Radu Silaghi-Dumitrescu

Brandon J Reeder

Peter Nicholls

Chris E Cooper

Michael T Wilson
email: wilsmt@essex.ac.uk

FERRYL HAEM PROTONATION GATES PEROXIDATIC REACTIVITY IN GLOBINS

Keywords: ferryl, globin, myoglobin, haemoglobin, peroxidase, Compound II

Ferryl (Fe(IV)=O) species are involved in key enzymatic processes with direct biomedical relevance; among others, the uncontrolled reactivities of ferryl myoglobin and haemoglobin have been reported to be central to the pathology of rhabdomyolysis and subarachnoid haemorrhage. Rapid scan stopped-flow methods have been used to monitor the spectra of the ferryl species in myoglobin and haemoglobin as a function of pH. The ferryl forms of both proteins display an optical transition with pK~4.7 and this is assigned to protonation of the ferryl species itself. We also demonstrate for the first time a direct correlation between haemo(myo)globin ferryl reactivity and ferryl protonation status, simultaneously informing on chemical mechanism and toxicity and with broader biochemical implications.

INTRODUCTION

Ferryl species, formally [Fe 4+ =O 2-] 2+ , are formed in haem and non-haem iron proteins upon treatment with peroxides or related dioxygen species, as part of their respective catalytic cycles/functions and/or as significant side-reactions under physiological and pathophysiological conditions. [START_REF] Groves | High-valent iron in chemical and biological oxidations[END_REF][START_REF] Silaghi-Dumitrescu | The nature of the high-valent complexes in the catalytic cycles of hemoproteins[END_REF][START_REF] Sono | Hemecontaining oxygenases[END_REF][START_REF] Dunford | Heme peroxidases[END_REF][5][START_REF] Schlichting | The catalytic pathway of cytochrome P450cam at atomic resolution[END_REF][START_REF] Reeder | Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage[END_REF][START_REF] Reeder | The radical and redox chemistry of myoglobin and hemoglobin: from in vitro studies to human pathology[END_REF][START_REF] Vollaard | A new sensitive assay reveals that hemoglobin is oxidatively modified in vivo[END_REF] Such species have long been known to contain an oxo atom bound to Fe(IV), as supported by Mössbauer, resonance Raman, NMR, ENDOR and X-ray absorption (EXAFS) spectroscopies, and by X-ray crystallography (as recently reviewed in Refs [START_REF] Groves | High-valent iron in chemical and biological oxidations[END_REF][START_REF] Silaghi-Dumitrescu | The nature of the high-valent complexes in the catalytic cycles of hemoproteins[END_REF][10][START_REF] Fujii | 17O NMR study of oxo metalloporphyrin complexes: Correlation with electronic structure of MO moiety[END_REF][START_REF] Bollinger | Stalking intermediates in oxygen activation by iron enzymes: Motivation and method[END_REF][START_REF] Behan | On the status of ferryl protonation[END_REF]). In haem proteins, the ferryl species is also referred to as 'Compound II'; the related 'Compound I' species contain an additional oxidizing equivalent located on the porphyrin ring or an adjacent amino acid residue.

Recent experimental and theoretical results have suggested that at least some ferryl species may in fact be protonated, i.e. Fe(IV)-OH rather than Fe(IV)=O. A hydroxo ligand has been implied for the histidine-ligated ferryl group in myoglobin (Mb) Compound II [START_REF] Hersleth | An iron hydroxide moiety in the 1.35 A resolution structure of hydrogen peroxide derived myoglobin compound II at pH 5.2[END_REF] at acidic pH and in cytochrome c peroxidase (CCP) Compound I, based on ~1.9-Å iron-oxygen distances determined by atomic-resolution X-ray diffraction crystallography [START_REF] Bonagura | High-resolution crystal structures and spectroscopy of native and compound I cytochrome c peroxidase[END_REF] (CCP Compound I is an atypical Compound I, in that the additional oxidizing equivalent is localized on a protein side-chain rather than on the porphyrin; in this respect, CCP Compound I is more akin to typical Compound II species). Yamazaki and co-workers have also shown that Compound II formation in peroxidases is accompanied by uptake of a proton (although they could not demonstrate the exact location of this proton). [START_REF] Hayashi | Heme-linked ionization in compounds I and II of horseradish peroxidases A2 and C[END_REF] Green and co-workers have more recently challenged the crystallographic findings on Mb and CCP, using EXAFS and resonance Raman spectroscopies. [START_REF] Behan | On the status of ferryl protonation[END_REF][START_REF] Green | IV) in chloroperoxidase compound II is basic: implications for P450 chemistry[END_REF][START_REF] Stone | X-ray absorption spectroscopy of chloroperoxidase compound I: Insight into the reactive intermediate of P450 chemistry[END_REF][START_REF] Stone | Evidence for two ferryl species in chloroperoxidase compound II[END_REF][START_REF] Green | Application of Badger's rule to heme and non-heme ironoxygen bonds: an examination of ferryl protonation states[END_REF][START_REF] Stone | Resonance Raman spectroscopy of chloroperoxidase compound II provides direct evidence for the existence of an iron(IV)-hydroxide[END_REF] Indeed, Green and co-workers have reported that the Fe-O bond in the quintessential peroxidase HRP (horseradish peroxidase) Compound II is ~1.65 Å, consistent with a non-protonated ferryl group. [START_REF] Green | IV) in chloroperoxidase compound II is basic: implications for P450 chemistry[END_REF] They have further shown that the Fe-O stretching frequency, as measured by resonance Raman, does not significantly change position in the range of pH from 4.5 to neutral and basic values for myoglobin Compound II. [START_REF] Stone | Evidence for two ferryl species in chloroperoxidase compound II[END_REF][START_REF] Stone | Resonance Raman spectroscopy of chloroperoxidase compound II provides direct evidence for the existence of an iron(IV)-hydroxide[END_REF] On the other hand, the same spectroscopic tools, together with Mössbauer spectroscopy and density functional calculations, have been used by Green and co-workers to establish that chloroperoxidase Compound II and cytochrome P450 Compound II are indeed protonated. [START_REF] Behan | On the status of ferryl protonation[END_REF][START_REF] Stone | Evidence for two ferryl species in chloroperoxidase compound II[END_REF][START_REF] Behan | Evidence for basic ferryls in cytochromes P450[END_REF] Chloroperoxidase and cytochrome P450 feature an endogenous anionic thiolate ligand to the haem, instead of the neutral imidazole ligand found in HRP (though some researchers have interpreted NMR and resonance Raman data for the latter as evidence for imidazolate character [START_REF] Silaghi-Dumitrescu | The nature of the high-valent complexes in the catalytic cycles of hemoproteins[END_REF][START_REF] Smulevich | Fifteen years of Raman spectroscopy of engineered heme containing peroxidases: what have we learned?[END_REF]) or in myoglobin. Precisely this difference in axial ligation has been proposed to be the reason why HRP Compound II would, unlike CPO Compound II, be unprotonated. [START_REF] Green | IV) in chloroperoxidase compound II is basic: implications for P450 chemistry[END_REF] A Mössbauer spectroscopy report, complemented by DFT calculations, was also interpreted as evidence that catalase Compound II is at least partially protonated; catalase, like CPO and P450, features an anionic ligand to the haem , namely, a tyrosinate. [START_REF] Horner | Mössbauer identification of a protonated ferryl species in catalase from Proteus mirabilis: Density functional calculations on related models[END_REF] The accumulated body of evidence from spectroscopic techniques thus currently favours the view that ferryl protonation does not occur within physiologically-relevant pH ranges with histidine-ligated haemoproteins, whereas the ferryl group is at least partially protonated in haemoproteins ligated by thiolate or tyrosinate. Nevertheless, as will be shown below, we believe that the entire body of spectroscopic evidence on histidine-ligated haemoprotein Compound II species may also bear an alternative interpretation.

Mb and haemoglobin (Hb) have long been known to form ferryl (Compound II) species upon treatment of the Fe(III) or Fe(II) state with peroxide. [START_REF] Svistunenko | reaction of haem containing proteins and enzymes with hydroperoxides: the radical view[END_REF][START_REF] Kelso King | The mechanism of metmyoglobin oxidation[END_REF][START_REF] Kobert | Beiträge zur Kenntniss der Methämoglobine[END_REF] Ferryl Mb and Hb are relatively stable at neutral and basic pH, but less so at acidic pH values. [START_REF] Reeder | The effects of pH on the mechanism of hydrogen peroxide and lipid hydroperoxide consumption by myoglobin: a role for the protonated ferryl species, (2001) Free[END_REF] This instability at acidic pH parallels an increased reactivity towards reducing substrates and it has been proposed on several occasions that protonation of the ferryl group could be responsible for these trends in reactivity. [START_REF] Reeder | Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage[END_REF][START_REF] Reeder | The radical and redox chemistry of myoglobin and hemoglobin: from in vitro studies to human pathology[END_REF][START_REF] Reeder | The effects of pH on the mechanism of hydrogen peroxide and lipid hydroperoxide consumption by myoglobin: a role for the protonated ferryl species, (2001) Free[END_REF][START_REF] Reeder | Characteristics and mechanism of formation of peroxide-induced heme to protein cross-linking in myoglobin[END_REF] A recent crystal structure of Mb ferryl at acidic pH has indeed shown a long Fe-O bond (1.9 Å), consistent with an OH ligand rather than the commonly-accepted Fe=O description. [START_REF] Hersleth | An iron hydroxide moiety in the 1.35 A resolution structure of hydrogen peroxide derived myoglobin compound II at pH 5.2[END_REF] Although subsequent spectroscopic work has led to the conclusion that the crystal structure of Mb ferryl must be in error and that Mb ferryl is not protonated, [START_REF] Green | IV) in chloroperoxidase compound II is basic: implications for P450 chemistry[END_REF], the reason for the pH dependence of ferryl Mb and Hb reactivity is still important, as this species has previously been detected in vivo and is known to be implicated in pathophysiological states involving acute oxidative stress, with this involvement being most manifest in compartments with lowered pH such as kidney and cerebrospinal fluid [START_REF] Reeder | Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage[END_REF][START_REF] Reeder | The radical and redox chemistry of myoglobin and hemoglobin: from in vitro studies to human pathology[END_REF] (although, notably, even normal blood contains detectable amounts of by-products derived from ferryl Hb [START_REF] Vollaard | A new sensitive assay reveals that hemoglobin is oxidatively modified in vivo[END_REF]).

