extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Chemistry Biology

text:
Structure of a xenon derivative of Escherichia coli copper amine oxidase: confirmation of the proposed oxygen-entry pathway

The structure of a xenon derivative of E. coli copper amine oxidase confirms the pathway of oxygen entry to the buried active site proposed for this class of enzymes.

Introduction
Copper amine oxidases (CuAOs) catalyse the oxidation of a wide range of amine substrates to generate a product aldehyde, ammonia and hydrogen peroxide (Mure et al., 2002). They are ubiquitous in nature and in humans are involved in diverse processes including cell adhesion and signalling Elmore et al., 2002;Yu et al., 2003). In bacteria, they play a role in general nitrogen catabolism (Parrott et al., 1987;Hacisalihoglu et al., 1997). They contain a protein-derived cofactor that is generated autocatalytically by posttranslation modification of a conserved endogenous tyrosine to generate 2,4,5-trihydroxylphenylalanine quinone (TPQ), requiring only the presence of copper and molecular oxygen (Cai & Klinman, 1994;Kim et al., 2002). TPQ is situated in the core of the protein at the end of a well defined substrate-access channel next to a type II copper coordinated by three histidines (Fig. 1;Parsons et al., 1995;Duff et al., 2006;Wilce et al., 1997;Lunelli et al., 2005;Kumar et al., 1996;Airenne et al., 2005;Li et al., 1998). The Escherichia coli enzyme (ECAO) shows a preference for small aromatic amine substrates such as phenethylamine and tyramine (Roh et al., 1994). The catalytic reaction proceeds via two half-reactions; the aldehyde product is released at the end of the reductive half-reaction before reduction of molecular oxygen in the oxidative half-reaction (Fig. 2).
Extensive structural and biochemical analyses of CuAOs from prokaryotic and eukaryotic sources have revealed that molecular oxygen binds on the opposite side of TPQ to the amine substrate (Fig. 2). There has been considerable interest in dissecting the pathway by which oxygen reaches and binds in the active site. Structural and mutagenesis studies have shown that oxygen does not enter via the substrate channel and that other possibly specific oxygen-access pathways must be present (Wilce et al., 1997;Li et al., 1998;Johnson et al., 2007;Wilmot et al., 1999;Goto & Klinman, 2002).
Several recent crystallographic studies of oxygen entry have been carried out using xenon as a probe to identify possible molecular oxygen-binding sites that may be part of the putative oxygen-entry pathway (Lunelli et al., 2005;Johnson et al., 2007;Duff et al., 2004).
Xenon-complex crystal structures are now available for the CuAOs from Hansenula polymorpha (HPAO), Pisum sativum (PSAO), bovine serum (BSAO), Pichia pastoris (PPLO) and Arthobacter globiformis (AGAO) (Lunelli et al., 2005;Johnson et al., 2007;Duff et al., 2004). These structures have revealed a consistent pattern of xenon binding that has implicated a major pathway for oxygen entry through a conserved -sandwich (Fig. 3). Very recently, in silico experiments that calculated likely low-energy pathways for oxygen entry in HPAO, PSAO, AGAO and PPLO were carried out that supported the major pathway identified by the structural studies (Johnson et al., 2007).
ECAO is distinct from Gram positive bacterial and yeast CuAOs in that it possesses an extra 9.3 kDa N-terminal domain, which gives ECAO a characteristic 'mushroom stalk and cap' shape (Parsons et al., 1995). Mammalian CuAOs also possess an N-terminal domain that is thought to act as a membrane anchor (Smith et al., 1998) but has not yet been visualized crystallographically (Airenne et al., 2005). Biochemical and structural characterization of ECAO indicate that the binding of oxygen in the active site is similar to that of the other family members (Smith & McPherson, 2008); however, it is possible that the presence of the additional N-terminal domain, which is located at one end of the structurally conserved -sandwich, could Schematic catalytic cycle of CuAOs identifying the key intermediates in the reaction pathway. Shown are the type-2 copper centre, the protein-derived cofactor TPQ and the off-metal O 2 -binding site located in the vicinity of a conserved active-site methionine (Met699 in ECAO). I is the resting state of the enzyme, intermediate II is the product Schiff base, intermediate III is the reduced TPQ or aminoquinol form, intermediate IV shows the proposed off-metal-bound dioxygen species prior to oxygen activation and reduction and intermediate V is the iminoquinone species which hydrolyses to release ammonia, completing the catalytic cycle.

Figure 1
The structure of ECAO (PDB code 1dyu) showing the substrate-entrance channel (magenta surface) and active site (Murray et al., 1999). The lower right panel is a more detailed view of the active site showing the TPQ, copper(II) ion, three coordinating histidine residues (sticks coloured by atom type) and the end of the substrate-entrance channel (magenta surface). The substrate-entrance channel was calculated using CAVER (Petrek et al., 2006) and the image was prepared using PyMOL (DeLano, 2002). alter or provide an alternate pathway for oxygen entry. In order to investigate this possibility, we have determined the crystal structure of ECAO in complex with xenon at 2.5 Å resolution.

Experimental
2.1. ECAO expression, purification and crystallization ECAO expression and purification was carried out as previously described (Murray et al., 1999). ECAO was concentrated to 8 mg ml À1 and crystals were grown by vapour diffusion at 291 K. Large pinkcoloured crystals grew over two weeks in 100 mM HEPES pH 7 and 1.2 M sodium citrate.

Xenon derivatization
ECAO crystals were harvested and transferred into a cryoprotectant solution comprising crystal-growth mother liquor containing 20% glycerol before being exposed to xenon for 8 min at 2.5 MPa in an Oxford Cryosystems Xcell. After derivatization, crystals were immediately flash-cooled in liquid nitrogen for X-ray data collection.

X-ray data collection and processing
A 2.5 Å diffraction data set was collected on beamline 14.1 at the SRS, Daresbury. Data collection was carried out at the highest beamline-accessible wavelength of 1.48 Å to maximize the anomalous signal from xenon (Xe f 00 = 6.9) and data were recorded using a Quantum4 ADSC CCD detector. The crystal was maintained at 100 K using a Cryostream (Oxford Cryosystems). 360 of data were collected with an oscillation angle of 1 and an exposure time of 30 s.   ECAO subunit showing the substrate-entry channel as a magenta surface and the putative oxygen-entry pathway as a green surface (generated by placing dummy atoms along the channel). The xenon sites observed in PPLO, HPAO, AGAO and PSAO are shown as cyan spheres. TPQ and copper ligands are shown as sticks coloured by element and copper is shown as a bronze sphere. This image was generated using PyMOL (DeLano, 2002). The diffraction data were processed using MOSFLM and scaled using SCALA (Leslie, 1992;Evans, 1993; Table 1).

Refinement and model building
All refinement and model building was carried out using the CCP4 software suite and Coot (Collaborative Computational Project, Number 4, 1994; Emsley & Cowtan, 2004). Initial maps were calculated by direct Fourier transform using the structure of native ECAO (PDB code 1dyu; Murray et al., 1999). An anomalous difference map was calculated and examined to locate possible xenon sites. 50 peaks were identified in the map with a level of 4 or above. Two of these (8.5 and 8.0 r.m.s.) were located at sites known to bind calcium in the native enzyme (Ca f 00 = 1.2 at 1.48 Å ). Weaker peaks (3.6-4.6 r.m.s.) were located at the copper site and on S atoms, which is expected at this wavelength (Cu f 00 = 0.55 at 1.48 Å , S f 00 = 0.5 at 1.48 Å ). The remaining peaks were assigned as xenon when they were higher than 4 r.m.s., did not overlap with copper, calcium or sulfur, corresponded to positive electron density in the F o À F c electron-density map and were located in mainly hydrophobic pockets. The occupancy of each xenon was modified such that after restrained refinement there was no further positive electron density in the F o À F c map and the B factors were similar to those of nearby atoms. The structure was further refined using iterative cycles of model building and restrained refinement to a final R factor of 16.1% and an R free of 21.9% (Table 2). Structure validation was carried out using SFCHECK and PRO-CHECK (Laskowski et al., 1993;Vaguine et al., 1999).

