 Chemical composition Chemical composition of PAE on supported photocatalyst samples for various experimental configurations was studied using XPS and EDS. Bearing in mind that pollen grains were disintegrated during preparation of the pollen core extract and the suspension containing the compounds from the pollen core was quite homogeneous, both of these surface sensitive techniques are suitable for characterization of chemical changes in a thin layer of PAE deposited on various substrates. The XPS measurements were performed in an ultrahigh vacuum (UHV) system with a base pressure of 1×10-8 Pa using Mg Kα line (1253.6 eV) of incident X-ray radiation. The angle between the hemispherical analyser and the plane of the sample surface was 60°. The survey spectra were recorded with pass energy of 40 eV and energy step of 0.25 eV. The high resolution scans were measured for the main elements (C 1s, O 1s, N 1s, Ti 2p and Au 4f) with energy pass of 15 eV. The binding energy (BE) scale was calibrated with respect to the Au 4f at 84 eV. Before the elemental surface composition was quantified, the contribution of Mg Kα satellite line was subtracted and the background was removed using Shirley’s routine. The relative elemental 3   

concentrations were determined from the areas of the corresponding peaks using sensitivity factors known from the literature [15-17]. While XPS analysis provides average elemental composition of the surface area of approx. 5 mm2, EDS coupled with SEM allows visualizing the distribution of chemical elements on a micrometric scale. To make the PAE conductive for EDS, an Au layer was deposited on the top of the samples.

2.2.3. Fluorescein diacetate probe (FDA) for enzymatic activity analysis The enzymatic activity of the PAE as function of various factors (Table 1) was determined using FDA. The fluorochromatic reaction yielded bright green fluorescence product, Fluorescein (excitation λexc=450 nm and emission λem=517 nm), being the result of enzymes activity related with hydrolysis reactions (Fig. 1) [8, 18]. This analysis was carried out for the experiments with photocatalyst in suspension. For this purpose, after the treatment and removal of PhC from the suspension by filtering, 2 mg fluorescent diacetate and 0.17 g sucrose were added to 1 ml of the PAE solution. After 3 hours stirring at room temperature the fluorescence intensity was measured using a fluorescence spectrophotometer LS55 and analysed in the range from 480 to 680 nm.

H2O +  sucrose  FDA  (Colorless )

Hydrolysis   

Fluorescein (yellow‐ green fluorescence )

 

Fig. 1. Reaction scheme of hydrolysis catalysed fluorescein diacetate 3.

Results

3.1 SEM-EDS Figure 2 displays SEM images (grey scale) and corresponding mapping of C distribution (red scale) (taken here as the indicative of the presence of PAE extract) over the sample surfaces for both species (Cup. and Pla.) at the four experimental configurations (I to IV). Cup. PAE has a fibre-like structure (Fig. 2 a) that remained almost unchanged after UV illumination (test II, Fig. 2 b). This structure disappeared when PAE was deposited on a PhC (Fig. 2 c), although carbon-rich zones can still be observed. After HPhC treatment, these zones nearly vanished and the C distribution became similar to that on the Al substrate. Pla. PAE has a granular microstructure on both Al substrate (Fig. 2 e) and PhC (Fig. 2 g). After UV illumination some carbon-depleted zones were formed, mainly on the periphery of the coated area 4   

(Fig. 2 f). After photocatalytic treatment the microstructure completely vanished (Fig. 2 h), whereas carbon mean concentration drastically decreased.

Fig. 2 (colour). Combined images of SEM (grey scale) and carbon mapping (red scale) measured by EDS for both PAEs and four experimental configurations. Looking at configuration IV for different elements, it is worth mentioning that the carbon decrease is clearly produced on the PhC surfaces (see figure 3, where element distribution on the sample of Pla. after the test IV is given). It can be seen that C distribution is almost the negative image of Ti distribution. Carbon was found mainly on the uncovered Al substrate. Similar analysis was carried out on both bare Al substrates and bare supported PhC coating showing no important variation in C distribution that was uniform on the whole surface. It can be concluded that without a PhC, UV irradiation has virtually no effect on the total amount of carbon even after 24 hours of exposure. However, when a PAE deposited onto a PhC was exposed to UV radiation for the same period of time the total amount of carbon significantly decreased.