Below we present data that demonstrate that the optical absorption spectrum of ferryl haem is distinctly pH-dependent. This optical transition is rapid (sub-millisecond) and, in our view, best interpreted as direct protonation of the ferryl haem species.

EXPERIMENTAL

Horse heart myoglobin (Sigma) and human haemoglobin were used as previously described. [START_REF] Reeder | Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage[END_REF][START_REF] Reeder | The radical and redox chemistry of myoglobin and hemoglobin: from in vitro studies to human pathology[END_REF][START_REF] Reeder | The effects of pH on the mechanism of hydrogen peroxide and lipid hydroperoxide consumption by myoglobin: a role for the protonated ferryl species, (2001) Free[END_REF][START_REF] Reeder | Characteristics and mechanism of formation of peroxide-induced heme to protein cross-linking in myoglobin[END_REF][START_REF] Reeder | Mechanism of reaction of myoglobin with the lipid hydroperoxide hydroperoxyoctadecadienoic acid[END_REF] Ferryl Mb and Hb were prepared as previously described; [START_REF] Reeder | The effects of pH on the mechanism of hydrogen peroxide and lipid hydroperoxide consumption by myoglobin: a role for the protonated ferryl species, (2001) Free[END_REF] briefly, a 50 µM solution of met Mb in 20 mM phosphate buffer at pH=8 was mixed with a 1.5-fold excess of hydrogen peroxide at room temperature; at this pH value, the only observable product is Compound II (as opposed to Compound I). [START_REF] Reeder | The effects of pH on the mechanism of hydrogen peroxide and lipid hydroperoxide consumption by myoglobin: a role for the protonated ferryl species, (2001) Free[END_REF] Ferryl formation was monitored in the Soret region as well as in the visible as previously described; [START_REF] Reeder | The effects of pH on the mechanism of hydrogen peroxide and lipid hydroperoxide consumption by myoglobin: a role for the protonated ferryl species, (2001) Free[END_REF] within ~20 minutes, the UV-vis spectrum converted to ferryl (Compound II) and remained unchanged throughout the time required for subsequent stopped-flow measurements. Stopped-flow measurements were performed using an Applied Photophysics model SX-18MV instrument (Applied Photophysics, Leatherhead, UK).

The buffers used for pH jump experiments were 200 mM phosphate (pH 2, 3, 7 and 8) and acetate (pH 4, 5 and 6); alternatively, for acidic pH's citrate buffers were also used.

Diode array-collected data were analyzed within the Pro-K II software (Applied Photophysics). Time courses recorded for up to 20 seconds following mixing of ferryl Mb/Hb with acidic pH solutions could be fitted as an A→B→C sequence. The spectra of A and B were very similar to each other and to the spectrum of Mb/Hb ferryl at pH 8, and also resemble the first spectrum recorded at 1.28 ms after mixing. Spectra of Species A are therefore shown in Figure 1. On the other hand, species C, which accumulates on a slower timescale, featured broadened bands throughout the UV-vis spectrum (cf Supporting Information), suggestive of protein unfolding, most likely accompanied by protonation and dissociation of the proximal histidine ligand. pH dependence data were fitted to the Henderson-Hasselbach equation assuming oneproton processes; for the kinetics data, initial guesses for maximal rates were chosen assuming a pKa of 4.5, after which both the maximal rates and the pKa values were optimized with a least-squares algorithm within Microsoft Excel 2003; calculated pKa values were ~4.7 for all data sets shown in Figure 2. The rates of decay of ferryl haem via reduction by ascorbate were measured by procedures identical to those used for the data in Figure 1, with the exception that 1 mM ascorbic acid was added at the time of mixing pH 8 ferryl with the respective buffers. Turnover experiments with ascorbate were performed under conditions similar to those used in a standard assay for ascorbate peroxidase, [START_REF] Lad | Role of histidine 42 in ascorbate peroxidase. Kinetic analysis of the H42A and H42E variants[END_REF] using 600 µM ascorbate, 1 mM hydrogen peroxide, and 5 µM myoglobin in 20 mM phosphate pH 7 at room temperature. Urate turnover experiments were similar to ascorbate turnover, except for the use of 500 µM urate instead of ascorbate, and 8 µM Hb instead of Mb. Turnover rates varied linearly with Mb/Hb concentrations in these ranges of concentrations (data not shown). Density functional calculations (UBP86/6-31G**) on unsubstituted iron porphyrin adducts listed in Table 1 were performed following protocols previously described; proton affinities are obtained in vacuo, but the trends are readily maintained in solvents; [START_REF] Silaghi-Dumitrescu | The nature of the high-valent complexes in the catalytic cycles of hemoproteins[END_REF]32] direct computational prediction of pKa values as opposed to simple proton affinities is possible but has inherent pitfalls as recently reviewed in Ref. [START_REF] Silaghi-Dumitrescu | Computational Inorganic Chemistry -a useful tool, and more[END_REF].

RESULTS AND DISCUSSION

Figure 1 shows optical spectra collected on rapidly mixing ferryl myoglobin (produced by standard procedures at basic pH, where it is relatively stable) with solutions of varying pH in a stopped-flow spectrophotometer. Following mixing, the time courses of spectral changes were recorded, from which the initial spectra (t=0) could be constructed (see Methods). The latter therefore report the spectra of the ferryl form at any given pH value before decay of the ferryl species or protein structural changes occurred. These spectra show a pH dependence with a pKa of ≤5. Likewise, a pKa of ≤5 is obtained for Hb ferryl in similar pH-jump experiments.

The optically-detected pKa values for the globin haem ferryl species are entirely consistent, as shown in Figure 2, with previously-measured [7, 8, 28-30, 34, 35] pH profiles for Mb ferryl reactivity in autoreduction, or in oxidative reactions such as HPODE consumption, isoprostane formation, or for ascorbate or urate reduction. These latter reactivity data hold direct physiological relevance; 13(S)-hydroperoxy-9,11(cis,trans)-octadecadienoic acid (HPODE), is generated in vivo from linoleic acid by lipoxygenases and has been used as a model lipid peroxide to explore the mechanism of met-globin-induced damage to lipid membranes; [START_REF] Reeder | Mechanism of reaction of myoglobin with the lipid hydroperoxide hydroperoxyoctadecadienoic acid[END_REF] isoprostane F 2 formation from low density lipoprotein (LDL) has been implicated in Mb-and acidosisdriven renal failure [START_REF] Moore | A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysisinduced renal failure[END_REF]; finally, ascorbate and urate are known to detoxify met and ferryl states of myoglobins and haemoglobins. [START_REF] Vollaard | A new sensitive assay reveals that hemoglobin is oxidatively modified in vivo[END_REF][START_REF] Cooper | On the formation, nature, stability and biological relevance of the primary reaction intermediates of myoglobins with hydrogen peroxide[END_REF][START_REF] Ames | Uric acid provides an antioxidant defense in humans against oxidant-and radicalcaused aging and cancer: a hypothesis[END_REF][39] These pH profiles are also identical to those previously measured by Skibsted and co-workers for the reactivity of ferryl Mb towards ABTS (a typical peroxidase substrate) as well as towards chlorogenate. [40,[START_REF] Carlsen | Protein binding in deactivation of ferrylmyoglobin by chlorogenate and ascorbate[END_REF] Free haemin in solution exhibits an identical pH dependence to myoglobin in the HPODE assay, [START_REF] Reeder | The effects of pH on the mechanism of hydrogen peroxide and lipid hydroperoxide consumption by myoglobin: a role for the protonated ferryl species, (2001) Free[END_REF] suggesting that this reactivity pattern is an intrinsic property of the haem rather than being due to protonation of a protein residue. Taken together, these data establish a direct link between biologically-relevant acid-induced reactivity of ferryl globins and a distinct spectral change in the optical spectrum of their haems.

The pH-dependent spectral changes shown in Figure 1 for ferryl Mb are consistent with spectra previously reported for the same species at basic pH vs. pH 3.5.[42] These differences were shown to be paralleled by changes in the MCD spectra that were irreconcilable with simple changes in hydrogen bonding patterns, and could only be explained as arising from protonation of an iron ligand at acidic pH.[42] This ligand was proposed by Thomson and co-workers[42] to be the proximal histidine, i.e.

[imidazole-haem-Fe 4+

-O 2-] 0 ↔[imidazolate-haem-Fe 4+ -O 2-] -1 . The pKa of this proximal histidine is ~11 in ferric Mb, i.e. [imidazole-haem-Fe 3+ -OH -] 0 ↔ [imidazolate-haem- Fe 3+ -OH -] -1 .
[42] Thomson and co-workers have pointed out that a pKa shift of several pH units for the imidazole ligand may be expected upon going from the ferrichydroxide state to the ferryl.[42] Nevertheless, especially in view of the recent crystallographic data on myoglobin Compound II, [START_REF] Hersleth | An iron hydroxide moiety in the 1.35 A resolution structure of hydrogen peroxide derived myoglobin compound II at pH 5.2[END_REF] an attractive alternative interpretation is that the myoglobin ferryl pKa may be due to protonation of the oxygen atom, rather than to protonation of an imidazolate axial ligand. Notably, in the myoglobin Compound II crystal structure, besides the iron-oxygen bond length being consistent with a protonated ferryl, the iron-imidazole bond length is consistent with a protonated imidazole (i.e., it is ~2.1 Å as opposed to the ~1.9 Å predicted for an imidazolate). [START_REF] Silaghi-Dumitrescu | The nature of the high-valent complexes in the catalytic cycles of hemoproteins[END_REF][START_REF] Hersleth | An iron hydroxide moiety in the 1.35 A resolution structure of hydrogen peroxide derived myoglobin compound II at pH 5.2[END_REF] In order better to estimate the relative likelihood of the two sites (imidazolate vs. oxygen atom) being protonated in Mb ferryl, Table 1 shows a list of similar [X-haem-Fe-Y] 0 ↔ [X-haem-Fe-Y] -1 processes (i.e. similar charge balances), where either one of the haem axial ligands X or Y undergoes deprotonation in Mb as well as in selected related proteins. Table 1 lists known pKa values or known protonation states where experimental data is available, and it also lists calculated proton affinities for models of the myoglobin active site as well as of the chloroperoxidase active site (see Experimental section for an account of how these models are treated computationally).