Results and discussion
After refinement, 11 xenon-binding sites were found in the structure (Fig. 4a). Although the occupancy of all sites was low (0.1-0.45), this is consistent with the xenon occupancies observed in other CuAO xenon complexes (Lunelli et al., 2005;Johnson et al., 2007;Duff et al., 2004). Xenons were numbered according to their occupancy (from high to low occupancy). The Xe atoms were bound in hydrophobic pockets located in either the amine substrate-entry channel or around the conserved -sandwich oxygen-entry pathway (Table 3). Nearly identical sites were observed in both subunits of ECAO present in the asymmetric unit 1 .
Two Xe atoms are bound in the amine substrate-entry channel. Xenon 4/11 occupies a site between Tyr381 and Tyr387 that is equivalent to that previously observed in AGAO and HPAO. Tyr381 is proposed to act as a gate regulating substrate access to the TPQ and the aromatic rings of Tyr381 and Tyr387 allow stacking interactions with the aromatic moiety of aromatic amine substrates (Wilmot et al., 1997(Wilmot et al., , 2004. Xenon 1/2 is bound further from the active site but is still close to the amine-substrate channel (Fig. 4b).
Two xenons, xenon 7/5 and xenon 9/-(only in monomer A), are bound inside the 'top' entrance of the -sandwich, close to the xenonbinding sites observed in PPLO and PSAO (Duff et al., 2004;Figs. 4a and 4b). We did not observe any xenon-binding sites inside the lower half of the -sandwich near the 'bottom' entrance to the putative oxygen-entry pathway. However, two xenon-binding sites (3/6 and 8/-) flank the lower half of the -sandwich, binding on its external face (Fig. 4a). This is consistent with the other copper amine oxidasexenon complexes, in which a xenon-binding site within the bottom half of the -sandwich channel has only been reported for AGAO (Fig. 3). The lack of xenon-binding sites in this region in PPLO, PSAO and HPAO suggests that xenon binding is not as favourable in this region of the -sandwich regardless of the presence or absence of an N-terminal domain. A final xenon, xenon -/10, is bound at the edge of ECAO in a hydrophobic pocket and is not close to any previously observed xenon position.
The xenon-complex ECAO structure reported here shows that molecular oxygen is likely to access the ECAO active site by very similar pathways to those proposed by experimental and in silico studies in other CuAOs (Lunelli et al., 2005;Johnson et al., 2007;Duff et al., 2004). No xenon-binding sites are observed in the ECAO N-terminal domain itself nor does the presence of this domain appear to affect xenon binding in the lower half of the -sandwich when compared with other CuAOs for which xenon complexes have been determined. These observations indicate that the N-terminal domain does not play a role in oxygen entry during ECAO catalysis, which is consistent with the lack of conservation of the N-terminal domain across the CuAOs.
where |F o | is the observed structure-factor amplitude and |F o | is the calculated structure-factor amplitude. ‡ R free is the R factor based on 5% of the data excluded from refinement. PP is the recipient of a Wellcome Trust Studentship. This work was also supported by the Biotechnology and Biological Sciences Research Council (Grant No. BB/C00468X/1) and the RCUK (ARP). We thank Dr Thomas Edwards, Dr Chi Trinh and the beamline 14.1 local contacts for assistance during data collection.


extfieldsofstudy:
Medicine
Computer Science

text:
Dimethyl 2-nitroterephthalate

In the molecule of the title compound, C10H9NO6, the two ester groups and the nitro group are inclined at 9.2 (2), 123.3 (6) and 135.2 (5)°, respectively to the mean plane of the benzene ring. In the crystal structure, molecules are stacked along the a axis, without any π–π interactions. The stacked columns are linked together by non-classical intermolecular interactions of the type C—H⋯O.

In the molecule of the title compound, C 10 H 9 NO 6 , the two ester groups and the nitro group are inclined at 9.2 (2), 123.3 (6) and 135.2 (5) , respectively to the mean plane of the benzene ring. In the crystal structure, molecules are stacked along the a axis, without anyinteractions. The stacked columns are linked together by non-classical intermolecular interactions of the type C-HÁ Á ÁO

S1. Comment
The title compound, (I), is useful as a raw material for the preparation of 2-amino-dimethyl-terephthalic acid, which is used as an important intermediate for dyes (Niu et al., 2002). The structures of dimethyl terephthalate (Brisse & Pérez, 1976) and dimethyl 2,3-dihydroxyterephthalate (Huang & Liang, 2007) which are closely related to the title compound have already been reported. In this article, we report the crystal structure of (I). A view of the molecule of (I) is presented in Fig. 1. The bond lengths and angles are within expected ranges. The C1/O1/C2/O2, C10/O6/C9/O5 and O3/N/O4 planes form dihedral angles of 9.2 (2), 123.3 (6) and 135.2 (5)°, respectively, with the C3-C8 plane. In the crystal structure, the molecules are stacked along the a axis, without any π-π interactions. The stacked columns are linked together by non-classical intermolecular interactions of the type C-H···O (Table 1).

S2. Experimental
A sample of commercial 2-nitro-dimethyl-terephthalic acid (Aldrich) was crystalized by slow evaporation of a solution in methanol.

S3. Refinement
Positional parameters of all the H atoms bonded to C atoms were calculated geometrically and were allowed to ride on the C atoms to which they are bonded, with H-C(aryl) = 0.93 Å and U iso (H) = 1.2U eq (C) or with H-C(methyl) = 0.96 Å and U iso (H) = 1.5U eq (C).  A view of the molecule of (I) with the atomic numbering scheme. Displacement ellipsoids were drawn at the 30% probability level.

Dimethyl 2-nitroterephthalate
Crystal data C 10 H 9 NO 6 M r = 239.18 Monoclinic, P2 1 /n Hall symbol: -P 2yn a = 6.9080 (14)  Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.


extfieldsofstudy:
Chemistry
Medicine
Computer Science

text:
N,N′-Bis(3-chloro-2-fluorobenzylidene)ethane-1,2-diamine

The molecule of the title centrosymmetric Schiff base compound, C16H12Cl2F2N2, adopts an E configuration with respect to the azomethine C=N bond. The imino groups are coplanar with the aromatic rings. Within the molecule, the planar units are parallel, but extend in opposite directions from the dimethylene bridge. An interesting feature of the crystal structure is the short intermolecular Cl⋯F [3.1747 (5) Å] interactions, which are shorter than the sum of the van der Waals radii of these atoms. These interactions link neighbouring molecules along the b axis. The crystal structure is further stabilized by π–π interactions, with a centroid–centroid distance of 3.5244 (4) Å.

The molecule of the title centrosymmetric Schiff base compound, C 16 H 12 Cl 2 F 2 N 2 , adopts an E configuration with respect to the azomethine C N bond. The imino groups are coplanar with the aromatic rings. Within the molecule, the planar units are parallel, but extend in opposite directions from the dimethylene bridge. An interesting feature of the crystal structure is the short intermolecular ClÁ Á ÁF [3.1747 (5) Å ] interactions, which are shorter than the sum of the van der Waals radii of these atoms. These interactions link neighbouring molecules along the b axis. The crystal structure is further stabilized byinteractions, with a centroid-centroid distance of 3.5244 (4) Å .

N,N'-Bis(3-chloro-2-fluorobenzylidene)ethane-1,2-diamine
H.-K. Fun and R. Kia Comment Schiff bases are among the most prevalent mixed-donor ligands in the field of coordination chemistry in which there has been growing interest, mainly because of their wide application in areas such as biochemistry, synthesis, and catalysis (Pal et al., 2005;Hou et al., 2001;Ren et al., 2002). Many Schiff base complexes have been structurally characterized, but only a relatively small number of free Schiff bases have had their X-ray structures reported (Calligaris & Randaccio, 1987). As an extension of our work (Fun, Kargar & Kia 2008;Fun, Kia & Kargar 2008) on the structural characterization of Schiff base ligands, the title compound (I), is reported here.
The molecule of the title compound ( Fig. 1), adopts an E configuration with respect to the azomethine C═N bond. The bond lengths (Allen et al., 1987) and angles are within normal ranges and are comparable with the values found in related structures (Fun & Kia 2008a,b;Fun, Kargar & Kia 2008;Fun, Kia & Kargar 2008). The two planar units are parallel but extend in opposite directions from the dimethylene bridge. The interesting feature of the crystal structure is the short intermolecular Cl···F interactions [symmetry code: x, -1/2 -y, -1/2 + z] with a distance of 3.1747 (5) Å, which is shorter than the sum of the van der Waals radii of these atoms. These interactions link neighbouring molecules along the b-axis.

Experimental
The synthetic method has been described earlier (Fun, Kargar & Kia, 2008). Single crystals suitable for X-ray diffraction were obtained by evaporation of an ethanol solution at room temperature.

Refinement
All of the hydrogen atoms were positioned geometrically with C-H = 0.95 or 0.99 Å and refined in riding mode with U iso (H) = 1.2 U eq (C). Fig. 1. The molecular structure of (I) with atom labels and 50% probability ellipsoids for non-H atoms. The suffix A corresponds to symmetry code (-x + 2, -y, -z + 1).

Special details
Experimental. The low-temperature data was collected with the Oxford Cyrosystem Cobra low-temperature attachment. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating Rfactors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.

Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å 2 )
x y z U iso */U eq


text:
Entrapment of Carbon Dioxide in the Active Site of Carbonic Anhydrase II*♦

The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO2-pressurized, cryo-cooled crystals to capture the first step of CO2 hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO2, for both the holo and the apo (without zinc) enzyme to 1.1Å resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO2 and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675–3679). These structures provide insight into the long hypothesized binding of CO2 in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO2. This method may also have a much broader implication for the study of other enzymes for which CO2 is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins.