5   

Fig. 3 (colour). Combined SEM-EDS images showing distributions of various elements for Platanus Hybrida (acerifolia) after the test (configuration IV: PAE/PhC + UV) : (a) C (red), (b) Ti (magenta), (c) Al (green). 3.2 XPS XPS wide spectra showed presence of C, O and N being the most relevant components. Also, Ti and Au from the substrate, and traces of P, S and Si were found. The analysis of binding energies of the elements determined from C 1s, O 1s, and N 1s core level spectra was done by peak deconvolution by using the minimum number of components required to obtain a quality data fit. Reasonable constancy in the peak position and full width at half maximum (FWHM) was considered to allow a comparison to be made between samples. From now on, these components will be referred with sequential numbers in order of increasing in the binding energy of the corresponding element. For correct assignment of these components to chemical functions the knowledge on the chemical structure of the constituents must be available. However, with exception of several proteins that could be identified in this work by proteomic analysis (See Supplementary materials), the literature data on the constituents of the PAEs are scarce. Generally, defatted pollen core extract is dominated by two major classes of compounds: (A) proteins and peptides, and (B) sugars and polysaccharides 6   

[13, 14]. Therefore, the peak components were assigned to the groups of the chemical functions present in these compounds.

Fig. 4. Reference XPS fine spectra of the bare substrates: a), c), and e) correspond to a gilded glass (Au); b), d) and f) refer to a PhC loaded gilded glass (PhC). Quantification of the components is shown in the graphs g), h) and i) for C 1 s, O 1s and N 1 s, respectively. The total heights of the bars represent surface concentration (at %) of the corresponding elements. The colour bars show surface concentrations (at. %) of the components with different binding states. 7   

Figure 4 shows fitted reference XPS fine spectra, the integrated total concentration of C, O and N and the concentrations of the different binding states of each element measured on bare substrates. Figures 4 a), c) and e) correspond to bare gilded glass, whereas figures 4 b), d) and f) refer to a PhC loaded gilded glass. Figures 5 to 7 present XPS fine spectra for C, N and O, respectively, for the cases I to IV for Cup. and Pla. For C four main components with the binding energies at approximately 284.5 (C1), 285.7 (C2), 287 (C3) and 289 eV (C4) were identified. C1 component owed to carbon only bound to C, H and S [19, 20]. C2 component corresponded to carbon singly bound to O or N (C-N-C=O, C=C-N, CCOO) [20, 21]. C3 was assigned to carbon making a double bound or two single bounds with O or N (N=C-N, O=C-NH-C, C-OH, C-O-C, C-O-C=O) [19] such as in histidine, tryptophan, and other amino acids. (See supplementary materials.) Other groups such as C-N, C-NH+, C-O with the binding energy at about 286.35 eV [20] can contribute to both C2 and C3. Finally, C4 was due to carbon making a double bound with O in esters, carboxylic acid and peptide link (C-O-C=O, O=COH, O=CN). Although the assignment of specific features in the O 1s peak has been intensively debated in the literature [22], there is still a lot of controversy. It is generally agreed on the discrimination of two organic oxygen species with the lowest energy: the double bonded oxygen at lower binding energies (531 eV) and the singly bonded oxygen at higher binding energies (533 eV). Additionally, a third species at higher binding energies is commonly assigned to adsorbed water or oxygen [23, 24]. Therefore, O 1s spectra were fitted with three organic components O2-O4. An additional inorganic component O1 at 530.4 eV was added in tests III and IV, associated to the photocatalyst (TiO2). O2 component at 532.4 eV owed to C-OH, O=C-O, O=C-N, C-O-C-O-C groups and chemisorbed oxygen on gold. O3 component at 533.4 eV corresponded to O=C-OH and O=C-O-C of proteins and peptides [19, 25]. O4 component at 534 eV was associated to singly bound O of cyclic [25] or carbonate esters (O-(C=O)-O) and carboxylic acid anhydride (C=O)-O-(C=O) [26]. For nitrogen three main components were identified: a weak component N1 at 398.9 eV owed to nitrogen adsorbed on gold, nitrogen of amine and imidazole (N=C-NH) [20] such as in histidine, tryptophan, etc. Component N2 at 401.0 eV corresponded to amides in peptide link, protonated amine and protonated nitrogen [19, 21]. N3 component at 402.5 eV was assigned to quaternary ammonium that, in part, could be associated with zwitterionic nature of amino acids.

