A High Throughput Method for the Detection of Metalloproteins on a Microgram Scale*
Martin Högbom
,
,
Ulrika B. Ericsson¶,
Robert Lam||,
M. Amin Bakali H.¶,
Ekaterina Kuznetsova**,
Pär Nordlund¶ and
Deborah B. Zamble
,
From the
Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, Box 596, SE-75124 Uppsala, Sweden, the ¶ Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden, || Affinium Pharmaceuticals, 100 University Avenue, 10th Floor, South Tower, Toronto, Ontario M5J 1V6, Canada, the ** Banting and Best Department of Medical Research, University of Toronto, 112 College St., Toronto, Ontario M5G 1L6, Canada, and the 
Department of Chemistry, Lash Miller Chemical Laboratories, University of Toronto, 80 St. George St., Toronto, Ontario M5S 3H6, Canada
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ABSTRACT
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Proteins that bind transition metals make up a substantial portion of the proteome, and the identification of a metal cofactor in a protein can greatly facilitate its functional assignment and help place it in the context of known cellular pathways. Existing methods for the detection of metalloproteins generally consume large amounts of protein, require expensive equipment, or are very labor intensive, rendering them unsuitable for use in high throughput proteomic initiatives. Here we present a method for the identification of metalloproteins that contain iron, copper, manganese, cobalt, nickel, and/or zinc that is sensitive, quick, robust, inexpensive, and can be performed with standard laboratory equipment. The assay is based on a combination of chemiluminescence and colorimetric detection methods, it typically consumes only 10 µg of protein, and most common chemical components of protein solutions do not interfere with metal detection. Analysis of 52 protein samples was compared with the results from inductively coupled plasma-atomic emission spectrometry to verify the accuracy and sensitivity of the method. The assay is conducted in a 384-well format and requires about 3 h for completion, including a 2-h wait; so whole proteomes can be assayed for metal content in a matter of days.
Proteomics is the biochemical and physiological characterization of all proteins and variants produced by a cell and includes determination of the structure and function of each protein, its interactions with other cellular factors, spatial and temporal location, and regulation (1, 2). This type of initiative is now possible on an unprecedented scale and motivates the challenge to develop technology and applications for systematic high throughput analysis (1, 3). One assay that would be a very useful addition to the existing battery of proteomic protocols is a quick and simple method for high throughput (HTP)1 identification of transition metal-containing proteins. Current estimates suggest that metalloproteins make up about one-third of the proteome (4, 5), so the identification of a metal cofactor would facilitate the functional assignment of a protein and help place it in the context of known cellular pathways (6). Furthermore this information would be useful for selecting attractive targets for high throughput structural genomic efforts (3, 7) because metal cofactors can often be used to phase x-ray crystallographic data by anomalous dispersion methods, and the protein structure can be solved without the need for secondary experiments such as the production of selenomethionine-containing protein or heavy metal soaking of protein crystals (811).
The currently available methods for metal analysis of proteins are not suitable for routine HTP efforts. These techniques, such as atomic absorption spectrometry, inductively coupled plasma coupled with either atomic emission spectroscopy (ICP-AES) or mass spectrometry (ICP-MS), or synchrotron techniques such as x-ray fluorescence spectrometry or extended x-ray absorption fine structure (EXAFS) either consume large amounts of protein or require specialized equipment that can be very expensive (1216). Ideally an assay for this purpose should be sensitive so that minimal amounts of protein are consumed, inexpensive, fast, adaptable to large numbers of samples, and feasible with common laboratory equipment. Here we describe such a method for the detection of proteins that contain the most common transition metals in biological systems: iron, copper, manganese, cobalt, nickel, and zinc (17). This robust assay was developed to meet all of the requirements listed above and is based on two consecutive reactions that are analyzed with luminescence and colorimetric detection methods.