REACTION 1
The second stage is the transfer of a proton from the zincbound water to bulk solvent to regenerate the zinc-bound hydroxide (Reaction 2). Here B is a proton acceptor in solution or a residue of the enzyme itself.
For hCAII (␣ class CA), this reaction is facilitated by a solvent molecule with a pK a near 7 that is directly coordinated to the zinc (6). This centrally located zinc exhibits a tetrahedral configuration with three histidines (His-94, His-96, and His-119) and either a water or a hydroxide molecule. The active site cavity can be loosely described as being conical in shape having a 15 Å diameter entrance that tapers into the center of the enzyme. The cavity is partitioned into two very different environments. On one side of the zinc, deep within the active site, lies a cluster of hydrophobic amino acids (namely Val-121, Val-143, Leu-198, Thr-199-CH 3 , Val-207, and Trp-209), whereas on the other side of the zinc, leading out of the active site to the bulk solvent, the surface is lined with hydrophilic amino acids (namely Tyr-7, Asn-62, His-64, Asn-67, Thr-199-O␥ 1 , and Thr-200-O␥ 1 ) (Fig. 1a).
Previously, molecular dynamics studies have implied that the hydrophobic region of the active site sequesters the CO 2 substrate and orients the carbon atom in readiness for nucleophilic attack by the zinc-bound hydroxide (Reaction 1) (1,8). Additionally, crystallographic studies have identified an ordered water molecule, positioned near the hydrophobic pocket, termed "deep water," W DW , that is stabilized by the amide nitrogen of Thr-199 and the zinc-bound hydroxide (Fig. 2a). It has been proposed that this water is likely displaced upon the infusion of CO 2 into the binding pocket (8,9). The hydrophilic wall of the active site has been shown, by x-ray crystallography, to create a well ordered hydrogen-bonded solvent network. It is hypothesized that this network is required to permit the transfer of a proton from the zinc-bound water to the bulk solvent via the experimentally identified proton shuttling residue His-64 (Reaction 2) (10 -13). Taken together, these two very different active site environments permit the sustained and rapid catalytic cycling of CO 2 to bicarbonate.

EXPERIMENTAL PROCEDURES
Protein Purification and Crystallization-hCAII was expressed in a recombinant strain of Eshcherichia coli (BL21(DE3)pLysS) containing a plasmid encoding the hCAII gene (14). Purification was carried out via affinity chromatography as described previously (15). Briefly, cells were enzymatically lysed with hen egg white lysozyme, and the lysate was placed onto an agarose resin coupled with p-(aminomethyl)-benzene-sulfonamide, an hCAII inhibitor. The protein was eluted with 0.4 M sodium azide, 100 mM Tris-HCl, pH 7.0, and the azide was removed by extensive buffer exchanging against 10 mM Tris-HCl, pH 8.0.
Preparation of Apoenzyme-The zinc from the holoenzyme was removed by incubation at 20°C in the chelation buffer (100 mM pyridine 2,6 dicarboxylic acid, 25 mM MOPS; pH 7.0) for 8 h. The enzyme was then buffer-exchanged against 50 mM Tris-HCl, pH 7.8, to remove the chelating agent (16). The complete loss of enzyme activity was verified through kinetic studies. The enzyme activity was revived through the addition of 1 mM ZnCl 2 , attributing the loss of activity to the absence of zinc rather than to the denaturation of the enzyme.
Crystallization and X-ray Data Collection-Crystals of holoand apohCAII were obtained using the hanging drop vapor diffusion method (17). 10-l drops of equal amounts of protein and precipitant were equilibrated against precipitant solution (1.3 M sodium citrate, 100 mM Tris-HCl, pH 7.8) by vapor diffusion at room temperature (ϳ20°C) (13). Crystals grew to ϳ0.2 ϫ 0.2 ϫ 0.5 mm in size after ϳ5 days. CO 2 Binding-To capture CO 2 in the active site of hCAII, it was essential to cryo-cool the crystals under CO 2 pressure. This was achieved using the high pressure cryo-cooling method that was originally developed for crystal cryoprotection (18). The crystals were first soaked in a cryo-solution containing 20% glycerol in precipitant solution. The crystals were then coated with mineral oil to prevent crystal dehydration and loaded into the bottom of high pressure tubes. In the pressure tubes, the crystals were pressurized with CO 2 gas at 15 atm at room temperature. 25 min later, without releasing CO 2 gas, the crystals were slowly frozen over 2 min by dipping the sealed end of the pressure tubes into liquid nitrogen. During the cooling process, it was noticed that the CO 2 gas pressure gradually dropped from 15 atm to below 1 atm due to CO 2 solidification.
X-ray Diffraction Data Collection-Diffraction data were collected at CHESS, beamline A1, at a wavelength of 0.9772 Å. Data were collected using the oscillation method in intervals of 1°step on an ADSC Quantum 210 CCD detector (Area Detector Systems Corp.), with a crystal to detector distance of 65 mm. A total of 624 and 360 images were collected for the holo and apo data, respectively. Indexing, integration, and scaling were performed using HKL2000 (19). The crystals of the CO 2 -bound holo-and apohCAII diffracted to 1.1 Å resolution and were processed to a completeness of 99.9% and an R sym of 8.8% and a  completeness of 93.1% and an R sym of 8.0%, respectively. Complete processing statistics are given in Table 1.
Structure Solution and Model Refinement-The structures of CO 2 -bound holo-and apohCAII were solved in a similar manner using the program SHELXL (20). Prior to refinement, a random 5% of the data were flagged for R free analysis (21). The previously determined 1.54 Å resolution crystal structure of holohCAII (PDB ID: 2CBA) (16) was stripped of all waters, the zinc, and any alternate conformers and used as the initial phasing model in a round of least squares, rigid-body refinement to 2.5 Å resolution to an R factor /R free of 31.3/33.2% for holo and 28.0/28.6% for apo enzyme. The data were then extended to 1.5 Å resolution, and the model was refined using conjugant gradient least squares refinement. After 20 cycles, the model and related -weighted electron density maps were read into the molecular graphics program Coot (22). Improperly built side chains and the zinc (in the holo structure only) were placed into their respective density, and the model was run through another round of conjugant gradient least squares refinement. Waters with positive density in the -weighted difference map were kept until all waters with reasonable density were built. The data sets were then extended to 1.1 Å resolution, and the final waters were built. Disorder was then modeled into the density by modeling all visible alternate conformations for both amino acid side chains and waters. Riding hydrogens were then placed on all residues except the imidazole nitrogens of the histidines. The weighting factor was then changed to 0.2 for one round followed by the use of all data for the final round. The final R final /R free for holo was 10.9/12.9%, and the final R final /R free for apo was 10.4/13.9%. Complete refinement statistics can be found in Table 1. The model geometries and statistics were analyzed by PROCHECK (23).
Additional Structural Features-There were no major changes in any amino acid atomic positions other than that mentioned for the phenyl ring of Phe-226 (due to CO 2 binding) (Fig. 2). There were a large number of dual amino acid conformations in these structures; however, this can be attributed to the near atomic resolution of the structure and not necessarily to the pressurized environment that the crystals experienced (the alternate conformations were Ile-22, Leu-47, Ser-50, Asp-52, His-64, Ser-152, Ser-217, and Val-223 for the holo, and Ile-22, His-64, Gln-103, Asp-162, Lys-172, Glu-214, Ser-217, and Val-223 for the apo structure). Many previous structural studies of hCAII have shown that His-64 occupies two conformations, termed "in" and "out." Generally it has been seen that the "in" conformation is favored, although there may be pH effects on the ratio of "in" to "out" (10,13). In both the holoCO 2 -bound and the apoCO 2 -bound structures, His-64 was seen to have a preference for the "out" conformation. Due to the presence of glycerol as a cryo-protectant, a well ordered glycerol molecule was observed at the mouth of the active site cavity. The oxygens of the glycerol overlap with the positions of the waters of the proton wire, thus indicating that the glycerol might be binding advantageously due to the "out" prevalence of His-64. Interestingly this glycerol molecule is seen only in the holo enzyme. A second molecule of glycerol is seen in the same posi-tion in both the holo and the apo structures on the surface of the protein located near residues 243-245 (Fig. 3).

RESULTS
Here we describe for the first time, to our knowledge, the experimental capture of CO 2 in the hydrophobic cavity of hCAII (Fig. 1). The holo-and apohCAII CO 2 -bound structures were refined to 1.1 Å resolution with final R factors of 10.90 and 10.35, respectively (Table 1) Table 2) and were refined, assuming full occupancy, and had final  a Values in parentheses are for the highest resolution shell. b R sym ϭ (⌺͉I Ϫ ͗I͉͘/⌺͗I͘) ϫ 100. c R factor ϭ (⌺͉F o ͉ Ϫ ͉F c ͉/⌺͉F obs ͉) ϫ 100. d R free is calculated the same as R factor , except it uses 5% of reflection data omitted from refinement. e The first number given is the average B-factor for the active site-bound CO 2 , the second is for the CO 2 bound near Phe-226. f The root mean square deviation of C ␣ positions as compared with the 1.1 Å resolution crystal structure of unbound holo hCAII (PDB ID: 2ILI) (13).

