research papers 16 # 2003 International Union of Crystallography  Printed in Great Britain ± all rights reserved J. Synchrotron Rad. (2003). 10, 16±22 Experimental aspects of biological X-ray absorption spectroscopy Isabella Ascone, a * Wolfram Meyer-Klaucke b and Loretta Murphy c a LURE, Ba à timent 209E, Universite  Paris-Sud, 91898 Orsay CEDEX, France, b EMBL, c/o DESY, Notkestrasse 85, Geb 25A, 22607 Hamburg, Germany, and c Daresbury Laboratory, Warrington WA4 4AD, UK. E-mail: isabella.ascone@lure.u-psud.fr Spectroscopic techniques, like X-ray absorption spectroscopy, will provide important input for integrated biological projects in genomics and proteomics. This contribution summarizes technical requirements and typical set-ups for both simple and complex biological XAS experiments. An overview on different strategies for sample preparation is discussed in detail. Present and future BioXAS spectrometers are presented to help potential users in locating the spectrometer required for their biological application. Keywords: BioXAS; metalloproteins; experimental set-up; macromolecules. 1. Introduction X-ray absorption spectroscopy (XAS) has been widely used in many areas of science during the last 20 years. The feasibility of using this technique for biological systems was demonstrated in the ®rst ¯uor- escence experiment (Jaklevic et al., 1977). Subsequently, several reviews highlighted its contribution to the characterization of metal centres in biological systems (Cramer, 1988; Garner & Charnock, 1993; Hasnain & Hodgson, 1999). Nevertheless, biological X-ray absorption spectroscopy (BioXAS) has demanding experimental requirements that until 1990 were available at only a few XAS beamlines worldwide. In 1985, Fiamingo & Alben (1985) pointed out the necessity to improve the experimental conditions in order to extract reliable information from BioXAS data; however, some of these have been developed only recently. In particular, there are two major technological advances that have improved BioXAS measurements: (i) The source intensity of second-generation machines has been increased by insertion devices or focusing optics while third- generation machines produce high-intensity and focused X-ray sources. This feature allows us to increase the signal, and to decrease the biological sample concentration and/or sample volume. (ii) Fluorescence detectors, which are essential for measurements on dilute samples, are now much more ef®cient and are continually improving. These technical improvements have increased the quality of BioXAS measurements: the information obtained is more reliable as the k-range of the EXAFS signal is extended. This paper will ®rstly present the typical biological XAS experi- mental set-up and then summarize the different elements such as source, monochromator, detectors and sample environment devices. Particular attention will be given to metalloprotein studies. The aim is to give the reader, interested in studying a protein metal site, some help in choosing the experimental set-up and the appropriate sample environment. 2. A typical biological XAS experiment Important scienti®c questions, like the structural and electronic characterization of metal sites, can be performed on BioXAS beamlines using a layout described in the following paragraphs. Synchrotron beamline. A basic X-ray spectroscopy station consists of a scanning high-resolution monochromator, a beam monitor and a signal detector. The signal is detected in transmission or in ¯uores- cence mode. In the ®rst case, two ionization chambers are generally used as detectors, to measure the X-ray ¯ux before (I o ) and after (I t ) the sample. In the second detection mode, speci®c for diluted samples (absorber concentration < 1%), a detector is placed perpendicular to the beam to measure the ¯uorescence signal (I f ). Sample. BioXAS can be measured on samples in any state, e.g. room temperature or frozen solution at liquid-nitrogen or helium temperatures, single crystals (Scott et al., 1982), crystalline slurry (Ascone et al. , 1997), all or part of an organism (Kramer et al., 1986). The choice of sample state depends entirely on the robustness of the sample and the scienti®c question you are trying to answer. However, typically frozen solutions are studied in order to lower the thermal disorder and minimize the radiation damage. Typically, the metallo- protein (metal concentration ca 1±5 mM, volume 10±120 ml) is loaded in a plastic cell with plastic ®lm covering the central aperture and frozen in liquid nitrogen. The sample is frequently measured at either liquid-nitrogen or helium temperatures via the use of cryostats. In some cases cryoprotectants need to be added to the protein solution. The materials used to construct the sample cell and sample envir- onment must be free of signi®cant metal content relative to the concentration of the sample itself. 3. Characteristics of the X-ray source The X-ray absorption cross section of a metalloprotein is particularly low compared with the absorption of samples frequently used for material science experiments. Metal is bound to one or more amino acid chains having a high molecular weight (10±100 kD or more). Moreover, the protein is diluted in an aqueous solution. The XAS signal (detected by ¯uorescence) is proportional to the intensity of the incident beam. Therefore BioXAS strongly depends on intense insertion-device sources which are available at second- and third- generation synchrotron beamlines. In contrast to other powerful techniques, like protein crystallography, BioXAS cannot be performed on laboratory X-ray sources such as rotating anodes or X-ray tubes. Thus, even the preliminary tests to prepare a BioXAS experiment have to be performed at a synchrotron centre. Dedicated beam time is necessary in order to optimize the conditions for sample concentration, its susceptibility to radiation damage or for kinetics. The intensity of the XAS signal depends on several parameters: the characteristics of the machine (critical energy, insertion devices, current etc.), the energy of the absorption edge of the metal of interest, the focusing optics and the quality of the ¯uorescence detector available. The intensity and size of the beam irradiating the sample will directly effect the minimum usable sample concentration and volume. At present, two major types of beamlines exist: (a) high- ¯ux beamlines on insertion devices (undulators, wigglers or wave- length shifters) (Gauthier et al., 1999; Sole  et al. , 1999; Tanida & Ishii, 2001), and (b) low-¯ux beamlines at bending magnets. Both types have advantages and disadvantages for BioXAS experiments. A high-¯ux beamline reduces considerably the quantity of protein required for XAS measurements (Ranieri-Raggi et al., 2003); sample volumes of 10±20 ml with metal concentrations of about 50±100 mM are feasible but with a risk of photoreduction altering the metal site.