For the cases where both experimental data and calculated proton affinities are available, theory and experiment appear to correlate well, without exception. Thus, calculated proton affinities in excess of 330 kcal/mol always correlate with a protonated state being observed at physiological pH, while calculated proton affinities of 250-260 kcal/mol appear consistent with the deprotonated state becoming populated in the physiological pH range. Table 1 thus notably shows that the proton affinity of [Imidazolate-Fe(IV)=O] -1 is very similar to those of other species, with the same overall charge and present in the same protein pocket, known to be protonated at neutral pH.

Computations thus offer no underlying electronic structural reason to assign the ferryl Mb/Hb pKa as due to protonation of the histidine ligand, rather than the ferryl itself.

Table 1 also shows that the proton affinity of the oxygen atom in an imidazole-ligated ferryl group, i.e. [Im-Fe(IV)-OH] 1+ -> [Im-Fe(IV)=O] 0 , would be slightly lower than the proton affinity of the ferric-hydroxo adduct, i.e. [Im-Fe(III)-OH 2 ] 1+ -> [Im-Fe(III)-OH] 0 (comparing two protonation processes with the same overall charge balance).

Ferric Mb and Hb feature an aqua ligand with a pKa of ~8-9; [START_REF] Antonini | Frontiers in biology[END_REF] the pKa of the ferryl Mb/Hb should therefore be expected to be lower than that of ferric Mb/Hb. This prediction is in good agreement with the experimental data in Figures 1 and2 (note, that our interpretation of the computational results focuses on trends and relative values, while the exact value of the difference in pKa values is not quantitatively addressed by our gas-phase calculations). It thus follows that the iron ligand protonating at pH~5 in ferryl Mb is most likely the 'oxo' oxygen. This now provides an atomic-level insight into the origin of the known [START_REF] Reeder | Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage[END_REF][START_REF] Reeder | The radical and redox chemistry of myoglobin and hemoglobin: from in vitro studies to human pathology[END_REF][START_REF] Reeder | The effects of pH on the mechanism of hydrogen peroxide and lipid hydroperoxide consumption by myoglobin: a role for the protonated ferryl species, (2001) Free[END_REF][START_REF] Reeder | Characteristics and mechanism of formation of peroxide-induced heme to protein cross-linking in myoglobin[END_REF] physiologically-relevant pH-dependence of the myoglobin ferryl reactivity. We have argued elsewhere that the alternative electronic description of Fe(IV)-OH as Fe(III)+hydroxyl radical would recommend protonated ferryl as a particularly reactive species. [START_REF] Reeder | The effects of pH on the mechanism of hydrogen peroxide and lipid hydroperoxide consumption by myoglobin: a role for the protonated ferryl species, (2001) Free[END_REF][START_REF] Reeder | Characteristics and mechanism of formation of peroxide-induced heme to protein cross-linking in myoglobin[END_REF]32] As stated in the introduction, a large body of spectroscopic evidence has recently been interpreted as evidence for a non-protonated ferryl in myoglobin, and for a protonated ferryl in anion-ligated haemoproteins (chloroperoxidase, cytochrome P450).

We feel that this spectroscopic evidence bears an alternative interpretation, which is also in line with our own data reported herein, as follows:

(1)

Green and co-workers find that using resonance Raman they can detect the Fe=O (non-protonated) stretching frequency for ferryl Mb even at pH values as low as 4.5. This is entirely consistent with our measured pKa value of 4.7: there should indeed be significant amounts of unprotonated ferryl present even at pH 5 [START_REF] Behan | On the status of ferryl protonation[END_REF][START_REF] Stone | Resonance Raman spectroscopy of chloroperoxidase compound II provides direct evidence for the existence of an iron(IV)-hydroxide[END_REF][START_REF] Behan | Evidence for basic ferryls in cytochromes P450[END_REF] (2) A history of decades of unsuccesful attempts with chloroperoxidase has shown that detection of the Fe-OH stretching frequency for a protonated ferryl can be extremely problematic. [START_REF] Silaghi-Dumitrescu | The nature of the high-valent complexes in the catalytic cycles of hemoproteins[END_REF][START_REF] Stone | Resonance Raman spectroscopy of chloroperoxidase compound II provides direct evidence for the existence of an iron(IV)-hydroxide[END_REF] Green and co-workers have recently remarkably succeeded to detect this Fe-OH frequency in chloroperoxidase Compound II. [START_REF] Stone | Resonance Raman spectroscopy of chloroperoxidase compound II provides direct evidence for the existence of an iron(IV)-hydroxide[END_REF] However, in parallel Mössbauer and DFT studies, they have also shown that ~30% of chloroperoxidase Compound II is in fact non-protonated; [START_REF] Stone | Evidence for two ferryl species in chloroperoxidase compound II[END_REF] neither they nor others have so far been able to detect this non-protonated CPO ferryl using resonance Raman spectroscopy. One lesson that we can therefore learn from CPO Raman spectroscopy is that detection of the ferryl ironoxygen stretching frequency by resonance Raman can be problematic even after sustained effort. The failure so far to detect a protonated ferryl group in myoglobin by resonance Raman is therefore not a strong argument against such protonation.

(3) Green and co-workers advocate that an anionic ligand (thiolate) in ferryl

Compound II results in protonation of the ferryl form at physiological pH values. [START_REF] Green | IV) in chloroperoxidase compound II is basic: implications for P450 chemistry[END_REF] If one were to interpret our measured pKa value in terms of the basic form of Mb Compound II containing an anionic imidazolate ligand, then we would also have to conclude, by analogy with thiolateligated CPO and P450 (and perhaps also by analogy with tyrosinateligated catalases [START_REF] Horner | Mössbauer identification of a protonated ferryl species in catalase from Proteus mirabilis: Density functional calculations on related models[END_REF]), that the basic, imidazolate-ligated, form of Mb ferryl contains a hydroxide ligand. This, however, would be at odds with the resonance Raman data, showing that even at pH 4.5 at least part of the sites contain a non-protonated ferryl.

(4) EXAFS data on horseradish peroxidase Compound II has recently been interpreted to show an iron-oxygen bond of 1.65 Å. [START_REF] Green | IV) in chloroperoxidase compound II is basic: implications for P450 chemistry[END_REF] This can be safely interpreted as proof for a non-protonated ferryl group. However, as previously discussed by us elsewhere, [START_REF] Silaghi-Dumitrescu | The nature of the high-valent complexes in the catalytic cycles of hemoproteins[END_REF] an older, independent EXAFS study cites a 1.9-Å distance for the same bond length, consistent now with a protonated ferryl. [START_REF] Chance | X-ray absorption studies of intermediates in peroxidase activity[END_REF] Furthermore, a ~0.1-Å difference in the other iron-axial ligand bond length (iron-histidine) also exists between the two EXAFS measurements. [START_REF] Green | IV) in chloroperoxidase compound II is basic: implications for P450 chemistry[END_REF][START_REF] Chance | X-ray absorption studies of intermediates in peroxidase activity[END_REF] In fact the earlier EXAFS measurement citing an Fe-O bond length at 1.9 Å and an ironhistidine bond length at 2.1 Å, is also entirely consistent with the atomic-resolution crystal structure of Mb ferryl, [START_REF] Hersleth | An iron hydroxide moiety in the 1.35 A resolution structure of hydrogen peroxide derived myoglobin compound II at pH 5.2[END_REF] while the more recent EXAFS study [START_REF] Green | IV) in chloroperoxidase compound II is basic: implications for P450 chemistry[END_REF] is at odds with crystallography both in terms of the Fe-O distance and in terms of the Fe-histidine distance; the latter is important to note, as the larger number of atoms in the histidine

6 Å

 6 ligand makes it less likely to be affected by crystallographic disorder or partial occupation compared to the Fe-O distance. There are now three reports of high-resolution crystal structures for three Compound II species (Mb -1.35-Å resolution,[14] CCP -1.3 Å,[15] HRP -1.[45]), all of which concur in indicating a protonated ferryl group and an ~2.1-Å Fe-histidine bond length. Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061421 Copyright 2007 Biochemical SocietyBased on arguments 1-4 presented above, our proposed interpretation of the Mb pKa as being due to ferryl protonation appears to be in reasonable agreement with other spectroscopic evidence as well as with X-ray crystallography.Thomson and co-workers have compared the MCD spectra of acidic and basic ferryl myoglobin, and found that the acid form closely resembled typical peroxidase Compound II species, whereas the basic form did not.[42] If we accept that acid ferryl Mb contains an Fe-OH bond, the implication is that most histidine-ligated Compound II species are also protonated. It has long been known that Compound II formation in peroxidases is accompanied by uptake of a proton;[START_REF] Hayashi | Heme-linked ionization in compounds I and II of horseradish peroxidases A2 and C[END_REF] our findings now seem to indicate that this proton is located on the ferryl moiety. In Mb/Hb, protonation brings about increased reactivity (cf Figure2); a non-protonated ferryl Mb/Hb at neutral pH presents an evolutionary advantage, limiting potentially deleterious reactivity in this dioxygen-carrying protein. It is also an evolutionary advantage that in peroxidases, enzymes the function of which is precisely to form ferryl and efficiently use its oxidizing power, the ferryl is protonated at neutral pH. In peroxidases, the ferryl pKa is likely modulated by a carboxylate side-chain hydrogen-bonded to the proximal imidazole[5]; this added negative charge should increase proton affinity and shift the pKa towards basic values. By contrast, the proximal imidazole/His ligand in Mb is hydrogen-bonded to a neutral backbone carbonyl group, implying a smaller proton affinity. The general conclusion is that Nature gates the reactivity of ferryl species by controlling their protonation state.

Figure 1 .

