Tables for
Volume I
X-ray absorption spectroscopy and related techniques
Edited by C. T. Chantler, F. Boscherini and B. Bunker

International Tables for Crystallography (2023). Vol. I. Early view chapter

Biological samples

Ritimukta Sarangia*

aSLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA
Correspondence e-mail:

X-ray absorption spectroscopy (XAS) is a powerful, high-resolution, local structure-determination technique that has been widely applied to studying metal sites in biological systems. The ability to simultaneously measure the geometric structure from the extended X-ray absorption fine-structure (EXAFS) region of the spectrum and the electronic/chemical structure from the pre- and near-edge XAS region has been applied to a wide range of metallo­enzymes, proteins and cofactors containing transition-metal active sites and to nonmetals that are present as ligands or other biological agents. Biological samples are temperamental to prepare, susceptible to beam-related radiation damage and are typically only available at low concentrations. Here, commonly employed methods of preparing and measuring biological samples for XAS measurements are discussed. These methods, along with advanced in-hutch instrumentation and detection modalities, aim to alleviate the challenges faced by these sensitive samples.

Keywords: biological samples; sample preparation.

1. Introduction

X-ray absorption spectroscopy (XAS) is a long-established synchrotron-based technique for investigating the geometric and electronic structures of molecular systems (Parsons et al., 2002[link]; Sarangi, 2018[link]; Strange & Feiters, 2008[link]; Ascone & Strange, 2009[link]). Its application to metal centres in biology has been slow due to the complexity of sample preparation, adverse interaction of the X-ray beam with sensitive biological samples, the need for low-temperature measurements and detector limitations in separating the signal of a dilute metal active site from the strong background absorption of the biological matrix (Penner-Hahn, 1999[link], 2005[link]). This measurement difficulty is accentuated by the fact that biological solution samples often suffer from structural disorder, from unknown ligand systems and from overall poor data quality that can complicate data analysis. In the last two decades, significant advances in biological X-ray spectroscopy have been achieved. Today, modern, high-brightness synchrotrons around the world have dedicated instrumentation for biological spectroscopy measurements targeted towards minimal beam exposure and damage and towards resolving the weak signal of dilute metallo-biological systems. Robust methodologies have been developed for the standardization of biological sample preparation, measurements and data analysis. It is important to make a distinction between sample-preparation requirements for biological near-edge XAS and extended X-ray absorption fine structure (EXAFS). While near-edge XAS has successfully been applied to biological samples prepared as purified solutions, whole animal and plant cells, tissues, tissue sections, organs and organelles, using micro-beam and nano-beam X-ray fluorescence methods, quantitative EXAFS measurements and analysis, especially on biological systems, is contingent on a relatively uniform sample with high molecular purity. The most widely applied methods for sample preparation for EXAFS measurements of different types of biological systems are discussed here.

2. Dilute solution samples

2.1. Static measurements

Sample preparation within the guidelines for the measurement is the critical first step for successful data collection. In contrast to certain optical spectroscopies, XAS requires a larger volume and a higher concentration of the metal centre of interest. This is especially challenging for biological systems, which inherently yield low-concentration/low-volume samples. Furthermore, biological samples often suffer from sample impurity due to adventitiously adsorbed metals, deficient loading of the metal active site, inadequate or incomplete purification methods and stochastic reasons associated with variability in vehicle growth (for example cell culture), sample isolation or additional metal sites that contribute to the EXAFS signal. Since XAS is an average technique, a thorough characterization of the metal content and an understanding of the behaviour of the metal centres in the biological system is critical for successful measurement and meaningful analysis.

The most interrogated biological samples are first-row transition-metal metalloprotein solutions. Other macromolecular systems, such as metal–DNA complexes and metal cofactors, for example molybdopterin complexes etc., have also been studied. Since metalloproteins are the dominant class of biological systems investigated using X-ray spectroscopy, for the purposes of this chapter the term metalloprotein is used to represent all metal-containing biological macromolecules. Metalloproteins amenable to study by EXAFS are typically limited to four or fewer unique absorbing metal sites of interest. EXAFS has also been applied to metalloproteins containing a much larger number of metal centres with limited success (Heath et al., 1996[link]). This number may be higher when considering metalloproteins that have metal clusters in their active sites. Metal concentrations typically range between 100 µM and 10 mM, depending on the purification protocol and solubility. Biological samples are almost invariably measured in a frozen aqueous matrix; therefore, glassing agents such as glycerol and glycol are added to suppress ice crystals and their diffraction spikes in the EXAFS data. To ensure a good glass, ∼20–40% glassing agent is used. This is factored into the final sample concentration. Sample-volume requirements are predominantly affected by the beamline characteristics: for measurements performed on a wiggler beamline with a broad focus (∼1 × 4 to 2 × 10 mm), the typical sample volume requirement is between 100 and 200 µl. For measurements on an undulator beamline (capable of biological EXAFS) with a smaller focus (∼0.02–0.1 mm), the typical volume requirements are ≤50 µl.