Comparison of holo-and apoCO 2 -bound hCAII-
The holohCAII structure shows, as modeled previously (1,8), that one of the oxygens of the CO 2 , O(2), interacts (3.5 Å) with the amide of Thr-199, and in doing so, causes a displacement of the water molecule W DW , whereas the O(1) is positioned between the zinc and Val-121. This arrangement places both CO 2 oxygens nearly equidistant from the oxygen of the zinc-bound solvent with distances of 3.0 and 3.1 Å, respectively, putting the carbon 2.8 Å from the zinc-bound solvent. This results in a side-on orientation of CO 2 with the zinc-bound solvent, at a distance that is well suited for the nucleophilic attack to take place on the carbon by the lone pair electrons of the oxygen in the zinc-bound hydroxide ( Table 2, Fig. 1b). Additionally, a new (or displaced) water molecule, W I , not previously observed in other holohCAII structures is seen to occupy a space between Thr-200-O␥ 1 and the O(2) oxygen of CO 2 (Figs. 1, b and c, and 4).
Interestingly, the CO 2 molecule in the apo enzyme shares a very similar geometry despite the absence of the zinc (Fig. 1c). A water is positioned near what would have been the zinc-bound solvent in the holohCAII, although it is ϳ0.6 Å closer to the histidine ligands. Both the CO 2 oxygens are positioned ϳ3.1 Å from this water molecule. The small shift of this water allows the CO 2 to pivot about the O(1) atom, shifting O(2) into a slightly tighter interaction with the amide nitrogen of Thr-199 (3.15 Å for apo when compared with 3.5 Å for holohCAII) (Fig.  1, b and c).
Secondary CO 2 Binding Site-In addition to the catalytic binding site, another CO 2 binding site (not believed to be involved in catalysis) was observed in a second hydrophobic pocket, ϳ11 Å away from the active site (Fig. 4a). In this pocket, the CO 2 displaces the phenyl ring of Phe-226, inducing a 30°tilt with respect to the plane of the ring (Fig. 4b).
Furthermore, this pocket lies next to Trp-97, a residue that biophysical analyses have shown acts as an initiator of proper folding of hCAII (24).

DISCUSSION
Catalysis of the hydration of CO 2 by hCAII at 10 8 M Ϫ1 s Ϫ1 approaches the diffusion-controlled limit and follows Michaelis kinetics with a maximal turnover near 10 6 s Ϫ1 and K m near 10 mM. The diffused CO 2 is expected to be loosely bound since it has no dipole moment, and the fact that CO 2 is more soluble in organic solvents is consistent with the observed hydrophobic binding site, which suggests that solvation is a significant contributor to binding. The dissociation constant of CO 2 at the active site of hCAII was estimated by infrared spectroscopy to be 100 mM (25), a value consistent with the kinetic properties of the catalysis. The constant of Henry's Law for the solubility of CO 2 in water under the conditions of these experiments (15 atm CO 2 ) indicates a maximal concentration of CO 2 near 0.45 M (26). These considerations suggest a nearly complete occupancy of CO 2 at the active site. With an energy barrier for catalysis near 10 kcal/mol, an insignificant reaction rate is expected at liquid nitrogen temperature. However, in our procedure, CO 2 was introduced to the crystal at room temperature, a procedure that surely decreased the effective pH of the crystal and surrounding solvent and promoted the forma-  (32). The binding modes of both the CO 2 substrate and the HCO 3 Ϫ product are similar, with the substrate favoring the hydrophobic side (green) and product favoring the hydrophilic side (orange). Note that the deep water (W DW , gray sphere) is displaced and a new water occupies the area between the side chain of Thr-200 and CO 2 (W I , cyan) upon CO 2 binding. The CO 2 is orientated so that the carbon is primed for the nucleophilic attack by the zinc-bound hydroxide (orange sphere). b, a superposition of the V143Y variant of hCAII (9,27). Note that the side chain of Tyr-143 (white) acts as a steric block to the CO 2 binding site. The figure was created using PyMOL.

TABLE 2 Distance (Å) geometry of CO 2 for holo-and apohCAII
The bond distances from the CO 2 molecule are given within a radial shell of 3.9 Å. The atoms of the CO 2 molecule are in bold text. The numbering of the CO 2 oxygens are in accordance to the text and figures.

Holo
Apo tion of the zinc-bound water at the active site. The observation of CO 2 at the active site is consistent with a zinc-bound water in our structures since this form would predominate at acidic pH and is unreactive toward CO 2 . The zinc-bound hydroxide form of the enzyme reacts with CO 2 ; however, the observation of no bound bicarbonate suggests that this form of the enzyme was not prominent. That the binding of CO 2 does not involve first-shell coordination to the zinc is consistent with previous spectroscopic studies (27,28). Moreover, the observed CO 2 binding site confirms previous kinetic and structural analyses of mutations made at Val-143 (9,29). From these studies, it was shown that bulkier substitutions led to significant decreases in activity. For example, a V143Y mutant had less than 0.02% the activity of the wild-type enzyme. A structural least squares superposition of V143Y with that of CO 2 -bound wild-type enzyme (C ␣ r.m.s.d. ϭ 0.26 Å) clearly shows that the tyrosine would directly interfere with CO 2 binding, thus blocking the substrate from binding in an orientation that is optimal for nucleophilic attack by the zinc-bound hydroxide (Fig. 2b).
The binding interactions of CO 2 determined here are very similar to those of the isoelectronic NCO Ϫ ion that is a potent inhibitor of hCAII. Crystallographic analysis of the complex of NCO Ϫ and hCAII shows that cyanate is bound on the hydrophobic surface of the active-site cavity and does not displace the zinc-bound water (30). Moreover, like bound CO 2 , the cyanate ion displaces the deep water and forms a hydrogen bond with the backbone amide of Thr-199; the tetrahedral coordination about the zinc is not disturbed in the complex. The distance between the carbon of bound cyanate and the oxygen of zinc-bound water is 2.4 Å, again similar to the corresponding distance for bound CO 2 . This comparison of the binding of CO 2 and the inhibitor NCO Ϫ supports our hypothesis that the observed binding site of CO 2 (Fig. 1) is a site of productive substrate binding. It is interesting to note that in studies of Co(II)-substituted carbonic anhydrase, cyanate appears to bind directly to the zinc (31).
The observed binding site of CO 2 aids in the interpretation of the next step of catalysis, the formation and subsequent release of the product, bicarbonate. Following the nucleophilic attack, two mechanisms have been proposed for the subsequent release of the HCO 3 Ϫ ion based on the theoretical free energy calculations of CO 2 /HCO 3 Ϫ intercoversion. The Lipscomb mechanism (32) propounds a monodentate Zn-HCO 3 Ϫ intermediate wherein a proton rapidly migrates from the original Zn-OH Ϫ to one of the other two oxygen atoms of the HCO 3 Ϫ ion. The zinc in this mechanism is held in a tetrahedral coordination (Fig. 5a). In contrast, the Lindskog mechanism (33) proposes a bidentate Zn-HCO 3 Ϫ intermediate that requires one of the two oxygen atoms of the original CO 2 molecule to coordinate directly with zinc, resulting in a penta-coordinated metal ion held in a trigonal bipyramidal geometry (Fig. 5b).
In addition, physical evidence is provided by previous structural work. Xue et al. (34) made a T200H mutant that displayed a higher affinity for HCO 3 Ϫ ion than the wild-type enzyme, and as a consequence, were able to capture bicarbonate in the active site and study the complex using x-ray crystallography (Figs. 2a  and 4). Least squares superposition of this structure on the wild-type hCAII CO 2 -bound structure (C ␣ r.m.s.d. ϭ 0.21 Å) shows that the CO 2 substrate molecule exists in the same plane as the Zn-HCO 3 Ϫ product (Fig. 2a). From a strictly structural perspective, the pseudo-bidentate nature of the captured Zn-HCO 3 Ϫ complex seems to favor the Lindskog hypothesis. Nevertheless, in both mechanisms, the release of the HCO 3 Ϫ product from the Zn-HCO 3 Ϫ intermediate is associated with the binding of a water molecule to the metal. The appearance of the previously unseen water, W I , in close proximity to the zinc was observed in the CO 2 complex structures of both the holo and the apo enzymes. This water could be either the displaced W DW water, seen prior to CO 2 binding (Fig. 2a), or a new ordered water, possibly arising due to a change in the local electrostatic environment. The position of this water with respect to the zinc leads us to suggest that this water may be the best candidate in the aforementioned water-associated displacement of product bicarbonate (Figs. 2 and 6).
In summary, this method of using pressurized gases, such as CO 2 , may be applicable to other enzymes to capture weakly bound substrates and/or identify hydrophobic pockets in enzymes that might play important roles in substrate binding or protein folding. The identification of a CO 2 binding site at the active site of hCAII, in combination with the previously known bicarbonate binding site and the location of ordered water molecules, W DW and W I , should now guide molecular dynamics studies to examine the catalytic event itself and decipher whether the Lipscomb (32) or Lindskog (33) mechanism is more feasible.


text:
Photodynamic effect of hypericin on the conformation and catalytic activity of hemoglobin.