 1 Figure 1. Upper panel: spectra of ferryl Mb collected at known pH values; the corresponding haemoglobin ferryl spectra are shown as inset. Lower panel: normalized pH dependence of A(529)-A(588) for Mb and Hb; fits to the Henderson equation yield pKa values of ~4.7 for both proteins.

Figure 2 .

 2 Figure 2. Comparison of the optically-detected pKa of Mb and Hb ferryl with pH dependant kinetics of decay with or without exogenous substrates added. The reactivity data points refer to reactions listed in text: 'iso-P' refers to isoprostane F 2 formation byMb,[START_REF] Moore | A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysisinduced renal failure[END_REF] 'HPODE' refers to HPODE (13(S)-hydroperoxy-9,11(cis,trans)octadecadienoic acid) consumption by Mb,[START_REF] Reeder | Mechanism of reaction of myoglobin with the lipid hydroperoxide hydroperoxyoctadecadienoic acid[END_REF] 'ascorbate+ferryl' referes to the rate of ferryl reduction by ascorbate, 'haemin' refers to HPODE consumption by free haemin,[START_REF] Reeder | The effects of pH on the mechanism of hydrogen peroxide and lipid hydroperoxide consumption by myoglobin: a role for the protonated ferryl species, (2001) Free[END_REF] and 'Mb, ascorbate turnover' and 'Hb, ascorbate turnover' refer to catalytic ascorbate and urate oxidation by Mb and Hb, respectively, in the presence of hydrogen peroxide.

Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061421

Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061421 Copyright 2007 Biochemical Society

Acknowledgements. Funding from the European Union-funded project

Eurobloodsusbtitutes and from BBSRC is gratefully acknowledged. Dr. I. Silaghi-Dumitrescu ('Babeş-Bolyai' University, Cluj-Napoca, Romania) is thanked for helpful discussions. Martine Rukengwa is thanked for assistance with preliminary experiments on ascorbate turnover.

protonated; [START_REF] Davydov | A Superoxo-Ferrous State in a Reduced Oxy-Ferrous Hemoprotein and Model Compounds[END_REF] protonated; [START_REF] Davydov | A Superoxo-Ferrous State in a Reduced Oxy-Ferrous Hemoprotein and Model Compounds[END_REF] protonated; [START_REF] Davydov | A Superoxo-Ferrous State in a Reduced Oxy-Ferrous Hemoprotein and Model Compounds[END_REF] alternative interpretation of Figure 2


Wera Teubner

Walter Meinl

Simone Florian

Michael Kretzschmar

Hansruedi Glatt
email: glatt@dife.de

Prof. H R Glatt

Identification and localization of soluble sulfotransferases in the human gastrointestinal tract

Keywords: RT, reverse transcription, SULT, sulfotransferase, UGT, UDPglucuronosyltransferase colon, intestinal biotransformation, SULT, sulfotransferases, stomach Biochemical

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Many drugs, secondary plant metabolites and other xenobiotics enter the human organisms via the gastrointestinal tract. Absorbed xenobiotics often have to be metabolized before they can be excreted. Biotransformation of some compounds may already occur in the intestinal mucosa. Lipophilic xenobiotics are usually absorbed in the small intestine and often metabolized in two phases, the introduction of a functional group in phase I and the usage of this functional group for the conjugation with an anionic moiety in phase II. The situation is somewhat different in the large bowel, where bacteria may convert xenobiotics (e.g. glycosylated phytochemicals) and xenobiotic metabolites (e.g. glucuronides excreted with bile) into products that may be better absorbed than the educts. In general, these metabolites already contain hydroxyl or other nucleophilic functional groups. Thus they may undergo conjugation reactions in the mucosa without need of prior phase-I metabolism. UDPglucuronosyltransferases (UGTs) and sulfotransferases (SULTs) represent the major classes of conjugating enzymes for nucleophilic substrates.

Biotransformation of xenobiotics is usually mediated by enzymes with very broad substrate tolerance and involves some risk of the formation of toxic metabolites, in particular of chemically reactive intermediates that may induce mutations and then lead to degenerative alterations, such as neoplasias. Indeed, stomach cancer is relatively common and colorectal cancer even is one of most prevalent neoplasias [START_REF]Food, Nutrition and the Prevention of Cancer: a Global Perspective[END_REF]. Among the phase-II enzymes, SULTs particularly are often involved in the formation of reactive intermediates [2,[START_REF] Glatt | Activation and inactivation of carcinogens by human sulfotransferases[END_REF]. They transfer the sulfo group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to acceptor molecules.

The sulfate moiety, resulting from O-sulfonation, is a good leaving-group in certain chemical linkages. Such sulfo conjugates are electrophilically reactive. Thus, various aromatic amines, alkenylbenzenes and hydroxymethylated polycyclic aromatic hydrocarbons are examples for SULT-dependent rodent carcinogens [2]. We have expressed individual human and rodent SULTs in target cells of standard mutagenicity tests. Using these tools, we demonstrated for more than 100 chemicals a bioactivation by human SULTs to genotoxic metabolites directly or after phase-I metabolism [START_REF] Glatt | Activation and inactivation of carcinogens by human sulfotransferases[END_REF]. The list of these chemicals comprises heat-induced food constituents (heterocyclic aromatic amines, 5-hydroxymethylfurfural), drugs (oxamniquine, cyproterone acetate), plant metabolites (safrole, aristolochic acids), environmental contaminants (alkylated polycyclic hydrocarbons, 3-nitrobenzanthrone) and industrial chemicals (2-nitropropane, nitro-and aminoarenes). When used at low concentrations, each promutagen was only activated by one or two human SULT forms, but different forms were involved in the activation of different compounds. In other cases, sulfo-conjugation can prevent the activation of promutagens [START_REF] Glatt | Activation and inactivation of carcinogens by human sulfotransferases[END_REF]. SULTs are also important in the regulation of the levels of various hormones, such as steroids, catecholamines and iodothyronines [START_REF] Visser | Pathways of thyroid hormone metabolism[END_REF][START_REF] Eisenhofer | Dopamine sulphate: an enigma resolved[END_REF][START_REF] Strott | Sulfonation and molecular action[END_REF]. The corresponding SULTs show high affinities for their endogenous substrates and could form an efficient defense system against food-borne hormones, if expressed in gut.

For these reasons, it would be interesting to know which SULT forms are expressed in various sections and cells of the human alimentary tract. However, most studies on gastrointestinal SULTs focused on the determination of the conjugation of a specific substrate or the detection of an individual SULT protein in a specific section of the gastrointestinal tract [START_REF] Sundaram | Human intestinal phenol sulfotransferase: assay conditions, activity levels and partial purification of the thermolabile form[END_REF][START_REF] Her | Human jejunal estrogen sulfotransferase and dehydroepiandrosterone sulfotransferase: immunochemical characterization of individual variation[END_REF][START_REF] Harris | Activity of phenolsulfotransferases in the human gastrointestinal tract[END_REF][START_REF] Chen | Human gastrointestinal sulfotransferases: identification and distribution[END_REF][START_REF] Pacifici | Inhibition of human liver and duodenum sulfotransferases by drugs and dietary chemicals: a review of the literature[END_REF]. Dooley et al. [START_REF] Dooley | Expression profiling of human sulfotransferase and sulfatase gene superfamilies in epithelial tissues and cultured cells[END_REF] attempted to systematically study the expression of the various SULTs in human tissues using reverse transcription (RT) PCR. However, this study focused on skin tissues, with less intense investigations of other tissues for comparison. Moreover, mRNA and protein levels may not correlate in many cases. We aimed to identify the SULTs as proteins and/or by way of their enzyme activities in various sections of the human alimentary tract. The expression in colon and rectum was of particular interest, as these are common tumor sites.

A total of 11 human SULT forms have been characterized on the gene, message and protein levels. Two additional possible SULT genes (SULT1C3 and 6B1) were mined in the human genome [START_REF] Freimuth | Human cytosolic sulfotransferase database mining: identification of seven novel genes and pseudogenes[END_REF]. We have constructed the putative SULT1C3 cDNA and demonstrated that it encodes a protein with SULT activity (W. Meinl and H. Glatt, unpublished results). Several nomenclatures have been proposed for SULTs. We use the same names in the present study as in our preceding papers. The names are identical with the names proposed by Blanchard et al.

[14] for all forms except the SULT1C subfamily. We are using the names introduced by Freimuth et al. [START_REF] Freimuth | Human sulfotransferases SULT1C1 and SULT1C2: cDNA characterization, gene cloning, and chromosomal localization[END_REF], who detected two members of this subfamily. Blanchard et al. [14] suggested the designations SULT1C2 for Freimuth's SULT1C1, and SULT1C4 for Freimuth's SULT1C2.

EXPERIMENTAL

Tissue samples

Tissue samples were obtained from Caucasian patients who underwent clinically indicated surgery. This material was anonymized and used following the recommendations of the central ethical committee of the German Medical Association. SULT expression was studied in histologically normal tissue resected together with diseased tissue. Rectal tissue was from 11 patients (3 male, 8 female; age 37 to 75 yr) with rectal tumors. Colon tissue was from 24 patients (13 male, 11 female; age 50 to 76 yr) with colon tumors and from 4 patients (3 male, 1 female; age 36 to 74 yr) with sigma diverticulitis. Cecal tissue was from a male patient (age 68 yr) with a rectal tumor. Ileal tissue was from 3 patients (2 male, 1 female; age 59 to 75 yr) with rectal tumors and from one male patient aged 76 yr with a colon tumor. Jejunal tissue was from a 33-yr old male patient suffering from a pancreas carcinoma. Stomach and liver tissues were biopsies from male patients; no pathological changes were found in these biopsies.

None of the tumor patients had received radio-or chemotherapy prior to the surgery.

Treatment with osmotic laxatives preceded all intestinal surgeries, which were conducted under anesthesia (midazolam for premedication; propafenol, sufetanil, rocuronium and isofluvane/O 2 /air for the operation). There is no indication that any of these agents might affect the expression of xenobiotic-metabolizing enzymes). Some patients were treated with additional drugs. However, the number of patients treated with a given drug was too small for studying a possible influence on SULT expression.