The first test relies on the well known fact that luminol, in the presence of certain catalysts, will produce chemiluminescence when mixed with a base and an oxidant (Fig. 1). This sensitive reaction is commonly used by forensic scientists at crime scenes to detect small amounts of blood because the heme iron in hemoglobin is a very efficient catalyst of this reaction (18). Many other transition metals also catalyze this reaction (for example, see Refs. 1927). The second test is based on the spectrophotometric detection of the metal complexes of the chelator 4-(2-pyridylazo)resorcinol (PAR). PAR forms colored complexes with several metals with a maximal absorbance around 500 nm (2830).

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FIG. 1. Light-producing luminol reaction. Certain transition metals can act as the catalyst for this reaction.
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The entire assay is performed in a 384-well format in about 3 h, including a 2-h wait, making the method truly high throughput. To verify the accuracy of this method we examined 52 protein samples that were also analyzed by ICP-AES, which is a standard method for metal analysis and quantification (12), and there is good agreement between the results of the two methods. Many of the commonly used components of protein solutions were also tested, and these do not appear to interfere with the assay signals. Because of its high capacity and simplicity and the fact that it can be performed on microgram quantities of proteins in standard buffers, this assay is a valuable addition to the methods used to analyze the many unknown and unclassified proteins now coming through the proteomic pipeline.
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EXPERIMENTAL PROCEDURES
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Chemicals
Luminol, PAR, urea, bovine carbonic anhydrase, bovine copper-zinc superoxide dismutase, bovine heart cytochrome c, and all metal salts were purchased from Sigma. H2O2 was purchased from Merck. All the samples were prepared with Milli-Q water, 18.2 megaohms-cm resistance (Millipore). The buffer solutions were treated with Chelex-100 (Bio-Rad) to minimize trace metal contamination. The proteins used to verify the method are from the structural genomic initiatives at the universities of Stockholm and Toronto.
Metal Analysis
Urea (4 µl of an 8 M solution) was added to the wells of a non-transparent, 384-well plate with optical bottom (Nunc). One protein (10 µg) was added to each well in no more than 2 µl of solution followed by the addition of 10 µl of a freshly prepared luminol solution (11 mM luminol, 500 mM Na2CO3, 230 mM H2O2) to all the wells. The first luminescence data were acquired within a few minutes on a Bio-Rad Fluor-S Multimager with an exposure time of 10100 s, although the acquisition time will vary between instruments. Subsequently 1 µl of a 2 mM PAR solution was added to each well, and the luminescence reading was repeated. The plate was incubated at room temperature for 2 h and then centrifuged at 3000 x g for 2 min to minimize interference from N2 bubbles produced by the luminol reaction. The absorbance at 492 nm was measured on a Fluostar Galaxy plate reader (BMG Labtech); the positive samples in this test are also distinguishable by visual inspection due to the reddish color compared with the yellow-to-clear negative samples.
ICP-AES analysis was performed on a PerkinElmer Life Sciences Optima 3000 DV system with GemTip Cross-Flow nebulizer equipped with a Scott double pass spray chamber with a segmented array, charge-coupled device detector. Plasma gas flow rate was 15 liters/min, auxiliary gas flow rate was 0.5 liters/min, and nebulizer gas pressure was 0.8 x 105 pascal. The software used was ICP Win Lab Version 3. The samples were diluted to
10 µM in Chelex-treated 20 mM HEPES, pH 7.5, and 50 mM NaCl. For some samples 5% glycerol (v/v) was used to minimize precipitation; our previous studies of standard metal solutions demonstrated that this concentration of glycerol did not affect quantitation by ICP-AES (16).
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RESULTS
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Metal Assay
To detect transition metals bound to proteins, a set of sequential tests was developed for analysis of samples in 384-well plates. The presence of transition metals is indicated by two signals, the luminol luminescence that is catalyzed by certain metals and the change in color of PAR solutions when particular metal complexes are formed. The tests were performed in a consecutive manner on the same samples to provide information not only about the presence of metals but also their identity.