Biological samples can denature with time, temperature fluctuations or contact with reactive metal sample cells such as aluminium. Typically, an inert plastic sample cell is employed; such cells have proven to be structurally robust over a large temperature range, ensuring that samples prepared at room temperature remain intact in the liquid-helium cryostat required for the measurement of biological samples. Measurements at liquid-helium temperature (4 K) reduce beam damage and improve the experimental data by eliminating noise arising from dynamic Debye–Waller disorder.

An important consideration during preparation for biological EXAFS measurements is the potential for beam-related damage and/or photoreduction (George et al., 2012[link]). Biological spectroscopy beamlines are therefore equipped with multiple-filter and fast-shutter systems to minimize these effects (Yano et al., 2005[link]). Despite this, samples that suffer very fast beam damage must be replaced several times during the course of measurement in order to obtain a valid low-noise data set. This can often be achieved by translating a sample in the X-ray beam, moving the beam spot to an unexposed area (Krest et al., 2015[link]). Beam damage poses a greater threat on high-intensity undulator beamlines; however, the significantly smaller beam footprint permits more measurement areas on a sample, compensating for the enhanced sample damage and the need for fresh spots. Unfortunately, certain of the glassing agents noted above hasten or catalyze photoreduction, with glycerol being a notorious culprit (Nienaber et al., 2018[link]). For very sensitive samples, lower concentrations of polymeric glassing agents are appropriate. With the advent and routine use of multi-element germanium detectors, the use of glassing agents is sometimes avoided altogether. This results in lower statistics (some detector channels become unusable due to ice-diffraction spikes) but higher quality data (less photoreduction). Beam-induced photoreduction can also be slowed by the use of in situ chemical oxidants. These must be tested to determine that they are otherwise unreactive with the protein.

2.2. Flow systems and dynamic sample delivery

While the majority of biological samples are measured as frozen or cooled liquids, new sample-delivery methods have been developed for XAS measurements that include microfluidic devices that create a liquid jet, a flow system and droplet delivery and stopped-flow methods. Microfluidic-based sample delivery is popular at X-ray free-electron lasers for simultaneous XRD and X-ray emission spectroscopy (XES)/XAS measurements (Chatterjee et al., 2019[link]). However, significant advancement in these newer sample-delivery techniques must be made to decrease the amount of sample consumed during measurement for widespread applicability to biological samples, especially for EXAFS measurements, which require higher signal-to-noise statistics. With the development of continuous scanning and fast-scanning measurements (Müller et al., 2016[link]; Fonda et al., 2012[link]), millisecond to second time resolution can be achieved and coupled with a stopped-flow mixer to study biological transformations as they occur. This method is also limited to higher metalloprotein concentrations, and the use of a stopped-flow mixer for metalloenzyme XAS has only been demonstrated for freeze-quenched samples (see below; Solomon et al., 2007[link]; Lee et al., 2002[link]).

3. Solid-phase samples

3.1. Freeze-quenched snow

While most biological measurements are made on frozen solutions, as described above, special sample-preparation methods are employed to trap sensitive and/or transient species. A popular technique is the freeze-quench method, in which bio-reactants are rapidly mixed and then microsecond quenched by ejection onto a deeply cryogenic surface (Yosca et al., 2013[link]). The mix/quench traps transient reaction intermediates whose presence has been detected by stopped-flow kinetics experiments. A freeze-quenched sample resembles the texture of soft snow and can be packed under cryogenic conditions into an open-face EXAFS sample cell. Some drawbacks of freeze-quenched samples include ice-diffraction interference and uneven sample packing. The latter can lead to inhomogeneity in sample concentration (sparse and dense signal regions in the sample holder), which could lead to nonlinearity in the absorption coefficient.