After resection, gut samples were opened and washed with ice-cold mucosa buffer, which contained Tris-HCl (40 mM, pH 7.8), KCl (120 mM), glycerol (200 g/l) and the protease inhibitor aprotinin (60,000 kallikrein inhibitory units per liter, purchased under the trade name Trasolyl from Bayer, Leverkusen, Germany). Tissue samples were frozen in liquid nitrogen and stored at -80 °C until further use. For histological evaluation and immunohistochemistry, tissue specimens were fixed in phosphate-buffered formaldehyde (4 %) and embedded in paraffin.

SULT1A1, 1A3 and 1B1 standards from inclusion bodies

The plasmid hST1B2/pKK233-2 [START_REF] Wang | Expression and characterization of a novel thyroid hormone-sulfating form of cytosolic sulfotransferase from human liver[END_REF] (hST1B2 is another designation for human SULT1B1) served as a template in the PCR procedure modifying the 5´-and 3´-flanking regions for subcloning SULT1B1 cDNA into pET24a(+) (Novagen, Madison, USA) using the restriction sites for NdeI and HindIII. Restriction sites were introduced using the following primers from BioTeZ (Berlin, Germany): 5´-GAA TTC CAT ATG CTT TCC CCA AAA G-3´ and 5´-GAA TTC AAG CTT TAA ATC TCT GTG CGG-3´. After an initial denaturing step for 5 min at 96 °C, amplification was carried out for 30 cycles of each 1 min at 94 °C, followed by 1 min at 60 °C and 2 min at 72 °C, and a final elongation step for 10 min at 72 °C. The reaction mixture (100 µl) contained 4 U Combi-Pol polymerase, 20 pmol of each primer, 50 ng of template, 2 mM dNTPs, 1.5 mM MgCl 2 and Combi-Pol reaction buffer diluted according to the manufacturer's protocol. All PCR reagents were from InViTek (Berlin-Buch, Germany).

For construction of pET-SULT1A1 and pET-SULT1A3, cDNAs were subcloned from pKK233-2-derived plasmids that contained either human SULT1A1 (also termed hP-PST) [START_REF] Falany | Steroid sulfation by expressed human cytosolic sulfotransferases[END_REF] or SULT1A3 (also termed hM-PST) [START_REF] Ganguly | Bacterial expression and kinetic characterization of the human monoamine-sulfating form of phenol sulfotransferase[END_REF] cDNA, into pET28b(+) (Novagen) using the restriction sites for NcoI and HindIII.

Escherichia coli BL21 (DE3) (Novagen) was transformed with pET-SULT plasmids to yield E. coli BL21-SULT1A1, -SULT1A3 and -SULT1B1 strains. Overexpression of SULT in these strains was achieved by induction with isopropylthio-b-D-1-galactopyranoside as described in the pET system manual (Novagen). More than 90 % of the soluble protein was SULT as determined by SDS-PAGE and staining with Coomassie blue. When inclusion bodies were used, a single band (representing SULT) was detected even when excessive protein levels were loaded.

For preparation of inclusion bodies, bacteria harvested from 50 ml culture were lysed in 1.25 ml 40 mM Tris-HCl (pH 8) with 4 % (w/v) saccharose, 1 mM EDTA and 2 mg/ml lysozyme and incubated on ice for 30 min. DNAseI was added to a final concentration of 90 µg/ml as well as MgCl 2 and MnCl 2 each at a final concentration of 20 mM. Incubation on ice was continued for another 30 min. Then 3 ml of a detergent buffer [1 % (w/v) deoxycholic acid, 1 % (w/v) Nonidet-P40, 2 mM EDTA, 200 mM NaCl and 20 mM Tris-HCl (pH 7.5)] was added. The mixture was vigorously shaken and centrifuged at 10,000 g at 4 °C for 10 min. The insoluble fraction was resuspended in 3 ml of a second detergent buffer [0.5 % (w/v) Triton X-100, 1 mM EDTA and 20 mM Tris-HCl (pH 7.4)] and centrifuged. The washing steps were repeated, then the insoluble fraction was resuspended in 3 ml of 50 mM Tris-HCl (pH 7.5) containing 1 mM EDTA, and centrifuged. This step was repeated twice, then the inclusion bodies were resuspended in 1 ml of 0.1 % SDS and stored at -80 °C. We have expressed all human SULTs in S. typhimurium TA1538 at levels amounting to 0.5 to 10 % of the cytosolic protein [START_REF] Meinl | Human sulfotransferases are involved in the activation of aristolochic acids and are expressed in renal target tissue[END_REF]. SULTs in these preparations are more stable and easier to handle than purified enzymes and inclusion bodies, and therefore were normally used as standards in immunoblotting.

SULT standards from cytosolic preparations of recombinant S. typhimurium strains

Preparation of cytosolic fraction from human tissues and bacteria

Surgical specimens were thawed from -80 °C on ice overnight. Opened intestinal samples were placed on an ice-cooled glass plate, rinsed with ice-cold PBS, and mucosa was scraped off with a rubber scalpel. Mucosa and liver were homogenized using a Potter-Elvehjem tissue grinder.

The homogenization medium (3 to 5 ml per g tissue) contained sodium phosphate buffer (10 mM, pH 7.4), KCl (150 mM), EDTA (1 mM) and Complete Protease Inhibitor Cocktail diluted according to the manufacturer's protocol (Boehringer Mannheim, Mannheim, Germany). One milliliter of the same buffer was used for sonication of bacteria harvested from 100 ml overnight culture. The homogenates were centrifuged at 100,000 g for 1 h at 4 °C. The resulting supernatant is termed cytosolic fraction. Protein concentrations were determined according to the method of Lowry using BSA as a standard.

Antisera

Antisera raised in rabbits against human SULT1B1 (previously termed ST1B2) [START_REF] Wang | Expression and characterization of a novel thyroid hormone-sulfating form of cytosolic sulfotransferase from human liver[END_REF] and human SULT2A1 (previously termed DHEA-ST) [START_REF] Comer | Immunological characterization of dehydroepiandrosterone sulfotransferase from human liver and adrenal[END_REF] were kindly provided by C. Falany (Birmingham, Alabama, USA) and are designated as "anti-1B1-antiserum" and "anti-2A1antiserum", respectively. The antisera named "anti-r1E1-antiserum" and "anti-1C1-antiserum" were raised in a rabbit against rat liver estrogen sulfotransferase [START_REF] Forbes-Bamforth | Identification of a new adult human liver sulfotransferase with specificity for endogenous and xenobiotic estrogens[END_REF] and in a sheep against human SULT1C1 [START_REF] Stanley | Expression profiling of human fetal cytosolic sulfotransferases involved in steroid and thyroid hormone metabolism and in detoxification[END_REF] (where it was named SULT1C2), respectively. Both antisera were generous gifts of M. Coughtrie (Dundee, UK).

For generation of anti-1A1-antiserum, two female New Zealand White rabbits (Tierzucht Schönwalde, Schönwalde, Germany) were immunized with 250 µg of human SULT1A1 inclusion bodies dissolved in PBS with 1 % (w/v) SDS and mixed with complete Freund´s adjuvant. Immunization was repeated after four weeks using incomplete Freund´s adjuvant.

After six additional immunizations at four-week intervals, sera were collected. Antisera from both rabbits were probed with various human SULT forms by immunoblot analysis, and the one chosen for further experiments was termed "anti-1A1-antiserum". In some cases immunoabsorption was used to increase the specificity of antisera. Anti-1A1-and anti-1C1-antisera were incubated with SULT1B1-sepharose to remove antibodies binding to SULT1B1. Likewise, anti-1B1-antiserum was incubated with SULT1A1-sepharose to enhance its specificity. For generation of SULT1A1-and SULT1B1-sepharose, isopropylthio-b-D-1-galactopyranoside-induced E. coli BL21-1A1 and -1B1 were sonicated in 50 mM NaHCO 3 (pH 8.3) and the 100,000 g supernatant was coupled to CNBr-Sepharose (Amersham Pharmacia Biotech, Braunschweig, Germany) according to the manufacturer's protocol. Antisera were incubated with SULT-sepharose at 4 °C overnight. These pretreated antisera showed somewhat lower immunoreactivities in immunoblot analyses than the native antisera. Since cross-reacting SULTs could normally be distinguished due to their differing electrophoretic mobilities, the native antisera were used in immunoblot analyses, unless specified otherwise. However, the pretreated antisera with enhanced specificity were consistently used for immunohistochemistry. Moreover, negative control antisera were used in immunohistochemistry; to this end specific antibodies were removed from anti-1A1-and anti-1B1-antisera by incubation with SULT1A1-and SULT1B1-sepharose, respectively.

Immunodetection of SULT forms in cytosolic fractions and characterization of antisera

Cytosolic fractions were separated using SDS-PAGE (11 % polyacrylamide) according to the method of Laemmli. Cytosolic fractions of S. typhimurium TA1538 expressing various human SULTs [START_REF] Meinl | Human sulfotransferases are involved in the activation of aristolochic acids and are expressed in renal target tissue[END_REF] as well as purified SULT inclusion bodies were included as standards. After electrophoresis, proteins were transferred to Hybond ECL membrane (Amersham Pharmacia Biotech) and probed with primary antisera diluted in Tris-buffered saline (50 mM Tris-HCl, pH 7.6, 150 mM sodium chloride) containing Tween 20 (0.1 %) and BSA (1 %). For anti-1A1antiserum, BSA was replaced by milk powder (1 %) as blocking reagent. Anti-1A1-and anti-1B1-antisera were diluted 1:10,000 in blocking reagent; for the other antisera, the dilution was 1:2000. Goat anti-rabbit IgG-peroxidase conjugate (Sigma, Deisenhofen, Germany), at a dilution of 1:2000, was used as secondary antibody. The immunoreactive bands were visualized using the enhanced chemoluminescence system together with Hyperfilm ECL (Amersham).