To determine the metal selectivity of these tests, they were first applied to aqueous solutions of pure divalent metal salts. Upon the addition of luminol, 1 nmol of iron, copper, or cobalt each produced very bright signals, whereas manganese, zinc, and nickel did not (Fig. 2A). The weak light apparent in the top of the manganese and nickel rows in Fig. 2A is due to light contamination from the extremely bright cobalt signal of the wells next to these positions. Fortuitously the subsequent addition of PAR has an effect on the luminescence signal of the different metals: it quenches the copper signal but activates manganese luminescence, allowing both of these metals to be clearly identified (Fig. 2B). Spectroscopic analysis at 492 nm revealed a strong absorption signal for copper, manganese, cobalt, nickel, and zinc but not iron (Fig. 2C). The maximum absorption for the PAR-metal complexes vary slightly between 490 and 510 nm (30); we chose to monitor the absorption at 492 nm because it is an appropriate wavelength to identify nickel and zinc, the two metals that are not detected in the luminol tests. Based on the results of these sequential tests the identity of each transition metal can be determined except for the distinction between nickel and zinc. Fig. 3 shows a flow chart that can be followed to determine the identity of individual metals. Magnesium was also analyzed but did not produce a signal at any step of the assay. It should be mentioned that the results in both tests were the same for Fe2+ and Fe3+ as well as for Mn2+ and Mn7+.

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FIG. 2. Serial dilutions of pure metal salt solutions. A, luminescence test. B, luminescence test after incubation with PAR. Note especially the disappearance of the copper signal and the appearance of the manganese signal in comparison with A. C, absorbance at 492 nm of the same samples as in B; nickel and zinc give the strongest signals.
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FIG. 3. Flow chart used to determine the metal identity based on the results in the three parts of the assay. In the simple case with only one metal in the sample, all metals except nickel and zinc can be distinguished.
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For the HTP generation of purified proteins for proteomic analysis only small amounts of proteins are usually produced (1). Ideally individual biochemical assays should only consume a small fraction of each sample, so a high degree of sensitivity is required. To examine the sensitivity of our metal assay, serial dilutions of pure metal stocks were tested (Fig. 2). In the PAR colorimetric test, 0.1 nmol of each transition metal except iron was clearly detected, and 10 pmol of copper, cobalt, and manganese and 50 pmol of iron were sufficient for detection with luminescence.
Protein Analysis
Fig. 4 shows an assay of five different proteins. The assignment (positive or negative) for the samples in each test is compared with the metal content determined by ICP-AES (Table I); the metals are grouped according to whether they are expected to produce a signal in each test. Protein A is a 38-kDa protein bound to 1.2 eq of manganese and 0.15 eq of iron as determined by ICP-AES; the low amount of iron produces a weak luminescence signal in the first test, and the manganese is clearly identified by the much stronger signal in the second luminescence test and a positive signal in the colorimetric test. Protein B is a 43-kDa protein containing 0.81 eq of iron and 0.11 eq of zinc; the low amount of zinc does not interfere with the identification of iron by a strong luminescence signal in both tests and only a weak signal in the colorimetric test. The non-negligible PAR signal suggests that a small amount of a second metal is present. Protein C is a 15-kDa protein containing 0.85 eq of zinc and 0.15 eq of iron; the iron produces a luminescence signal, whereas the zinc results in the strong signal in the colorimetric test. Protein D is a 31-kDa protein with 2.0 eq of copper and 2.0 eq of zinc bound; the copper is clearly identified by a bright signal in the first luminescence test but not in the second, and the strong signal in the colorimetric test indicates the presence of nickel or zinc. Finally, protein E is an 18-kDa protein with less than 0.01 eq of any metal and produces a negative result at each step. These data demonstrate that although all of the metals obey the rules established for positive or negative signals in the separate parts of the assay, certain combinations of metals will produce signals that can be interpreted in more than one manner. This weakness is clearly illustrated by the analysis of protein C. The combination of iron and zinc produces strong luminescent signals in both tests and a strong colorimetric signal, the same pattern of signals that is generated by cobalt alone. For this reason, the major strength of the assay is to determine whether or not a transition metal is bound to the protein rather than identifying which metal is present, although this can be achieved in many cases.