8   

Fig. 5. XPS C 1s fine spectra of the samples of Cup. (a-d) and Pla. (e-h) on a gold substrate (a, e), after UV irradiation (b, f), on an inactivated PhC (c, g) and on UVactivated PhC (d, h). Concentrations (at. %) of C components with different binding energies to the total concentration of C with respect to the sum of organic elements (C, O and N) are shown by colour bands: i) Cup. and j) Pla. 9   

Fig. 6. XPS O 1s fine spectra of the samples of Cup. (a-d) and Pla. (e-h) on a gold substrate: without UV irradiation (a, e) and after UV irradiation (b, f); on a PhC: without UV irradiation (c, g) and after UV irradiation (d, h). Concentrations (at. %) of O components with different binding energies to the total concentration of O with respect to the sum of organic elements (C, O and N) are shown by colour bands: i) Cup. and j) Pla.

10   

Fig. 7. XPS N 1s fine spectra of the samples of Cup. (a-d) and Pla. (e-h) on a gold substrate (a, e), after UV irradiation (b, f), on a PhC without UV irradiation (c, g) and on PhC after UV irradiation (d, h). Concentrations (at. %) of N components with different binding energies to the total concentration of N with respect to the sum of organic elements (C, O and N) are shown by colour bands: i) Cup. and j) Pla.

11   

On the basis of comparison of the C 1s spectra of the PAEs on Au substrate (Fig. 5 a and e) with the reference spectrum of bare Au substrate (Fig. 4 a), components C2, C3 and C4 were attributed mainly to the PAEs, while C1 could have contributions from both PAE’s carbon and adventitious hydrocarbon contamination of the substrate. Similarly, from the comparison between Fig. 6 a and e with Fig. 4 c and d, respectively, the components O3 and O4 were unambiguously corresponded to the PAEs, whereas O1 was assigned to inorganic oxygen from a PhC. O2 can have contributions from both the PAE and the substrate. For both PAEs (Fig. 7 i and j) N concentration was severalfold higher than on both bare substrates (Fig. 4 g and h). 3.3 Enzymatic activity The results of characterization of enzymatic activity of Cup. and Pla. PAE treated with the PhC in a form of suspension are given in Fig. 8. Figure 8 a) shows the fluorescence spectra for Cup. treated under the different test configurations. For both species, fluorescence spectra corresponding to test configurations I-III have similar shape that was identical to the spectrum of fluorescein, although with different intensity. In contrast, a fluorescein peak could not be found on the fluorescence spectra in configuration IV. These results suggested that the enzymatic activity of both PAEs was completely supressed. Figure 8 b) shows relative fluorescence intensity (%) at 517 nm emission wavelength for both PAEs. The fluorescence intensity under UV irradiation or PhC chemical reactivity decreased nearly two-fold, but under photocatalysis it dropped almost two orders of magnitude.  

Fig.8. (a) Fluorescence intensity emission spectra of Cup. for four test configurations; (b) relative fluorescence intensity (%) at 517 nm emission wavelength. 4. Discussion  

The results of SEM, XPS, and FDA were coincident in the experiment IV. In all the cases high degradation of organic materials associated with pollen was observed. However, there were some discrepancy between the results on enzymatic activity and XPS and EDS-SEM data in the experiments II and III. In fact, while the enzymatic activity of PAES was reduced in both experiments, XPS and SEM morphology did not show significance variation as compared with the 12   

untreated PAEs (test I). These differences can be explained when considering that XPS and FDA techniques provide complementary information on different features of the components of pollen extracts. FDA was used to characterize the enzymatic activity of allergic pollen proteins that is tightly related with their structural organization in four levels: 1 – order of amino acids in the polypeptide chain, 2 – structure of alfa and beta helixes, 3 - folded structure of alfa and beta helixes and 4 - overall three-dimensional structure. In turn, XPS is sensitive only to the chemical environment of elements, i.e. the first structural level. In addition, analysis of the bonding structures for the fine XPS spectra of C, O and N allowed distinguishing between the species associated with PAEs and others related with the substrates. In the further analysis of XPS data we will focus mainly on the evolution of the components that have been unambiguously attributed to PAEs: C2-C4, O3-O4 and N2. 4.1. The effect of UV irradiation on the PAEs For Pla. the total C and O concentrations were almost unchanged between the tests I and II (Fig. 5 j and Fig 6 j). For Cup. C slightly increased, and O slightly decreased (Fig. 5 i and Fig. 6 i). From Figs. 5 a), b) and i) it is evidenced that the increase in C under photolysis of Cup. (test II) was mainly due to C2 associated with polypeptides. Relative concentrations of various C and O components of Cup. PAE in tests I and II are shown on Table 2. Table 2. Relative percentage (%) of the components C2–C4 and O2–O4 in the tests I and II for Cup. Binding energy (BE) and possible associated chemical groups. C2