In order to remove bound antibodies for reprobing with other antisera, membranes were incubated with 62.5 mM Tris-HCl (pH 6.7) containing 100 mM mercaptoethanol and 2 % SDS at 50 °C for 1 h. Afterwards they were washed twice with Tris-buffered saline containing 0.1 % Tween 20 for 15 min before new blocking reagent was applied. Then membranes were probed with the next antiserum as described above. All blots contained various controls (cDNAexpressed SULTs), whose reactivity with the various antisera is known. Incomplete removal of an antibody or washing out of antigen before reprobing would have led to unspecific or insufficient signals, respectively, of these controls -what never was the case under the conditions used. Likewise, comparable results were obtained when we repeated the analysis with the antiserum used for the initial probing at the end of the reprobing series.

Immunohistochemistry

Slices of 4 µm were dewaxed in toluene and rehydrated in alcohol, and then washed sequentially in water and PBS containing 0.1 % Tween 20. They were then treated with 0.3 % H 2 O 2 for 10 min and incubated with PBS containing 0.1 % Tween 20 and 1 % BSA (or milk powder in case of anti-1A1-antiserum) for 30 min at room temperature. Antisera raised against SULT1B1 (1:1500 diluted in PBS containing 0.1 % Tween 20 and 1 % BSA), SULT2A1

(1:800 in PBS containing 0.1 % Tween 20 and 1 % BSA) or SULT1A1 (1:1500 in PBS containing 0.1 % Tween 20 and 1 % milk powder) were applied at 4 °C overnight. Unbound antibody was removed by washing with PBS containing 0.1 % Tween 20. Bound antibodies were detected using Vectastain ABC kit (Vector Laboratories, Burlingame, California, USA) in combination with the diaminobenzidine chromogen system (Immunotech, Marseilles, France).

Nuclei were counterstained with methylgreen (Sigma), 0.8 % in 20 % ethanol.

Enzyme activity measurements

Enzyme activity of SULT2A1 and 1E1 was determined by turnover of [1,2,6,7-

Genotyping of SULT1A1

Genomic DNA was prepared from mucosa samples using the Qiagen Tissue kit. A polymorphism, encoding an Arg/His exchange in codon 213, was analyzed by PCR and enzymatic restriction of PCR products as described previously [24].

RESULTS

Characterization of antisera

Cytosolic fractions were prepared from S. typhimurium strains expressing the individual human SULTs. Varying levels of each preparation were electrophoresed, blotted and probed with the various antisera (Figure 6 in Supplementary Data). Each antiserum recognized several SULT forms with varying sensitivity. SULT1A1, 1A3, 1B1, 1C1 and 2A1 showed strong signals with at least one antiserum even at the lowest amount of bacterial cytosol used (1 µg total protein, corresponding to 10-60 ng SULT protein). Somewhat higher amounts of protein were required for detecting SULT1A2 and 1E1. The remaining forms were only recognized when relatively high SULT levels were used. Semiquantitative data on the reactivity of each antiserum with each SULT form are presented in Table 2 in the Supplementary Data.

Tissue and cellular distribution of SULT1A forms

Immunoblot analyses. SULT1A1 differed in its electrophoretic mobility (32 kDa) from all other forms except SULT4A1. Anti-r1E1-antiserum as well as anti-1A1-antiserum readily detected SULT1A1 standard but showed negligible immunoreactivity with SULT4A1.

Therefore, the identification of SULT1A1 protein was unambiguous. It was well detected in all samples from gastrointestinal tissues using anti-r1E1-antiserum (Figure 1, top panel) and anti-1A1-antiserum (Figure 2, lower panel). Using cDNA-expressed SULT1A1 as a standard, tissue levels of this protein were estimated (Table 1). They were particularly high in ileum and liver.

The next highest levels were observed in large bowel. The differences between cecum, colon and rectum were smaller than those among different colonic or rectal samples.

The SULT1A1 level varied more strongly among the 28 colon samples studied (11-fold) than among the 11 rectal samples (4-fold). SULT1A1 shows a common genetic polymorphism involving an Arg/His exchange in codon 213. Using platelets as an enzyme source, Raftogianis 3 in Supplementary Data). However, the highest mean levels were observed in the *His/*Arg rather than the *Arg/*Arg genotype, and the levels of the individual samples strongly overlapped between all genotypes. Thus, the Arg/His polymorphism does not appear to be the major factor determining SULT1A1 expression in large bowel. The expression was not associated with the sex, the only other factor analyzed (data not shown).

SULT1A2 co-migrated with SULT1B1 (32.5 kDa) but not with any other SULT form. Both antisera that detected SULT1A2 with good sensitivity (anti-1A1-antiserum and anti-r1E1antiserum) cross-reacted with SULT1B1. Preincubation with SULT1B1-sepharose abolished this cross-reactivity. Using the pretreated antiserum, a weak signal at 32.5 kDa was obtained with some samples from colon, cecum and ileum but not with SULT1B1 standard (Figure 2, lower panel). It was also seen in liver when the amount of protein was increased to 200 µg (data not shown), in agreement with previous results with three other liver samples [START_REF] Meinl | Human sulfotransferases are involved in the activation of aristolochic acids and are expressed in renal target tissue[END_REF].

SULT1A3 co-migrated (34 kDa) with several other SULTs (1C2, 1C3 and 2A1). Using anti-r1E1-antiserum (Figure 1, top panel) and anti-1A1-antiserum (Figure 2, lower panel), a protein co-migrating with these SULTs was observed in rectum, colon, cecum, ileum, jejunum and stomach, but not in liver. Since the immunoreactivity of both antisera was high for SULT1A3 but low for the other 34-kDa forms, it was likely that the signal represented SULT1A3. This notion was corroborated by results of analyses using antisera that preferentially detect the other forms. Thus, anti-1C1-antiserum, which recognized the SULT1C2 and 1C3 standards, failed to produce any 34-kDa signal in gastrointestinal samples (Figure 2, upper panel). The remaining 34-kDa form, SULT2A1, showed much higher expression in liver than in any gastrointestinal sample (see below). This high hepatic level of SULT2A1 did not produce any signal with the anti-r1E1-and anti-1A1-antisera recognizing a 34-kDa protein in the extrahepatic samples. Thus, the latter signals appeared to be exclusively due to SULT1A3. Expression of SULT1A3 was very high in all ileal samples studied (Table 1). Levels amounting to approximately 1/5 of those in ileum were detected in the remaining intestinal sections (jejunum, cecum, colon and rectum). However, an 8.8-fold variation was observed among the colonic samples (Table 4 in Supplementary Data). It was not associated with gender (data not shown). Variation was less in rectum and small intestine (3.7-and 1.4-fold, but in smaller numbers of samples). The gastric sample showed a lower SULT1A3 level than the intestinal samples. SULT1A3 was not detected in liver. observed when the SULT1A-specific antibodies were removed from the antiserum by preincubation with SULT1A1-sepharose. In colon, SULT1A protein was detected in mature enterocytes (luminal half of the crypts) and endothelial cells (Figures 3A-C). Immunostaining of the enterocytes was restricted to the nuclei in the middle of the crypt but extended to the cytoplasm and clearly intensified in more luminal cells (Figures 3B and3C). Endothelial staining was particularly strong in the capillaries of the lymphoid follicles (Figure 3B). In ileum, staining was observed in mature enterocytes, starting at the crypt villus border and in endothelial cells (Figures 3D and3E). Anti-1A1-antiserum does not distinguish between the different SULT1A forms. Since the predominating amount of the antigen is localized in the mature enterocytes and since SULT1A1 as well as SULT1A3 are abundant in colon and ileum (as shown in the immunoblot analyses), both forms must be present in these cells. The data do not make it possible to state which SULT1A forms are expressed in endothelial cells.

Tissue distribution and cellular localization of SULT1B1

Immunoblot analyses. SULT1B1 co-migrated with SULT1A2 (32.5 kDa) but not with any other form. Anti-1B1-antiserum detected SULT1B1 with high sensitivity, but was inactive towards SULT1A2. Using this antiserum, SULT1B1 protein was detected in stomach, jejunum, ileum, cecum, colon and rectum (Figure 1, middle panel). In that blot, the immunoreactive protein in the jejunum sample migrated slightly faster than that in the neighboring lanes.

However, this difference disappeared in a repeat experiment, suggesting that it had accidental technical rather than biological reasons. SULT1B1 was not detected in liver when 50 µg cytosolic liver protein was loaded (Figure 1, middle panel). This amount was used in order to avoid an excessive signal with anti-2A1-antiserum (Figure 1, top panel). When the amount of hepatic protein was increased to 200 µg, the presence of SULT1B1 was clearly manifested (data not shown) in agreement with previous results with three other liver samples [START_REF] Meinl | Human sulfotransferases are involved in the activation of aristolochic acids and are expressed in renal target tissue[END_REF].

Anti-r1E1-antiserum clearly recognized SULT1B1 in addition to the SULT1A forms and SULT1E1. This antiserum was useful in comparing the expression levels of several SULT forms in human tissues (Figure 1, top panel). A nine-fold variation in the SULT1B1 level, which was not associated with gender (data not shown), was observed among colonic samples, whereas variability was less (nearly 5-fold) in rectum and small intestine (Table 4 in Supplementary Data). As described in preceding sections, colon also showed the highest variation in the levels of SULT1A1 and 1A3. However, the variation of these three SULT forms was not concurrent. As shown in Figure 4, the SULT1A1 signal was stronger in some Immunohistochemical localization. Anti-1B1-antiserum preincubated with SULT1A1sepharose (to increase the specificity) stained mature enterocytes in colon (upper third of the crypt; Figure 5A) and ileum (starting at the transition from crypt to villus; Figures 5B andC).

Staining was cytoplasmatic. In addition, a small number of cells of the lamina propria, possibly leukocytes, were stained. Staining was abolished when the anti-SULT1B1-antiserum was preincubated with SULT1B1-sepharose to remove the anti-SULT1B1-antibodies.

Tissue distribution of SULT1C forms

SULT1C1 differed in its electrophoretic mobility (33 kDa) from the other SULT forms.

However, the difference was small with regard to SULT1B1 (32.5 kDa). Using anti-1C1pretreated with SULT1B1-sepharose, SULT1C1 protein was readily detected in stomach mucosa, but was not found in rectum, jejunum and cecum (Figure 2, upper panel). Low levels close to the limit of detection appeared to be present in a few samples of colon and ileum.