Hypericin, extracted from H. perforatum, can induce the generation of reactive oxygen species by visible light irradiation, which may consequently induce the conformational change of hemoglobin. We have not only employed UV-vis spectroscopy to observe the changes of UV-vis spectra of the protein, which reveals the conformational changes of the protein, but also employed electrochemical method to obtain its enhanced peroxidase activity. The photodynamic effect of hypericin on the conformation and catalytic activity of the protein has also been proven to be strongly dependent on the irradiation time, the hypericin concentration and the presence of oxygen. This work is beneficial not only to the fabrication of more sensitive hydrogen peroxide biosensor, but also to the guidance of the usage of this medicinal herb molecule, since the conformational change of the protein and the enhanced peroxidase can be easily obtained only by visible light irradiation on hypericin, the process of which is so common to happen.

Introduction
Study on photosensitizers has received more and more attention. However, the effect of the photosensitization on DNA, protein, cell, or even an organ should be given more attention, no matter whether the effect is harmful or helpful for the potential clinical application. Hypericin (HY, Scheme 1) is a phenanthroperylene quinine derivative. It can be extracted from H. perforatum, a popular medicine for the treatment of depression [1][2][3][4][5][6][7][8]. It has been used as a conventional medication for the treatment of depression and wound healing for a long time [9]. Recently, more and more interest has been given to some other important pharmaceutical potentials of this species, such as antivirus activity, anti-HIV, antibacterial activity and antitumor activity [10][11][12]. Most of the clinic functions are proposed to be related to the phototoxicity of HY. For instance, the cytotoxic activity can be greatly enhanced after light activation [9,[13][14][15][16]. The mechanism how it works is still to be explored, however, it is proposed that after being exposed in visible light at the wavelength of 540-600nm, HY will transfer light energy to oxygen to generate reactive oxygen species (ROS), which may further induce the apoptosis of cells [17]. Some other studies report that HY preferentially accumulates in the cell membranes, especially the mitochondria membrane, which might be the target in the photodynamic therapy [18]. Both the type I and type II photodynamic reactions can take place in the photoactivation of HY, resulting in the formation of radicals, such as singlet oxygen and superoxide radical [9]. Scheme 1. Chemical structure of hypericin.
In the paper, we report our studies of the photodynamic effect of HY on the structure and function of a protein by employing UV-vis spectroscopy and electrochemical methods. Hemoglobin (Hb), a kind of heme protein, is used as the target protein for this work. On one hand, the structure and function of Hb are relatively very clear, which is easier to get to know the information of the conformational and functional changes. On the other hand, it is relatively easier to design the experimental protocols, since its electrochemistry has been somewhat largely studied in our lab [19,20]. Besides, we have found that the reactive molecules, such as hydroxyl radical, superoxide anions, may influence the peroxidase activity of the protein, which may be ascribed to the conformational transformation of the protein [21]. Based on the data obtained in this study, we found that visible light irradiation of the protein in the presence of HY would cause the change of its conformation. And, the peroxidase activity of the protein towards hydrogen peroxide (H 2 O 2 ) can be obviously enhanced. Since the structural and formational changes can take place after the treatment of the protein with visible light, the process of which is so common to occur, this study should be very interesting to lots of scientists in different research areas.

Results and Discussion
UV-vis spectroscopy is a very helpful technique to study the conformational changes of heme proteins, since the Soret band of the heme which is located at 407nm can provide very useful information on the secondary structure of heme proteins [23][24][25][26]. The position of the Soret band will shift or the absorption will decrease if the structure of a heme protein is transformed. Figure 1 shows that the absorption of the Soret band remains almost unchanged even after 3h irradiation by visible light in the absence of HY, however, the peak will decrease evidently only after 1 hour treatment if this medicinal species is in the presence. The longer the irradiation time is, the smaller the absorption peak is. Therefore, the heme moiety of the protein can be hardly influenced only by visible light. Nevertheless, irradiation treatment on Hb together with HY, the structure of the protein, especially the microenvironment of the heme ring, will be changed or even be badly damaged. We propose that the ROS, generated by the irradiation of HY, may react with the protein at the heme position, and change the microenvironment of the heme ring. It can be also observed from Fig.1 that the decrease of the absorption is much less significant for the case of lower HY concentration. So, the photodynamic effect of HY on Hb is not only a time dependent but also a concentration dependent process. We have further employed electrochemical method to check whether the dynamic activity of HY may have any effect on the peroxidase activity of the protein. As is well known, the main physiological function of Hb is to carry oxygen. In the meantime, some other activities of this protein have also been revealed. Especially in recent years, the peroxidase activity of Hb has been largely studied, and these findings have been employed for the development of third-generation biosensors [27]. Based on our previous work of the preparation of protein-film modified electrodes and the fabrication of Hb-based H 2 O 2 biosensors with this protein [19][20][21], we have first prepared an Hb-HY film modified electrode to study the catalytic activity of Hb towards H 2 O 2 . As is shown in Figure 2, the reduction peak of Hb will increase gradually with the addition of H 2 O 2 in the test solution. Further studies reveal that a linear relationship between the increase of the reduction peak and the H 2 O 2 concentration can be obtained from 1×10 -5 mol/L to 5×10 -4 mol/L, and the linear regression equation is: y = 2.15459 + 0.27599x, r = 0.999.
In order to obtain an obvious contrast, and to more clearly show the change of the peroxidase activity of the protein, the value of the reduction peak with the addition of 1×10 -5 mol/L H 2 O 2 is chosen as the baseline, and is set to be 0. As is shown in Figure 3, the slope of the catalytic linearity is enhanced obviously after the irradiation of visible light on Hb together with HY. So, the catalytic activity of Hb towards H 2 O 2 has been obviously improved. And, the slope increases with the irradiation time, which suggests that a longer time treatment on the samples will result in a higher catalytic ability of the protein. Meanwhile, concentration of HY is found to be another key factor. As is shown in Figure 4, although the slopes will also increase for the case of low HY concentration, the changes are indistinct in the first two hours. An obvious increase can be observed only after a 3 hours irradiation. And the increase is much smaller than that for the case with high HY concentration. These experimental results obtained with electrochemical method coincide very well with the results obtained by UV-vis spectroscopy, which further reveals that the photodynamic effect of HY is not only a time-dependent, but also a concentration-dependent process.  From our further studies, this effect has also been known to be an oxygen-dependent process, although O 2 is not required for the antivirus activity of HY [28]. As is shown in Fig.5, if the samples are prepared and irradiated in an anaerobic situation, the catalytic slope will keep unchanged even if a 3h treatment has been made. This is reasonable, since O 2 is required in a photodynamic process to generate ROS, which is crucial in photoactivating HY. So, no influence of this medicinal species on the peroxidase activity of Hb can be obtained without O 2 being involved. These results have also confirmed that the phtosensitization of HY would be achieved only in the presence of oxygen. After absorbing the light energy, HY will be transformed to be an excited state, which may further transfer the energy to oxygen directly or indirectly, both of which induce the formation of the ROS, especially the singlet oxygen. ROS then react with Hb, which may induce the change of the microenvironment of the heme position. As a result, the catalytic ability of the protein towards H 2 O 2 is enhanced. Linear relationship between the increase of the reduction peak current and H 2 O 2 concentration after visible light irradiation of 0h, 1h, 2h, 3h, respectively in an anaerobic condition. Others same as in Figure 3.
In summary, with UV-vis spectroscopic and electrochemical techniques, we have found that irradiation on the mixture of Hb and HY by the very common visible light can activate HY to generate ROS, which will make the change of the structure of Hb and enhance the catalytic activity of the protein towards the H 2 O 2 reduction. This process depends not only on the irradiation time but also on the concentration of HY. Meanwhile, O 2 , which is required for the generation of ROS, is also found to be a vital element in the photoactive effect. This work has not only confirmed the photosensitization of HY and revealed the enhanced peroxidase activity of Hb, but also be helpful to the development of more sensitive H 2 O 2 biosensor and the usage of this medicinal herb molecule.

Reagents
HY (~99%) was purchased from Shanghai Healthjoy Chemical Co. Ltd. Stock solutions were prepared by dissolving known amounts of HY in 1 mL of dimethyl sulphoxide (DMSO). Hb from bovin blood was purchased from Sigma and used without further purification. Stock solutions were prepared by dissolving known amounts of Hb in 1 mL of double-distilled water. DMSO and H 2 O 2 (30% (w/v) solution) were provided from Shanghai Jinshan Tingxin Chemical Reagent Co. and Nanjing Chemical Reagent Co., respectively. Other chemicals were of analytical grade. Doubledistilled water, which was purified with a Milli-Q purification system (Branstead, Boston, MA, U.S.A.) to a specific resistance of >18MΩ cm, was used in all the experiments, and all the solutions were stored in the refrigerator at 4 °C.