C3

C4

O2

O3

O4

BE (eV)

285.7

287.7

289.0

532.4

533.0

534.0

Possible

C-N-C=O,

N=C-N,

C-O-C=O,

C-OH,

O=C-OH

O-(C=O)-O,

bonds

C=C-N,

O=C-NH-C, O=C-OH,

O=C-O,

O=C-O-C

(C=O)-O-(C=O)

C-COO

C-OH,

O=C-N,

O=CN

C-O-C,

C-O-C-O-C

C-O-C=O Test I

38.1

40.0

15.8

8.9

35.3

55.7

Test II

43.9

36.8

15.8

19.4

43.8

36.3

The decrease in O was associated mainly with O4 and was not compensated by a certain increase in O2 (Fig. 6 a, b and i). As already published, dialdehydes and carbonate esters associated with O4 can be formed during partial oxidation of heteropolysaccharides of intine [27, 28], but the oxidation 13   

products are relatively unstable and decompose through hydrolysis catalysed by water [29]. This could result in partial decomposition of dialdehides and carbonate esters and decrease of O4 peak in tests II and III. Other components of C 1 s and O 1 associated with polypeptides and polysaccharides remained generally unaltered after UV irradiation. N concentration that is directly associated with proteins was almost unaffected by UV irradiation in case of Cup. (Fig. 7 i) and only slightly decreased for Pla. (Fig. 7 j). All these factors seem to indicate that primary structure of proteins was not significantly affected in this test. However, enzymatic activity assayed in FDA test notably decreased. According to Neves-Petersen et al. [30] UV irradiation could be absorbed by side chains of tryptophan, tyrosine and phenylalanine resulting in their photoionization and generation of solvated electrons. The generated solvated electrons can subsequently undergo fast geminate recombination with their parent molecule, or they can be captured by electrophilic species (O2, H3O+) [31]. Generated superoxide (O2-) is known to cause pronounced degradation of proteins and denaturation of enzymes [32]. In case the solvated electron is captured by cystine, the result can also be the breakage of the disulphide bridge since tryptophane is the preferred spatial neighbour of the disulphide bridge. Disruption of disulphide bonds can significantly affect the enzymatic activity since they contribute to the tertiary and quaternary protein structures. From the analysis of the fine spectra of S 2p shown in Fig. 9 for Pla. (for Cup. the peak S 2p was insignificant), it is reasonable to suggest that photoxidation of thiols seems to occur. The components S1 and S2 were associated with unbounded thiol [33] and sulphonate [34], correspondingly. After UV irradiation of PAE (test II) the component S1 decreased while the ratio of S2 to S1 increased from 0.4 (test I) to 0.6 (test II). This result is in line with literature studies [35].

Fig. 9. XPS fine spectra of S 2p: (a) initial; (b) after UV irradiation

4.2. The effect of inactivated PhC on the PAEs No important variations in N 1s peak structure could be found after test III, but N2 was slightly shifted towards lower energies. For Cup. N concentration was nearly the same in both tests. For Pla. 14   

N significantly decreased, but still was much higher, than on the bare PhC. In test III N/Ti ratio was 0.4 and 0.6 for Cup. and Pla., respectively, whereas it was only 0.03 on the bare PhC. C decreased, while O increased for both PAEs. For Pla. this variation was more significant. It should be noted that for PAEs deposited onto a PhC O concentration was lower, than on the bare PhC, but C was higher. This can be an indication that although some chemical degradation of the PAEs on inactivated PhC could occur, the PAEs were not decomposed to such extent as they did under HPhC as it will be shown below. The structure of the components of C 1s was almost the same as in test I, although C2 for both PAEs and C3 for Cup. slightly shifted towards lower energies (Fig. 5 c, g). In addition, two weak shake up components at higher energies: 294 eV (C5) and 297 eV (C6) were found. Majumdar et al. associated these peaks to O-CO-O and C-CO-HN2, respectively [36]. The components C5 and C6 localized at energies higher than 294 eV were negligible in tests I and II, but grew up in tests III and IV. In contrast to C 1s, O 1s spectra experienced significant changes (Figs. 6 c and g vs. Fig. 6 a and e, correspondingly): O3 became predominant organic component, while O4 decreased. The ratio O3/O4 increased from 0.63 (test I) to 4.30 (test III) for Cup. and from 1.03 (test I) to 1.85 (test III) for Pla. In addition, an inorganic component O1, i.e. O bound to Ti, appeared. Furthermore, O3 and O4 slightly shifted towards higher energies. The observed shifts of some components can indicate changes of the chemical environment due to some kind of chemical interactions between the PAEs components and the PhC, e.g., chemical adsorption, etc. Conformational changes in secondary and tertiary protein structures and/or partial proteolysis associated with adsorption on TiO2 could explain the significant decrease in enzymatic activity [37]. This decrease was more significant, than after UV irradiation since the degree of conformational distortion should be larger in test III than in test II. Bouhekka et al. demonstrated that significant changes of protein conformation – decrease of the percentage of alpha helix and increase of random helix – could be induced by visible light irradiation of proteins on TiO2 supports [38]. On the other hand, polysaccharides being the main constituents of intine could irreversibly adsorb on TiO2 due to highly attractive Van der Waals forces as reported by Jucker et al. [39]. Shift of XPS peaks in test III could be, in part, explained by this interaction.