SULT1C2 and 1C3 proteins co-migrated with SULT1A3 and 2A1 (34 kDa) and were detected by all five antisera used, including anti-1C1-and anti-1B1-antisera. The latter antisera did not produce a 34-kDa signal on blots with the various tissue samples. Thus, SULT1C2 and 1C3 were not found in the samples, but the limit of detection was dissatisfactory (400 ng per mg cytosolic protein) due to moderate immunoreactivity of all antisera available.

Tissue distribution of SULT1E1

Immunoblot analyses. SULT1E1 protein was best detected using anti-r1E1-antiserum. This antiserum also recognized several other human SULTs, which however differed in their electrophoretic mobility from SULT1E1. A band with the characteristic mobility of SULT1E1 (33.5 kDa) was clearly detected in liver and ileum (Figure 1, top panel). Faint signals at the same position were observed in jejunum and cecum. Marginally faster-moving proteins were detected in stomach and -very weakly -some colon samples. Clarification was required whether this band represented SULT1E1 or another form, with SULT1C1 being a candidate.

Enzyme activity. Sulfation of estradiol at a low substrate concentration (20 nM) is a good marker for the presence of SULT1E1 enzyme [START_REF] Falany | Sulfation and sulfotransferases: 3. Enzymology of human cytosolic sulfotransferases[END_REF]. Substantial estradiol sulfotransferase activities were observed in liver and ileum, followed by jejunum and cecum (Table 5 in Supplementary Data). These findings agree with the results of the immunoblot analysis. Sulfation of estradiol, at a very low rate, was also detected in colonic, rectal and gastric samples. At such low rates it is important to examine the specificity of the reaction, as other SULTs can also catalyze the sulfation of estradiol, although they only reach their maximal activity when the concentration of estradiol is increased by several orders of magnitude, e.g. to 6 µM for SULT1A1 [START_REF] Falany | Sulfation and sulfotransferases: 3. Enzymology of human cytosolic sulfotransferases[END_REF]. In order to estimate the contribution of non-SULT1E1 forms to the sulfation of estradiol in colonic cytosol, we mixed bacterially expressed SULT1A1, 1A3 and 1B1 at the same levels as found in a pooled colon preparation. The estradiol sulfotransferase activity of this SULT pool was similar to that of colon cytosol (Table 5 in Supplementary Data). Thus, the activity cannot be used to demonstrate the presence SULT1E1 in colon.

Likewise, the estradiol sulfotransferase activity observed in rectum and stomach samples can readily be explained by the presence of forms other than SULT1E1.

Tissue distribution and cellular localization of SULT2A1

Immunoblot analyses. SULT2A1 co-migrated (34 kDa) with several other SULTs (1A3, 1C2 and 1C3). Anti-2A1-antiserum efficiently detected SULT2A1, did not give any signal with SULT1A3, and only showed very week immunoreactivity with SULT1C2 and 1C3. Moreover, the latter forms were not detected in the tissues investigated. Therefore, SULT2A1 protein could be unequivocally identified in immunoblot analyses. High levels of SULT2A1 were detected in liver (Figure 1, bottom panel). Lower levels were found in small intestine (jejunum and ileum). Expression was very low in cecum and not detected in the remaining tissues (stomach, colon and rectum).

Enzyme activity. DHEA is an excellent substrate for SULT2A1 [START_REF] Falany | Sulfation and sulfotransferases: 3. Enzymology of human cytosolic sulfotransferases[END_REF]. The only other human SULT forms that conjugate DHEA are SULT2B1a and 2B1b [START_REF] Meloche | Expression and characterization of the human 3b-hydroxysteroid sulfotransferases (SULT2B1a and SULT2B1b)[END_REF]. However, these forms show much lower activity with DHEA than SULT2A1 and were not detected in the tissues investigated (last paragraph of Results). DHEA sulfotransferase activity in tissue samples (Table 5 in Supplementary Data) correlated with the results of the immunoblot analyses, in the following order: liver > ileum, jejunum > cecum > rectum, colon, stomach (below limit of detection).

Immunohistochemistry. No immunohistochemical staining was observed in colon mucosa using anti-2A1-antiserum (Figure 5C), which agrees with the immunoblotting and enzyme activity results obtained with subcellular preparations. In ileum, mature enterocytes but no other cells, were stained (Figures 5D and5E). Staining primarily occurred in the cytoplasm, but the ileal enterocytes so compact that a minor staining in nuclei could not be ruled out.

Tissue distribution of the remaining SULT forms (2B1a, 2B1b and 4A1)

The electrophoretic mobilities of SULT2B1a (40 kDa) and 2B1b (42 kDa) were unique among the human SULT members. Anti-2A1-antiserum detected these forms, expressed in bacteria, with moderate sensitivity (limit of detection ~100 and 400 ng/mg protein, respectively).

Neither form was detected in hepatic or gastrointestinal samples. Recently, it was reported that the subcellular localization of SULT2B1b may vary between tissues; it was primarily detected in cytosol in prostate, but in the nuclei in placenta [START_REF] He | Different subcellular localization of sulphotransferase 2B1b in human placenta and prostate[END_REF]. Since we only used the cytosolic fraction, we would have overlooked nuclear enzyme in our immunoblot analysis. However, we found no staining of nuclei (or other cellular compartments) in our immunohistochemical analyses of colon with anti-2A1-antiserum, which cross-reacts with SULT2B1 forms. Ileal enterocytes were stained with anti-2A1-antiserum, but staining was primarily observed in the cytoplasm.

No appropriate antiserum was available for detecting SULT4A1. Others have detected SULT4A1 mRNA or protein in brain, but not in any other tissue [START_REF] Falany | Molecular cloning and expression of novel sulphotransferase-like cDNAs from human and rat brain[END_REF].

DISCUSSION

Overlap of the substrate specificity [START_REF] Falany | Sulfation and sulfotransferases: 3. Enzymology of human cytosolic sulfotransferases[END_REF][START_REF] Fujita | Enzymatic characterization of human cytosolic sulfotransferases; identification of ST1B2 as a thyroid hormone sulfotransferase[END_REF] limits the possibility of elucidating the expression pattern of the various SULTs solely based on activities. Therefore, we focused on the identification of SULT forms by immunoreactivity. We put much effort into a good electrophoretic resolution of SULT forms and the characterization of a series of antisera. In general, we were able to unambiguously identify the SULT proteins detected in immunoblots.

In individual cases, the results were further corroborated using enzyme reactions with substrates (estradiol and DHEA) that are characteristic for SULT1E1 and 2A1, respectively.

We detected seven SULT forms in the alimentary tract. SULT1A1, 1A3 and 1B1 were found in all parts of the alimentary tract. They showed their highest levels in ileum. The ileal level of SULT1A1 was slightly above the level of the hepatic sample used. SULT1A3 was present in ileum at a somewhat higher level than SULT1A1, but was absent in liver (with a detection limit of 5 % of the ileal level). Likewise, expression of SULT1B1 was 17 times higher in ileum than in liver. All three forms were also present at relatively high levels in large bowel. Differences in the levels were minor between cecum, colon and rectum, but substantial between colo-rectal samples from different subjects. A genetic polymorphism that affects the expression of SULT1A1 in platelets did not appear to be an important factor for the variation of Other SULT forms (1A2, 1C1, 1E1 and 2A1) were only detected in some parts of the gastrointestinal tract. SULT1A2 appears to be much less abundant in the human organism than the other SULT1A forms. Others detected SULT1A2 on the mRNA level [START_REF] Dooley | Expression profiling of human sulfotransferase and sulfatase gene superfamilies in epithelial tissues and cultured cells[END_REF], but not on the protein level despite an intensive search [START_REF] Nowell | Examination of human tissue cytosols for expression of sulfotransferase isoform 1A2 (SULT1A2) using a SULT1A2-specific antibody[END_REF]. Under our electrophoretic conditions, SULT1A2 separates from SULT1A1, although these proteins differ in only 12 amino acid residues. While SULT1A2 was detected in some gastrointestinal samples and in liver, its level was always much lower than that of SULT1A1. SULT1C1 appears to be primarily expressed in various tissues in the fetal stage [START_REF] Stanley | Expression profiling of human fetal cytosolic sulfotransferases involved in steroid and thyroid hormone metabolism and in detoxification[END_REF][START_REF] Her | Human sulfotransferase SULT1C1: cDNA cloning, tissue-specific expression, and chromosomal localization[END_REF][START_REF] Sakakibara | Molecular cloning, expression, and characterization of novel human SULT1C sulfotransferases that catalyze the sulfonation of N-hydroxy-2acetylaminofluorene[END_REF]. In addition, its mRNA was found in adult kidney, thyroidal gland and stomach [START_REF] Her | Human sulfotransferase SULT1C1: cDNA cloning, tissue-specific expression, and chromosomal localization[END_REF]. Now, we unambiguously detected SULT1C1 protein in stomach. A low level, at the limit of detection, appeared to be present in some colonic and ileal samples, whereas no signals were obtained in the remaining tissues investigated. From previous immunoblot analyses it is known that SULT1E1 and 2A1 are expressed in jejunum [START_REF] Her | Human jejunal estrogen sulfotransferase and dehydroepiandrosterone sulfotransferase: immunochemical characterization of individual variation[END_REF] and liver [START_REF] Song | Estrogen sulfotransferase expression in the human liver: marked interindividual variation and lack of gender specificity[END_REF][START_REF] Aksoy | Human liver dehydroepiandrosterone sulfotransferase: nature and extent of individual variation[END_REF]. We confirmed these findings. In addition, we demonstrated the presence of SULT1E1 and 2A1 protein and enzyme activity in ileum and, at a low level, in cecum. Both forms were absent in stomach, colon and rectum. The negative results for SULT2A1 in all three tissues and for SULT1E1 in stomach agree with negative results reported for the mRNA by Dooley et al. [START_REF] Dooley | Expression profiling of human sulfotransferase and sulfatase gene superfamilies in epithelial tissues and cultured cells[END_REF]. However, these authors detected SULT1E1 mRNA in colon and colo-rectal tissue. Perhaps, SULT1E1 protein is degraded so rapidly in colon and rectum that it does not reach a detectable level, or it is expressed at sites other than the mucosa (we only used the mucosa, whereas Dooley et al. [START_REF] Dooley | Expression profiling of human sulfotransferase and sulfatase gene superfamilies in epithelial tissues and cultured cells[END_REF] did not specify their "colon" and "colorectal" tissue). In contrast to us and Dooley et al. [START_REF] Dooley | Expression profiling of human sulfotransferase and sulfatase gene superfamilies in epithelial tissues and cultured cells[END_REF], Tashiro et al. [START_REF] Tashiro | Expression and activity of dehydroepiandrosterone sulfotransferase in human gastric mucosa[END_REF] detected SULT2A1 mRNA, SULT2A1 protein and DHEA activity in gastric mucosa. Their enzyme assay, involving a chromatographic step to reduce background signals, was more sensitive than our assay. They determined activities of 0.05 to 0.93 pmol ⋅ mg -1 ⋅ min -1 , which is much lower than those we found in other parts of the intestine (up to 121 pmol ⋅ mg -1 ⋅ min -1 in an ileal sample). In that study, SULT2A1 immunoreactivity and mRNA hybridization was localized in parietal cells. In contrast, ileal expression of SULT2A1 was exclusively observed in mature enterocytes in the present study.