Preparation of UV-vis spectroscopic samples and light treatment
Sample I preparation: 1µL HY solution (0.1 mol/L), 5µL Hb (8mg/ml, pH 7.0) and 4µL Tris-HCl buffer ( pH 7.0) were mixed in a microcentrifuge tube. Sample II preparation: 0.5µL HY solution (0.1 mol/L), 5µL Hb (8mg/ml, pH 7.0) and 4µL Tris-HCl buffer ( pH 7.0) were mixed in a microcentrifuge tube. Sample III preparation:1µL HY solution (0.1 mol/L), 5µL Hb (8mg/ml, pH 7.0) and 4µL Tris-HCl buffer ( pH 7.0) were mixed in a microcentrifuge tube. This sample was thoroughly deoxygenated by blowing high-purity nitrogen for at least 1 min, and then sealed by parafilm. The samples were exposed in the visible light emitted by a 200W filament lamp, which was filtered by an orange filer to get a wide band illumination above 580nm, for 1h, 2h, 3h, respectively, at a distance of 20cm.

Spectrocopic measurements
UV-vis spectroscopy was performed using a UV-2550 spectrophotometer (Shimadzu, Japan). After the treatment with visible light irradiation, the samples were then twentyfold diluted and analyzed immediately with the spectrophotometer. The blank was a 0.1M tris-HCl solution (pH 7.0).

Preparation of the modified electrode
The substrate pyrolytic graphite (PG) electrode (A = 6.28 mm 2 ) was prepared by inserting a PG rod in a glass tube and fixing it with epoxy resin. Electrical contact was made by attaching a copper wire to the rod with the help of Wood's alloy (a fusible bismuth-based alloy). Before the modification of the substrate electrode with HY and the protein, the substrate PG electrode was first polished using rough and fine sand papers. Its surface was then polished to mirror smoothness with alumina (particle size of approx. 0.05 µm)/water slurry on silk. Finally, the electrode was thoroughly washed by ultrasonicating in both double-distilled water and ethanol for approx. 5 min. 10µL of HY and Hb mixture with their concentration being 1×10 -2 mol/L and 6.2×10 -5 mol/L, respectively, was mixed with 10µL DMSO (20%) and then spread evenly on the surface of the substrate PG electrode. The HY and Hb mixture had been previously treated with visible light irradiation before its immobilization onto PG electrode surface. The modified electrode was dried overnight at room temperature in the dark. After that, this electrode was thoroughly rinsed with double-distilled water and could be ready for use.

Electrochemical Apparatus
Cyclic voltammetry (CV) was performed with a PARC 263A Potentiostat/Galvanostat (EG&G; Princeton, NJ, USA), using a three-electrode configuration at 25 ± 0.5 °C. The reference electrode was a saturated calomel electrode (SCE) and the counter electrode was a platinum electrode. Potentials are reported with respect to SCE unless specially stated. All the test solutions were thoroughly deoxygenated by bubbling high-purity nitrogen through the solution for at least 10 min. A stream of nitrogen was blown gently across the surface of the solution in order to maintain the solution anaerobic throughout the experiments.


s2fieldsofstudy: Chemistry Medicine

text:
Solid-phase extraction and reverse-phase HPLC: application to study the urinary excretion pattern of benzophenone-3 and its metabolite 2,4-dihydroxybenzophenone in human urine.

Background: Benzophenone-3 (BZ-3) is a common ultraviolet (UV) absorbing compound in sunscreens. It is the most bioavailable species of all UV-absorbing compounds after topical application and can be found in plasma and urine. Objectives: The aim of this study was to develop a reverse-phase high performance liquid chromatography (HPLC) method for determining the amounts BZ-3 and its metabolite 2,4-dihydroxybenzophenone (DHB) in human urine. The method had to be suitable for handling a large number of samples. It also had to be rapid and simple, but still sensitive, accurate and reproducible. The assay was applied to study the urinary excretion pattern after repeated whole-body applications of a commercial sunscreen, containing 4% BZ-3, to 25 healthy volunteers. Methods: Each sample was analyzed with regard to both conjugated/non-conjugated BZ-3 and conjugated/non-conjugated DHB, since both BZ-3 and DHB are extensively conjugated in the body. Solid-phase extraction (SPE) with C8 columns was followed by reverse-phase HPLC. For separation a Genesis C18 column was used with an acethonitrile-water mobile phase and the UV-detector was set at 287 nm. Results: The assay was linear r2 > 0.99, with detection limits for BZ-3 and DHB of 0.01 μmol L−1 and 0.16 μmol L−1 respectively. Relative standard deviation (RSD) was less than 10% for BZ-3 and less than 13% for DHB. The excretion pattern varied among the human volunteers; we discerned different patterns among the individuals. Conclusions: The reverse-phase HPLC assay and extraction procedures developed are suitable for use when a large number of samples need to be analyzed and the method fulfilled our objectives. The differences in excretion pattern may be due to differences in enzyme activity but further studies, especially about genetic polymorphism, need to be performed to verify this finding.

Introduction
Ultraviolet (UV) radiation from the sun has adverse effects on human skin; it causes photoaging, sunburn and most seriously, skin cancer. The protective ozone layer has been subject to destruction and the areas where the ozone layer is thin have higher incidence of skin cancer.
Malignant melanoma is one of the most rapidly increasing types of cancer in Sweden and non-melanoma skin cancer is also increasing. UV-radiation from the sun is the most important etiological factor.
Sunscreens are widely used to protect us against harmful radiation. Benzophenone-3 (BZ-3) has been a commonly used fi lter in sunscreens for the last few decades; it has both UVA and UVB protecting properties. It is an organic chemical absorber with a molecule weight (MW) of 228.25 and CAS number 131-57-7. The structure is shown in Figure 1a (ChemIDplus). The ultimate sunscreen stays on the skin and its protective mechanism functions there. However, BZ-3 is relatively lipophilic (log P 3.64 ± 0.37) and several reports have shown that BZ-3 penetrates the skin and is excreted in urine (Hayden, Roberts et al. 1997;Gustavsson Gonzalez, Farbrot et al. 2002;Janjua, Mogensen et al. 2004;Sarveiya, Risk et al. 2004;Gonzalez, Farbrot et al. 2006). Urine is the major excretion pathway and 2,4-dihydroxybenzophenone (DHB), one of the major metabolites in rats (Okereke, Kadry et al. 1993). DHB has a MW of 214.22 and CAS number 131-56-6. The structure is shown in Figure 1b. BZ-3 is the most bioavailable compound of all UV-absorbing chemicals, (Nash, 2006) and is extensively conjugated in the human body.
We wanted to develop an assay to measure the amounts of BZ-3 and DHB in human urine. The method had to suit our needs. For example, it had to be possible to handle a large number of samples easily; hence it had to be rapid and simple but still sensitive, accurate and reproducible. Several methods using high performance liquid chromatography (HPLC) and BZ-3 have been described (Abdel-Nabi, Kadry et al. 1992;Jiang, Hayden et al. 1996;Vanquerp, Rodriguez et al. 1999;Chisvert, Pascual-Marti et al. 2001) and this work has been developed to some extent on the basis of previously described method (Abdel-Nabi, Kadry et al. 1992). However, there are few methods for extraction of BZ-3 and DHB in human urine after it has been metabolized by the human body (Sarveiya, Risk et al. 2004). Abdel-Nabi et al. used urine from rats (Abdel-Nabi, Kadry et al. 1992). Several other methods are designed for product evaluation and not for extraction from biological media. None of the methods fi tted our needs completely and for that reason we developed this method. More than 1000 urine samples were collected and each was analyzed for conjugated/non-conjugated BZ-3 and conjugated/non-conjugated DHB. More than 4000 analyses were performed.
The method we developed was used to study the excretion pattern of BZ-3 and DHB in urine after repeated topical whole-body applications of a sunscreen containing BZ-3 to 25 human volunteers.

Chromatography
A gradient HPLC-system with a pump PU-1580 (Jasco), a 50 μl loop injector and a SPD-10A VP uv-vis detector (Schimadzu) were used. Chromatographic separation was achieved on a Genesis C18 ID (4.6 mm × 150 mm) column. A Genesis C18 (20 mm × 4.0 mm) was used as the precolumn.
The mobile phase was acethonitrile:dionized ultra fi ltered water (UF) (0.45 μm) 44:66 (v/v) with 1 mL TFA to 10000 mL mobile phase with gradient elution according to Table 1. UV absorption was done at 287 nm. The runtime was 31.5 min.

HO
Sample pre-treatment Pre-treatment before extracting conjugated samples The urine samples were centrifuged at 2100 g for 10 minutes. 1 mL urine was used. 100 μL glucuronidase/arylsulphatase and 25 μL internal standard were added. The sample was incubated at 37 °C 16 hours. The sample was diluted with 2 mL phosphate buffer (50 mmol L −1 , pH 6.5). Most samples were diluted 1:20 with 0.9% NaCl-solution.