3.2. Lyophilized proteins

Another convenient method of biological sample preparation is lyophilization, which removes the ice from a frozen solution sample by vacuum sublimation. This can minimize sample degradation and maximize ease of storage. A lyophilized sample is a dry powder that can be packed into an open-face EXAFS sample cell similar to that used for freeze-quenched samples. The advantage of this preparation is the absence of ice interference in the EXAFS data coupled with a greater concentration than is achievable in solution. Several studies have been performed on metalloproteins to understand the role of the aqueous hydration sphere by comparing the XAS of dissolved and lyophilized protein systems (Noth et al., 2015[link]).

3.3. Single crystals

The synchrotron X-ray beam lends itself beautifully to the measurement of single-crystal polarization-dependent XAS, which has successfully been applied to obtain EXAFS on single-crystal metalloproteins (Yano et al., 2006[link]). By aligning a specific metal–ligand bond to the polarization vector of the beam, the electronic absorption due to that bond can be enhanced, while that of bonds along other axes is relatively suppressed. With EXAFS data along several axes of interest, a complete picture of bonding can be developed with resolution of each individual contribution. This is contingent on the crystal packing and the alignment of the molecular axis with the space group of crystallization. This technique requires focused beam optics, a goniometer for sample alignment, a liquid-helium cryojet for sample cooling and simultaneous/sequential measurement of fluorescence and diffraction using high-capacity solid-state and CCD detectors, respectively. In addition, since the signal intensity from single crystals is often lower than that of solution samples, the technique can only be applied to larger crystals or those with a smaller unit cell. The goniometer setup can also be used for isotropic fluorescence measurements on polycrystalline samples packed into metal-loop sample holders. This method is useful for measuring intermediates trapped in crystalline form and for investigating spectroscopic differences between solution and crystalline phases (Wilson et al., 2013[link]).

4. Tender and soft X-ray measurements

Tender XAS refers to spectroscopy in the ∼2–5 keV energy window. Of the biological elements of interest in the tender X-ray regime, S K-edge XAS (2.4 keV) dominates the literature and has been extensively applied to biological systems to gain insight into metalloprotein active-site bonding from the perspective of sulfur-containing ligands (Anxolabéhère-Mallart et al., 2001[link]). The method is dominantly used to investigate the pre- and near-edge region of the spectra for electronic structure information. X-ray spectroscopy has also been applied to other elements in the tender X-ray regime, such as Mo L edges in molybdopterin enzymes (Corbett et al., 2005[link]; George et al., 1990[link]; Bjornsson et al., 2015[link]) and the P K edge in DNA (Czapla-Masztafiak et al., 2016[link]; Yamada et al., 1993[link]). Irrespective of the element, sample preparation is largely determined by the experimental setup. Samples are prepared as dilute solutions (∼1 mM of the element of interest or higher) and are either kept fixed during measurement or continuously replenished by a flow system (Czapla-Masztafiak et al., 2016[link]). Since tender XAS focuses on the electronic aspect of the metal/ligand centres in biology and is often limited to the near-edge region of the spectrum, samples can also be prepared as lyophilized solids (Dey et al., 2007[link]). Most measurements in the tender X-ray regime are performed under helium or in evacuated chambers to minimize the impact of air absorption and pose additional considerations for sample preparation due to increased radiation damage in this energy range. This issue is made worse by the fact that most measurements are performed at higher temperatures, either without any cooling (RT measurements; Czapla-Masztafiak et al., 2017[link]; Ha et al., 2017[link], 2019[link]) or using low-flow liquid-helium cryostreams (Hackett et al., 2012[link]) or conduction cooling methods (Dey et al., 2007[link]). One method for averting or slowing photoreduction is the inclusion of chemical oxidants such as K3[Fe(CN6)] or K2IrCl6 (DeBeer George et al., 2001[link]; Basumallick et al., 2005[link]). Complexes used as models for enzyme active sites are typically solid state and therefore can be prepared for ambient temperature measurements.

The soft X-ray regime (500–1000 eV) can be exploited to measure first-row transition-metal L-edge XAS, which is used to investigate the detailed electronic structure of the metallo­protein active site (Grush et al., 1996[link]; Baker et al., 2017[link]). In this regime, the samples are typically in one of the solid phases discussed in Section 3[link]. In addition, a partially dehydrated thin-film method has also been used in which the solution metallo­protein sample layered on a thin silicone substrate is partially evaporated. L-edge XAS can then be measured on this substrate-adhered film under ultrahigh-vacuum conditions (Kubin, Kern et al., 2018[link]; Kubin, Guo et al., 2018[link]). Recently, relatively ambient pressure measurement methods have been developed using a differentially pumped vacuum chamber and a low-volume sample-delivery system (Smith & Saykally, 2017[link]).


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