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FIG. 4. The assay performed on serial dilutions of proteins. A, luminescence test. B, luminescence test after the addition of PAR. C, colorimetric test. D, absorbance at 492 nm of the data shown in C. The molecular masses and metal content of the proteins as determined by ICP-AES were: a 38-kDa protein with 1.2 eq of manganese and 0.15 eq of iron (A), a 43-kDa protein with 0.81 eq of iron and 0.11 eq of zinc (B), a 15-kDa protein with 0.85 eq of zinc and 0.15 eq of iron (C), a 31-kDa protein with 2.0 eq of copper and 2.0 eq of zinc (D), and an 18-kDa protein with less than 0.01 eq of any metal (E). 0 indicates no protein present.
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TABLE I Assay on protein dilutions
Lum1 and Lum2 indicate luminescence detection before and after the addition of PAR, respectively, and Col indicates the absorption measurement at 492 nm.
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Serial dilutions of these proteins reveal that if the metal content is between 0.85 and 2 eq of metal per protein, a signal in the luminescence tests is observed with as little as 0.1 µg of protein, especially after the addition of PAR, which lowers the background and allows less intense signals to be detected (Fig. 4, A and B). The absorbance test is, as mentioned previously, less sensitive, but in the cases where the proteins are close to fully loaded with metal, as little as 1 µg of protein is sufficient for a detectable positive signal (Fig. 4, C and D). However, given that most recombinant metalloproteins in our experience contain less than 1 eq of metal, we suggest that at least 1 µg of protein should be used for the luminescence test and 10 µg if the colorimetric PAR test is to be performed as well. It should be mentioned that the reddish color of the positive samples in the PAR absorbance tests, compared with the yellow-to-clear negatives, is actually more easily detected by eye than with a plate-reading spectrophotometer. This is due to several factors that influence the absorbance of any particular sample in the plate. For example, if bubbles or a precipitate is present the weakly positive signals are difficult to distinguish from the background noise. Furthermore the presence of protein elevates the base line to between 0.1 and 0.2 absorbance units (Fig. 4D), so the signal should be compared with the negative protein controls and not to wells that do not contain any protein. The proteins used in this experiment were Corynebacterium ammoniagenes ribonucleotide reductase R2 expressed in manganese-supplemented medium (A), Escherichia coli ribonucleotide reductase R2 (B), E. coli methionine sulfoxide reductase B (C), bovine copper-zinc superoxide dismutase (D), and E. coli lactoylglutathione lyase, apo form (E).
Interferences
Many protein purification protocols include a variety of reagents that are used to stabilize the protein. For this reason, several common components of protein solutions were added to this assay to determine whether they interfere with the detection of transition metals. The addition of 1 µl of the following solutions to the protein-containing wells prior to the addition of luminol did not interfere with the assay (data not shown): 10 mM CaCl2, 10 mM MgCl2, 20 mM DTT, 50 mM ß-mercaptoethanol, 20% Me2SO, 200 mM ammonium sulfate, 100 mM Tris, 100 mM HEPES, 100 mM Na2HPO4, and 100 mM imidazole. The strong metal chelator EDTA (1 µl of a 20 mM solution) inhibits both reactions for all metals; EGTA (1 µl of a 20 mM solution) also disturbs the analysis, although iron (but not copper, manganese, and cobalt) can still be identified in the luminol test, and copper, nickel, and zinc (but not manganese and cobalt) can still be identified in the colorimetric test. Thus, if EDTA or EGTA is present in the protein buffer, it should be removed prior to analysis. The weaker chelator citrate, however, does not interfere (1 µl of a 100 mM solution added to the sample). Previous studies suggest that several other metals may produce signals in this assay; for example gold, rhodium, chromium, vanadium, and titanium have been reported to catalyze the luminol reaction (2124, 26), and niobium, vanadium, cadmium, titanium, silver, uranium, tin, and lead are also known to give a color change with PAR. None of these metals are very common in most organisms, however, and the sensitivity, optimal wavelength for detection, and whether conditions are compatible with the present assay have not been examined.