Pre-treatment for non-conjugated samples
The urine samples were centrifuged at 2100 g for 10 minutes. 1 mL was used. 25 μL internal standard was added. The sample was diluted with 2 mL phosphate buffer.
1. The column was activated with 2 mL methanol.
The vacuum was turned off when the methanol reached the top of the sorbent, to prevent the column from drying. The procedure was repeated once. 2. The column was rinsed with 2 mL phosphate buffer. The vacuum was turned off when the buffer reached the top of the sorbent bed, to prevent column from drying. The procedure was repeated twice. 3. The sample was transferred to the column with a pasteur pipette and the sample was then drawn slowly through the column. The column was dried under full vacuum. The procedure was repeated once. 4. The column was rinsed twice with 1 mL phosphate buffer. After each rinse it was dried under full vacuum. 5. The sample was eluted four times with methanol: TFA 99:1 (v/v) 0,25 mL. After each eluation it was dried under full vacuum. 6. The samples were transferred to glass vials.

Laboratory-made extraction controls
One healthy volunteer applied 2 mg cm −2 of a sunscreen containing 4% BZ-3, a total of 33 g of sunscreen containing 1.32 g of BZ-3. All urine was collected for 24 hours after the application. The urine was diluted with urine from four healthy volunteers into two different concentrations, 7.0 μmol L −1 and 45 μmol L −1 for BZ-3 and 0.86 μmol L −1 and 6.3 μmol L −1 for DHB.

Internal standard
BZ was used as internal standard at the concentration 5 mmol L −1 (0.091 g). BZ was diluted in 100 mL methanol. Volumetric fl asks, class A were used. The internal standard was prepared fresh every month and stored in a refrigerator at a temperature between 2 and 8 °C.
Urine collection and urine samples 25 volunteers (16 women and 9 men; mean age 27 years, range 22-42) participated in the study. Height and weight were measured. Their body surface area (BSA) was calculated with the DuBois formula: BSA = 0.007184 × [height (cm)]0.725 × [weight (kg)]0.425). The sunscreen used was a commercially available sunscreen, SPF 14 containing 4% BZ-3. Before the fi rst application of sunscreen each volunteer gave a urine sample to confi rm that no BZ-3 was found in the urine prior to this investigation. Each volunteer received 2 mg cm −2 according to his or her BSA. The sunscreen was distributed in plastic containers, one for each application. The amount of sunscreen per application varied between the participants from 26 g to 47 g. The total amount of BZ-3 varied between 10.4 g to 18.8 g. BZ-3 was measured in the urine. One volunteer was excluded because the written instructions were not followed accurately.
The volunteers were instructed to apply the sunscreen evenly over the entire body, with the exception of the scalp and genital area, morning and night for 5 days, a total of 10 times. They were allowed one shower/day before the second application. During the fi ve days the sunscreen was applied, all urine was collected, the volume measured and 10 mL from each sample saved and stored at −70 °C. Hence, each volunteer produced a different n umber of urine samples. They collected the urine for 5 days. After the last appli- cation they continued to collect urine for another 5 days, making a total of 10 days. The time of day, number and volume for each urine sample were recorded. A total of 1234 urine samples were collected and analyzed.

Minimum detectable limits
The minimum detectable limit was defi ned as three times the baseline noise level. The detection limits for BZ-3 and DHB were 0.01 μmol L −1 (0.1 ng per 0.05 mL sample) and 0.16 μmol L −1 (2 ng per 0.05 mL sample), respectively.
Chromatography and selectivity Figure 2 shows chromatograms of the HPLCseparation of BZ-3, DHB and internal standard.
It was more diffi cult to achieve separation for DHB since there was more interference in the beginning of the chromatogram.
A calibration curve was made for each sample series, which also consisted of extraction controls at low and high concentrations before each of the 9 urine samples. Each sample series consisted of 60 samples.

Precision and linearity
Within-day precision showed relative standard deviations (RSD) 10% and 11% for the low concentrations of BZ-3 and DHB and 7.2% and 6.6% for the high concentration of BZ-3 and DHB, respectively. Between-days precision showed RSD 10% and 13% for the low concentrations of BZ-3 and DHB and 8.0% and 8.7% for the high concentration of BZ-3 and DHB respectively. There was no difference regarding precision between BZ-3 and DHB, except for the lowest concentration of DHB, which exhibited greater variation (13%) owing to interferences in the beginning of the chromatogram (Fig. 2).
BZ-3, DHB and internal standard showed excellent linearity with correlation coeffi cient (r 2 ) Ͼ 0.99 for all concentrations.

Excretion pattern
BZ-3 and DHB were extensively conjugated and only a small proportion was excreted in the nonconjugated form, mean values 5.9% and 8.8% respectively (Fig. 3).
The excretion pattern varied among the individuals. We discerned two patterns and three groups. Nine of the volunteers showed a pattern of rapid excretion of conjugated BZ-3 on the days when the sunscreen was being applied, followed by evenly decreasing excretion on the 5 consecutive days when the sunscreen was not being applied. This is exemplifi ed in Figure 4a. Seven of the volunteers showed a slow and even increase of conjugated BZ-3 during the days the sunscreen was being applied and a slow and even decrease during the days the sunscreen was not applied, resembling a Gaussian curve. This is exemplifi ed in Figure 4b. Eight of the volunteers did not fi t into any of the patterns above (Fig. 4c), forming a third group.
The excretion of conjugated DHB followed the excretion of conjugated BZ-3 regardless of pattern.

Discussion
A total of 1234 samples were collected and each sample was analyzed for conjugated/non-conjugated BZ-3 and conjugated/non-conjugated DHB. More than 4000 samples were analyzed and we found this method very suitable for handling large numbers of samples.
Methanol/UF water was used instead of urine for the standard solutions because in high concentrations, the added BZ-3 was precipitated in the urine. For this reason we also included the laboratory-made extraction controls that where extracted with the SPEcolumns in the same procedure as the other urine samples. They were also used to control the withinday and between-days variations, which were found to be suffi ciently low for the method to be considered stable.
As internal standard BZ was used. It is a closely related structural analogue of BZ-3. The use of an internal standard provides a more correct quantitation.
The detection limits were at the same levels as in other methods, (Abdel-Nabi, Kadry et al. 1992;Sarveiya, Risk et al. 2004;Schakel, Kalsbeek et al. 2004) and they were sufficient to analyze the samples on day 10.
Previous studies in rats have shown that BZ-3 is extensively conjugated and excreted in the urine; although a small part is metabolized by cytochromes P-450 to DHB. DHB also undergoes extensive conjugation. A minor route of elimination of BZ-3 is faeces, which were not included in the present study. The metabolite 2,2´-dihydroxy-4-methoxybenzophenone (DHMB) was not investigated since it was detected only in trace amounts in the urine, but it was the major metabolite found in faeces (Okereke, Kadry et al. 1993).
2,3,4-trihydroxybenzophenone (THB) is also a metabolite of BZ-3. However, Sarvieya et al. have shown that THB is only found in trace amounts in human urine (Sarveiya, Risk et al. 2004). In our experimental set-up THB had unstable properties and it was therefore not analyzed.
Reverse-phase HPLC is a reliable method for detection and quantitation. Compared to other methods such as gas chromatography and mass spectronomy, reverse-phase HPLC is in general more available, most laboratories have access to this method.
Pharmacogenetics is the study of how genes infl uence an individual's response to drugs. There are several examples of when pharmacogenetics is of importance. In dermatology, hydroxychloroquine is used to treat severe cases of polymorphic light eruption and discoid lupus erythematosus. Inherited defi ciency of glucose-6-phosphate dehydrogenase should be excluded before treatment with hydroxychloroquine is started.
There are also large inter-individual differences in the capacity to conjugate, for example 10% of the North American population is homozygous for the UGT1A1*28 allele. FDA has guidelines about the anti-cancer drug Irinotecan, stating that reduction in the starting dose should be considered for patients known to be homozygous for the UGT1A1*28 allele (FDA, 2005).
This could be one explanation why there are so pronounced differences in the excretion pattern between the subjects.
In the future the genotype might play a role in determining when to decide what sunscreen is best suited for an individual. If the person's ability to conjugate is low, a sunscreen containing BZ-3 may not be the sunscreen of choice. There have been speculations about the estrogenic effects of some sunscreens, including BZ-3. It might not have any potent effect by itself but if a person is medicated with estrogens there might be a more potent effect from an endocrine active sunscreen in some individuals due to saturation of the conjugation enzymes. It seems that sunscreens containing BZ-3 are being phased out. For example, no sunscreens sold at the Swedish pharmacies contain BZ-3.
Future studies about genotype and sunscreens are needed to evaluate the importance of our fi ndings. . Excretion patterns of conjugated BZ-3 and conjugated DHB during the 10 days urine was collected. Regardless of pattern DHB showed a similar curve to BZ-3. Pattern 1 (9/24) shows rapid excretion on the fi rst days and then evenly decreasing excretion (a). Pattern 2 (7/24) shows a slow and even increase followed by a decrease resembling a Gaussian curve (b). The third group did not fi t into patterns 1 or 2 (8/24) (c).