In additional experiments, we studied the cellular localization of SULTs in ileal and colonic mucosa. The results indicate that SULT1A1, 1A3, 1B1 (in both tissues) as well as 2A1 (detected only in ileum) are primarily expressed in differentiated enterocytes. This localization supports the idea that the enzymes are part of the metabolic barrier of gut. In addition, we detected SULT1A protein(s) in endothelial cells. Localization of SULT1A in enterocytes of the luminal surface of colon has been described [START_REF] Windmill | Localisation of aryl sulfotransferase expression in human tissues using hybridisation histochemistry and immunohistochemistry[END_REF], but not staining of intestinal capillaries.

Sulfotransferase activity towards phenols (typical substrates of SULT1A forms) has been reported for human aortic endothelial cells [START_REF] Yang | Phase II biotransformation of carcinogens/atherogens in cultured aortic tissues and cells: I. Sulfation of 3-hydroxybenzo[a]pyrene[END_REF] and bovine brain microvessel endothelial cells [START_REF] Baranczyk-Kuzma | Substrate specificity of phenol sulfotransferase from primary cultures of bovine brain microvessel endothelium[END_REF]. It has been suggested that SULTs in brain vessels may form a metabolic component of the blood-brain barrier [START_REF] Baranczyk-Kuzma | Substrate specificity of phenol sulfotransferase from primary cultures of bovine brain microvessel endothelium[END_REF]. Thus, they may have a similar barrier function in gut endothelial cells.

Xenobiotics (or their phase-I metabolites) containing a nucleophilic group usually are substrates not only for SULTs, but also for UGTs. Expression of both enzyme classes shows common characteristics in the human alimentary tract, as shown in the present study for SULTs and in that of Strassburg et al. [START_REF] Strassburg | UDPglucuronosyltransferase activity in human liver and colon[END_REF] for UGTs: Various SULTs and UGTs are highly expressed in the gastrointestinal tract, including some forms that are not found in liver. Moreover, the pattern of the forms expressed differs between various parts of the gastrointestinal tract.

However, sulfo-conjugation involves a much higher risk for the formation of reactive intermediates than glucuronidation [START_REF] Glatt | Activation and inactivation of carcinogens by human sulfotransferases[END_REF]. Nevertheless, it is difficult to assess whether SULTs in the human alimentary tract represent a risk factor for carcinogenesis. It is important to notice that SULTs were only detected in mature enterocytes, but not in the stem cells of the epithelium. If a reactive sulfo conjugate remained in the SULT-proficient cells, then the specific expression of SULTs in terminally differentiated cells might be a protective measure rather than a risk factor for carcinogenesis. Unfortunately, the animal species that are normally used in carcinogenicity studies, mouse and rat, only show low expression of most SULT forms in gut. For example, we only detected SULT1B1 and an unidentified form (putatively a member of the SULT1C subfamily, based on its immunoreactivity) in rat colon [START_REF] Teubner | Stable expression of rat sulfotransferase 1B1 in V79 cells: activation of benzylic alcohols to mutagens[END_REF]. Moreover, the level of SULT1B1 in colon mucosa only amounted to 30 % of the hepatic level in the rat [START_REF] Teubner | Stable expression of rat sulfotransferase 1B1 in V79 cells: activation of benzylic alcohols to mutagens[END_REF], whereas the level of corresponding human enzyme was five times higher in colon mucosa than in liver (present study). Likewise, sulfotransferase activity towards the promutagen 1hydroxymethylpyrene was nearly 1000 times higher in cytosolic fraction from liver compared to colon mucosa in the rat, whereas this variation only was eightfold for corresponding preparations from humans [START_REF] Glatt | Metabolic formation, distribution and toxicological effects of reactive sulphuric acid esters[END_REF]. The low SULT protein and activity levels in the gastrointestinal tract of the rat are reflected in low mRNA levels for many SULT forms in gastrointestinal and other extrahepatic tissues compared to liver [43]. In agreement with this tissue distribution, liver was the target organ of all SULT-dependent carcinogens identified in mice and rats [2]. We just succeeded in constructing transgenic mouse models that express human SULT1A1 and 1B1 in colon at levels similar to those found in man (W. Meinl, G. Dobbernack, H. Himmelbauer and H. Glatt, unpublished results). We will use these models to

  3 H(N)]dehydroepiandrosterone ([ 3 H]DHEA; 2.2 TBq/mmol, NEN DuPont, Bad Homburg, Germany) and [6,7-3 H(N)]17b-estradiol (1.48 TBq/mmol), respectively, as described previously[START_REF] Forbes | Human fetal adrenal hydroxysteroid sulphotransferase: cDNA cloning, stable expression in V79 cells and functional characterisation of the expressed enzyme[END_REF]. The reaction mixture (250 µl) contained 5 to 200 µg cytosolic protein, 7 mM MgCl 2 , 20 µM PAPS, 3 µM [ 3 H]DHEA or 20 nM [ 3 H]estradiol, and 50 mM Tris-HCl (pH 7.4). Preliminary experiments using varying amounts of cytosolic fraction of each tissue were performed to determine the linear range. Blanks contained water instead of PAPS. After 20 min at 37 °C, 250 µl 100 mM Tris-HCl (pH 8.7) and 3 ml of chloroform were added for termination of the reaction. Following a single extraction, 250 µl of the aqueous layer was subjected to liquid scintillation counting. Biochemical Journal Immediate Publication. Published on 5 Mar 2007 as manuscript BJ20061431 © 2007 Biochemical Society Teubner et al. page 10

  et al.[START_REF] Raftogianis | Phenol sulfotransferase pharmacogenetics in humans: association of common SULT1A1 alleles with TS PST phenotype[END_REF] found 7.7-and 6.[START_REF] Visser | Pathways of thyroid hormone metabolism[END_REF]-fold higher SULT1A1 activity in *Arg/*Arg and *Arg/*His subjects than in *His/*His subjects. The *His/*His genotype was associated with the lowest Biochemical Journal Immediate Publication. Published on 5 Mar 2007 as manuscript BJ20061431 © 2007 Biochemical Society Teubner et al. page 11 mean SULT1A1 protein level in colon as well as rectum, 88 and 58 % compared to the to the *Arg/*Arg genotype (Table

©

  Immunohistochemical localization. Anti-1A1-antiserum pretreated with SULT1B1-sepharose (to increase the specificity) showed strong signals in both colon and ileum. No staining was Biochemical Journal Immediate Publication. Published on 5 Mar 2007 as manuscript BJ20061431

  the SULT1A3 and 1B1 signals (e.g. C3). In other samples, the SULT1A3 (C4) or 1B1 (C1) signal was strongest.

  gut. At present, we can only speculate that dietary habits or microbial colonization may modulate the expression of SULTs.

  of these enzymes in the activation and inactivation of carcinogens and other toxicants, in particular those affecting the alimentary tract. 43 Dunn, R. T. and Klaassen, C. D. (1998) Tissue-specific expression of rat sulfotransferase messenger RNAs. Drug Metab. Dispos. 26, 598-604 Biochemical Journal Immediate Publication. Published on 5 Mar 2007 as manuscript BJ20061431 © 2007 Biochemical Society

Figure 1 ©Figure 2 25 Figure 3 26 Figure 4 27 Figure 5

 12253264275 Figure 1 Immunodetection of SULTs in various parts of the gastrointestinal tract using antisera raised against rat SULT1E1 (top panel), human SULT1B1 (middle panel) and human SULT2A1 (bottom panel)

Biochemical Journal Immediate Publication. Published on 5 Mar 2007 as manuscript BJ20061431 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 5 Mar 2007 as manuscript BJ20061431 © 2007 Biochemical Society

	Teubner et al.	page 6

Biochemical Journal Immediate Publication. Published on 5 Mar 2007 as manuscript BJ20061431 © 2007 Biochemical Society

	Teubner et al.	page 9

Biochemical Journal Immediate Publication. Published on 5 Mar 2007 as manuscript BJ20061431 © 2007 Biochemical Society

ACKNOWLEDGMENTS

The authors thank Elisabeth Meyer and Sabine Braune for excellent technical assistance and Prof. Frank Dombrowski, Magdeburg, Germany, for histopathological evaluation of samples. This work was financially supported by Deutsche Forschungsgemeinschaft (INK 26) and Bundesministerium für Bildung und Forschung (grant BIO/0313028A).

page 28

Table 1 Summary on SULT forms detected in cytosolic fractions from liver and various sections of the gastrointestinal tract

SULT levels were determined semi-quantitatively using cDNA-expressed proteins as standards.

This method may involve inaccuracies in the absolute value (we estimate by a factor of up to 3), but should be more accurate for comparison of the same protein in different samples (within rows). Values are means of n samples. An extended version of this table is presented in Supplementary Data (Table 4). -, below the limit of detection.