Accuracy of the Assay
To evaluate this assay, 52 different protein samples were collected from the ongoing structural genomic initiatives at the Universities of Stockholm and Toronto. Some were selected because of suspected metal content (due to color or other indicators), and some were selected at random. A few known metalloproteins were also purchased from Sigma (listed under "Experimental Procedures"). The protein concentrations of the stocks varied between 5 and 20 mg/ml. The assay was performed on 10 µg of each protein sample in duplicate in a 384-well plate. The samples were also subjected to analysis by the established method of ICP-AES to validate the assay results. The results were summarized as follows. Each sample was rated as positive or negative in the two tests. Detection in the luminol test only before or after the addition of PAR (as in the cases of manganese and copper) counted as positive. Positive signals in the PAR colorimetric test were identified by absorbance measurements at 492 nm as well as by eye; more positives could be identified by eye as mentioned above. The metal content of each sample was independently determined by ICP-AES, and the amounts of all of the transition metals that should give a signal in any one of the tests were added together. For example, a complex sample with 0.15 eq of iron, 0.80 eq of copper, and 0.10 eq of zinc results in 0.95 (0.80 + 0.15) eq of metal that should give a luminescence signal and 0.90 (0.8 + 0.10) eq of metal that should give an absorbance signal. The assay results (positive or negative) were then plotted on the same chart as the amount of the appropriate metals in each of the samples as determined by ICP-AES.
Fig. 5 shows the two charts, one for the luminescence test and one for the colorimetric test. The data are also summarized in Tables II and III. These results demonstrate that for the luminescence tests all of the proteins containing more than 0.2 eq of the metals that are detected in these tests produce a positive signal, and all proteins with 0.02 eq or less exhibit no signal. The samples that fall in the "gray area" of between 0.03 and 0.2 eq generate mixed results. The colorimetric test is less sensitive, and for our data the corresponding gray area is 0.080.58 eq (Table II). In short, samples that contain close to stoichiometric amounts of metal produce a positive signal in all cases, and samples that contain less than a few percent of metal always have negative results.
The type of analysis just described is biased because the molecular weights of the proteins differ, but the assay was run on a constant mass of protein (10 µg). So even if two proteins have the same stoichiometry of metal bound, the sample of a larger protein will contain less metal than that of a smaller one. Still we present the data in this manner because we believe this is the most practical way to run the assay. If the size of the protein is taken into account the results are as follows. Positive signals are always detected in the luminescence test for protein samples containing 30 pmol or more of the metals detected, and samples with less than 6 pmol always show up negative, so the "gray" range is between 6 and 30 pmol. For the colorimetric test the corresponding numbers are 21 and 150 pmol (Table III). One major reason for the existence of this gray area is likely that the metals vary in the strength of the signal produced; for example all five negative samples in the gray area of the luminescence test have copper as their main metal component, which is also true for three of the four negative samples in the gray area of the colorimetric test.