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Synthesis of thienyl analogues of PCBM and investigation of morphology of mixtures in P3HT

Novel [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) analogues containing benzo[b]thiophene (3a, 3b) and thieno[3,2-b]thiophene (3c, 3d) were synthesized and characterized. The morphology of the thin films prepared from the mixtures of these methanofullerenes with regioregular poly(3-hexylthiophene) (P3HT) was investigated by AFM measurement and UV-Vis absorption spectroscopy. A solubility test of these methanofullerenes was performed by using dichloromethane as a solvent. es-TThCBM (3d) exhibited 1.4 times greater solubility in dichloromethane than PCBM.

Introduction
The demand for inexpensive, renewable energy sources continues to stimulate new approaches to the production of efficient, low-cost photovoltaic (PV) devices [1][2][3]. In particular, the use of polymers for the fabrication of solar cells would offer considerable advantages such as the low-cost production of large-area, flexible, and light solar cells [4][5][6][7]. Recently, bulk heterojunction solar cells consisting of a conjugated polymer (donor) and fullerene derivatives (acceptors) have inspired much scientific research owing to these reasons [8,9]. In this research, it has been demonstrated that the PV cells based on regio-regular poly(3-hexylthiophene) (P3HT, Figure 1) with [6,6]-phenyl-C61-butyric acid methyl ester (PCBM, Figure 1) are highly efficient and promising devices [10]. Many efforts have been devoted to improve device efficiency, for example, changing the solvent for the spin coating of the active layer [11,12], annealing the device to improve the crystallinity of the polymer [13,14], and controlling the growth rate of the organic film [15,16]. These studies reported that the morphology of the active layer is a critical factor that determines the device efficiency.
In particular, there is no research focusing on how the structure of fullerene derivatives affects the morphology of the active layer of a PV device. However, it is essential to explore novel fullerene derivatives in order to achieve a significant improvement in the efficiency of PV cells. Recently, Hummelen et al. reported a device fabricated with a thienyl analogue (ThCBM, Figure 1) [21] for the purpose of improving the miscibility with P3HT. However, there is no obvious explanation related to the advantages of ThCBM over PCBM. In this study, we prepared novel PCBM analogues having various types of thienyl groups and investigated their structural effect on solubility and the morphology of their mixtures with P3HT.

Results and Discussion
Synthesis of thienyl analogues of PCBM Thienyl-substituted methanofullerenes were synthesized by a general procedure [22], as shown in Scheme 1. First, benzo[b]thiophene and thieno[3,2-b]thiophenes were acylated by using SnCl 4 ; the acylation was performed at low temperature as a safeguard against the high reactivity of the thienyl groups (yields: 1a; 15%, 1b; 46%, 1c; 74%, 1d; 33%). Glutaric acid monomethyl ester chloride was used as an acylation reagent in order to form the same structure as that of the butyric acid methyl ester contained in PCBM. The acylation of benzo[b]thiophene yielded 2-and 3-substituted compounds in a 1:3 ratio. These isomers were isolated with a recycling HPLC system using chloroform as an eluent and the compounds 1a and 1b were easily obtained. Then, the carbonyl group of 1 was converted to hydrazone by mixing 1 with p-toluenesulfonyl hydrazide. The compounds 2 were obtained as white to pale yellow solids in good yields (2a; 85%, 2b; 90%, 2c; 74%, 2d; 70%). The methanofullerenes were prepared from p-tosylhydrazones (2) by a one-pot method. A slight excess of p-tosylhydrazone (1.2 equiv) was mixed and heated with C 60 in the presence of sodium methoxide. The reaction was monitored by TLC and stopped within 3 to 4 h. As expected, the compound 3d exhibited an extremely low R f value because of the high polarity of the additional ethyl ester group. During the reaction, mono, di, or highly substituted methanofullerenes were obtained simultaneously; therefore, the monosubstituted methanofullerenes were separated from the mixtures by column chromatography. The products were further purified by reprecipitation from methanol.
In general, the resulting products of the above synthesis process contain the isomers of [5,6]fulleroid (methanoannulene-type bonding) and [6,6]methanofullerene (cyclopropane bonding) [23]. These isomers are clearly distinguishable by the 13 C NMR chemical shifts of bridging units. It is well known that [5,6]fulleroid has low solubility and is thermodynamically unstable; hence, it cannot be used in a photovoltaic device. However, except for 2b, the thienyl-substituted hydrazones 2a, 2c, and 2d yielded only [6,6]methanofullerenes. The hydrazone 2b yielded a mixture of [5,6] and [6,6] isomers, probably because of the steric hindrance of benzo[b]thiophen-3-yl or the difference in the electron-donating property between benzo[b]thiophene-3-yl and 2-yl. Therefore, the product 3b needed heat treatment to convert to [6,6]methanofullerene. In short, thienyl analogues have an advantage over PCBM that they do not need the isomerization step at a temperature as high as 180 °C. The structures of the obtained methanofullerenes (3) were determined by 1 H and 13 C NMR, IR, and FD-MS spectra. The reaction conditions and product yields are summarized in Table 1. a Thermal conversion from [5,6]fulleroid to [6,6]methanofullerene in ODCB.

Solubility of methanofullerenes
Good solubility of fullerenes is important for fabricating a photovoltaic device with good morphology [24]. In fact, PCBM is one of the most soluble fullerene derivatives and this is the main reason why it is used for the fabrication of PV cells. Chlorobenzene is normally used as a solvent of P3HT and fullerenes to fabricate a bulk hetero layer by spin coating; however, chlorobenzene almost completely dissolves methanofullerenes and hence cannot be used to determine and compare the solubility of different methanofullerenes at a small scale; therefore, in this study, dichloromethane was used as a solvent in the solubility test. As shown in Table 2, the solubilities of the obtained methanofullerenes (3) and ThCBM were significantly different despite little difference in their structures. Surprisingly, es-TThCBM (3d) exhibited the best solubility of 1.4 times that of PCBM; in contrast, TThCBM (3c) exhibited the lowest solubility. This result plainly indicates that an ester group is a key structure to yield a good solubility. It is very interesting that the solubilities of regioisomers of 2-BThCBM (3a) and 3-BThCBM (3b) were extremely different; however, the reasons related to the difference of structures are unknown. ThCBM exhibited a lower solubility than those of PCBM and 2-BThCBM (3a).
It is important that the solubility of es-TThCBM (3d) exceeds that of PCBM, which is promising to obtain better and thick active layers for PV cells.

Investigation of morphology
Bulk hetero films of these novel methanofullerenes were spin coated on glass substrates and were characterized by using AFM and UV-Vis absorption spectroscopy in order to study and compare their morphological features. 3-BThCBM (3b) and TThCBM (3c) were excluded because their high-concentration solutions could not be prepared in the solvents used in this study. The bulk hetero films were prepared as follows. Regioregular P3HT and methanofullerene (either 3a or 3d) were dissolved in chlorobenzene at a 1:0.8 wt ratio, followed by filtration. Then, the solution was directly spin coated on a freshly rinsed glass plate and dried for 24 h at room temperature under vacuum.
Tapping-mode AFM measurements were performed on a film area of 500 × 500 nm. and (D) es-TThCBM (3d) showed smooth slopes without such shoulders. As is generally known, the peak at 500 nm is derived from P3HT, almost independent from methanofullerene. Thus, the difference in the shapes of the peaks should be attributed to the variation of the solid state of P3HT [26]. When P3HT is in a crystal state, additional peaks are observed at a higher wavelength because of the π-π interaction between its polymer chains. Therefore, the P3HT contained in the films of PCBM and ThCBM is in a more crystalline state than that contained in the films of 2-BThCBM (3a) and es-TThCBM (3d), which is almost amorphous.
Assuming that the solubility trend of the methanofullerenes is the same in chlorobenzene and dichloromethane, PCBM and es-TThCBM (3d) seem to disperse very well in the mixture and result in more fine morphology. However, the variation of the  film morphology had no correlation with the solubility trend of methanofullerenes and was therefore attributed to the structures of the methanofullerenes: 2-BThCBM (3a) and es-TThCBM (3d) are slightly larger than PCBM and ThCBM. The methanofullerenes (3a, 3d) seem to interfere the aggregation of P3HT, which leads to a more amorphous phase. Comparing PCBM and ThCBM, there is no evident difference in their morphology. The improvement in miscibility between P3HT and PCBM by replacing the phenyl group of PCBM by a thienyl group was not clearly confirmed at this stage.

Conclusion
Novel PCBM analogues containing thienyl groups were synthesized. The morphology of the bulk hetero films of the obtained methanofullerenes was different from that of PCBM.
To some extent, the aggregation of P3HT was restricted by the large volume of 2-BThCBM (3a) and es-TThCBM (3d). Among the obtained methanofullerenes, es-TThCBM (3d) exhibited the best solubility. It is a rare methanofullerene that has greater solubility than PCBM. The improvement in solubility by just adding an ester group is very informative and promising for further development of methanofullerenes. Bulk heterojunction solar cells using newly synthesized methanofullerenes are being fabricated and evaluated.