4.3 The effect of photocatalysis on the PAEs The most striking changes of the structures of C 1s and O 1s occurred in test IV, i.e. after HPhC treatment. For both PAEs C3 and C4 significantly decreased and C2 became the dominant component (Fig. 5 d, h, i and j). These changes were especially pronounced for Cup. It is noteworthy that C 1s was very different from that of the original PAE, but except small differences in shake up components it was almost identical to the spectrum of the reference bare PhC (Fig. 4 b). 15   

Similar trend could also be observed for O 1s (Fig. 6 d, h, i and j). Ratio of organic/inorganic components, i.e. (O2+O3+O4)/O1, decreased from 1.75 (test III) to 0.32 (test IV) for Cup. and from 1.64 (test III) to 0.68 (test IV) for Pla. The final values of the ratio were close to those for the bare PhC substrate – 0.23. Concerning N 1s, significant decrease in the total amount, but not in the peak shape was observed. Ratio N/Ti in test IV decreased to 0.05 and 0.1 for Cup. and Pla., correspondingly, that is much lower, than in test III and only slightly higher, than on bare PhC. The observed variations in C 1s, O 1s and N 1s core spectra gave strong evidences of decomposition of PAEs under photocatalysis. Although gas emission from PAEs during photocatalysis was not measured in this study, we suggest that the products of photocatalytic degradation were rather small volatile molecules such as CO2, NH3, alcohols, etc. that could left the PAEs during the test. This conclusion is based on the finding that mineralized C and O in the form of carbonates (C4 and O2 [21]) and/or nitrates (component of N 1s at 408 eV [40]) were not significant in the XPS spectra after HPhC, while no lixiviation could occur during the test. These results are in line with the general reactions pathways of amino acid and saccharide photocatalytic degradation [1] which showed that indeed gases and volatiles rather than salts are the main products of photocatalytic decomposition of amino acids, peptides and saccharides. Therefore, results of XPS were consistent with SEM-EDS mapping and showed the decrease of the total amount of carbon. In addition XPS evidenced significant reduction in the C, O and N species associated with PAEs. It should be reminded that XPS provides chemical information for all components of pollen core extract, whereas FDA is specific for enzymatic proteins. On the basis of these findings a general scheme of possible processes associated with enzymatic protein degradation/decomposition has been proposed (Fig. 10).

16   

Fig. 10. Tentative scheme of possible mechanisms of pollen allergen degradation. The geometrical structure of the pollen allergen is represented by pectate lyase C (See supplementary materials) [41].

5 Conclusions The effect of photocatalysis on pollen core extracts containing pollen allergenic proteins of two species: Cupressus Arizonica and Platanus Hybrida (acerifolia) was studied. The results of XPS showed that carbon and nitrogen concentrations associated with the PAEs drastically decreased after HPhC treatment. Chemical elements mapping that was carried out using EDS corroborated this finding. XPS results provided strong evidence that the total amount of organic matter significantly decreased as a result of HPhC. Complete deactivation of enzimatic activity of allergen extract after HPhC treatment was confirmed by fluorescein diacetate probe analysis. In order to discriminate photocatalytic degradation from photolysis and chemical reactions between PAE and PhC without UV irradiation four different tests were carried out. These tests showed different behaviour for Cup. and Pla., although for both species the effects of UV irradiation and chemical reactivity of PhC were much less significant than photocatalysis. Decrease in enzymatic activity after UV irradiation and adsorption on PhC was tentatively associated with conformational changes of enzymes induced by photochemical processes or Van der Waals forces.

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