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DISCUSSION
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Because of the many parameters involved in the determination of the metal content of proteins, metal analysis is inherently difficult, and any approach will have strengths and weaknesses (16). Some of the common drawbacks of existing methods include the consumption of large amounts of protein, the complexity of the protocols, and specialized and expensive equipment (1215). These disadvantages make them particularly unsuitable for HTP proteomic efforts and problematic for routine analysis in smaller laboratories. Here we present a method for metalloprotein identification that has the advantage of being sensitive, quick, robust, cheap and can be performed with standard laboratory equipment. The method is based on consecutive luminescence and colorimetric tests performed in a multiwell format, which is commonly used for HTP proteomic analysis and is amenable to robotic manipulation (1). Iron, copper, manganese, and cobalt are detected in the luminescence test, and nickel, zinc, manganese, copper, and cobalt are detected in the colorimetric PAR test. This assay was optimized for the purpose of detecting metals in proteins, but it is very robust with few observed interferences, so it could also be used on other types of samples. Many studies have examined the use of luminol for the detection of transition metals; however, there is no uniformity in the methods or conditions used, and it is clear that a variety of factors can affect which metals, or even which oxidation states of these metals, are detected (23, 25, 27). For this reason, empirical analysis of standards must be performed for each new set of conditions. The present assay seems rather independent of the oxidation state of the metals; this is at least in part likely due to the Fenton reaction that cycles metal ion oxidation states in the presence of hydrogen peroxide. Indeed Fe2+ and Fe3+ give the same behavior in the assay, and this is also true for Mn2+ and Mn7+.
Conditions have been identified that allow both tests to be performed sequentially on the same protein sample to minimize the amount of protein needed. The chemiluminescence test is more sensitive, and in most cases 1 µg of protein is sufficient for clear results. The test based on the colored PAR-metal complexes is somewhat less sensitive, and if the additional information from this test is desired, in particular the detection of nickel and zinc and the distinction between iron and cobalt, 510 µg of protein are needed for reliable results.
The assay is divided into three steps, and each step provides different and sometimes complementary information on the type of metal present. In principle, all of the metals examined can be separately identified except for nickel and zinc. However, because proteins sometimes contain a mixture of several metals, clear identification may not be possible in all cases. Hence the major strength of this assay is to quickly identify metalloproteins out of a large number of unknowns by using just a small amount of sample. Furthermore the method, as described here, is not suitable for precise quantification of metal(s) in an unknown protein. The major reason for this is that if a sample contains different metals, their signals may "overlay" in the assay as discussed previously. In addition, although urea is included to denature the protein, metal release may not be quantitative, and the denatured protein could compete with the reactions. However, the method is suitable as a tool to compare the metal content of samples of a known protein that differ in activity or that have been prepared in different ways.
From the proteomic perspective, the present method has considerable advantages over existing means for metalloprotein detection with the main benefits being the low protein consumption and ability to simultaneously analyze several hundred samples cheaply and rapidly. The assay can be performed directly on protein samples in standard protein purification buffers with no need for further sample preparation. These features, combined with a compatibility with high throughput and automated methods, make it a powerful tool in the field of metalloproteomics. In practice, whole proteomes can be assayed for metal content in a matter of days. Such a study would undoubtedly yield a wealth of information that could have several applications from aiding in functional assignment of individual proteins to mapping metal homeostasis pathways in the organism to choosing successful targets in structural genomic efforts. The limitations of the method with respect to metal identification may lead to further analysis of the positive samples for some applications, but the number of samples will be reduced from the whole proteome to just the desired metalloproteins.
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ACKNOWLEDGMENTS
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Some of the protein samples used in this study were generously provided by Dr. Alexei Savchenko and Pål Stenmark. We also thank Dr. Aled Edwards for advice.
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FOOTNOTES
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Received, December 16, 2004, and in revised form, March 14, 2005.
Published, MCP Papers in Press, March 16, 2005, DOI 10.1074/mcp.T400023-MCP200
1 The abbreviations used are: HTP, high throughput; ICP-AES, inductively coupled plasma-atomic emission spectrometry; PAR, 4-(2-pyridylazo)resorcinol; eq, molar equivalent. 
* This work was supported in part by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) (to D. B. Z.), The Structural Biology Network of Sweden (to M. H.), and The Swedish Research Council (to P. N.); by an industrial research fellowship from NSERC (to R. L.); and by funding from the Structural Proteomics in Toronto Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence may be addressed. Tel.: 46-18-4715062; Fax: 46-18-530396; E-mail: hogbom{at}xray.bmc.uu.se

To whom correspondence may be addressed. Tel./Fax: 416-978-3568; E-mail: dzamble{at}chem.utoronto.ca
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