INSTRUMENTS
Getting Started
Although the primary literature usually provides the impetus for attempting a new measurement, this literature is often obscure or at least succinct regarding details involved in the interpretation and even execution of the experiment. Therefore, secondary sources are generally a more useful starting point when approaching a new form of measurement.
An introductory text focused on physical biochemistry and its methods can often clarify the basic theoretical underpinnings. There are many such texts that vary greatly in their level of detail and in the mathematical sophistication required, so find one at a level comfortable for your background. The following five volumes from the Practical Approach series published by Oxford University Press are useful, pragmatic starting places for many of the resources within BIF. Individual chapters focus on a specific measurement or class of measurements by presenting basic theory, experimental design and caveats often with "cookbook" style directions for getting started. The volumes are available in the Steenbock Library--click on the titles to check for availability. The volumes are also available in the Biochemistry Library (301 Biochemistry Addition).
Other reference materials and resources for specific measurements might be available and will be provided upon request.
Voyager DE-Pro MALDI-TOF Mass Spectrometer (Applied Biosystems)
In the 1980s, soft ionization techniques that minimized fragmentation were developed and led to a revolution in mass spectrometric analysis of intact proteins, peptides, and other biological molecules. In one method, matrix-assisted laser desorption ionization (MALDI), sample is mixed with a large excess of a low molecular weight, UV-absorbing molecule (the matrix) and dried onto a target. MALDI requires little material (<1 picomole is easily measured) and sample preparation is fast, simple, and requires little if any cleanup prior to analysis. In the mass spectrometer, flashes of light from a laser are absorbed by the matrix resulting in volatilization and ionization of both sample and matrix. In the Voyager the gas phase ions are briefly accelerated by an electrical field after which the time-of-flight (TOF) from target to detector is measured. TOF is directly related to the mass of the ion divided by its charge, which in the case of MALDI is usually +1; therefore, one essentially has a direct measurement of the ion's molecular weight.
The high resolving power of the Voyager (especially at masses <~5 kDa) facilitates accurate mass determinations on mixtures of peptides (e.g., derived from proteolytic digestion) without prior separation. Such "mass mapping" coupled with in silico digestions of sequences found in various sequence data bases is routinely exploited in proteomics to identify unknown proteins and to map sites of post translational modification. Mass mapping of the sites of chemical modifications (e.g., cross-linking or residue specific reagents) can be used to probe protein tertiary and quaternary structure.
The Voyager provides a means to determine the mass of a biological molecule at an accuracy previously unavailable by other methods. The simplicity of the method and ease of use of current instrumentation makes rapid determination of highly accurate masses by MALDI-TOF possible with only minimal training. After training the instrument is available on a walk-in basis.
XL-A Analytical Ultracentrifuge (Beckman Coulter)
The analytical ultracentrifuge remains one of the most reliable tools for studying macromolecular complexes in solution. In sedimentation equilibrium, the sample is spun in the ultracentrifuge until the distribution of macromolecules becomes constant. In the case of a single species, this equilibrium distribution is related to the molecular weight of the molecule under study. Interpretation of the distribution is more involved if there is self-association or more than one macromolecular species present. In either case the measurement is thermodynamically rigorous and requires no "calibration standards". Sedimentation velocity uses higher speeds than equilibrium and measures the rate at which macromolecules move. These data can help enumerate the number of species present and are a primary source of data on a molecule's hydrodynamic radius, the Stokes radius. The Stokes radius is dependent on the molecule's mass, shape, and solvent components that are part of it overall size.
The instrument can measure molecular weights from several hundred to millions, and is ideal for the study of weak, noncovalent associations, including both self-association and interactions between different macromolecules.
Cary Bio400 UV/Vis Spectrophotometer (Varian)
Routine use in quantification and characterization makes some form of UV/Vis spectrophotometer a workhorse instrument for most laboratories. Routine measurements of buffer and sample spectra can prove useful in diagnosing and confirming problems such as aggregation or contamination.
The Cary BIO 400 is a double monochrometer design and can operate in either true double-beam or single-beam mode. The instrument is capable of high resolution, sensitivity, and photometric accuracy over the wavelength range of 175 to 900 nm. It is equipped with a multiposition cell holder (up to 12 samples in single-beam operation) with Peltier-based temperature control (-10 to 100 ?C) and a rear beam attenuator, facilitating measurements to high absorbances. In addition to spectroscopy and quantitation, it is well suited for kinetic studies and following spectral changes that accompany perturbations such as temperature (i.e., melting).
Model 202SF Circular Dichroism Spectrophotometer (Aviv Biomedical)
In circular dichroism (CD) spectroscopy, the difference in the absorbances of left- and right-handed circularly polarized light impinging on a solution is measured. Chiral molecules in the solution will absorb one polarization to a greater extent than the other, such that D- and L-tryptophan have CD spectra that are mirror images. For proteins and peptides, the spectra in the far UV region (<260 nm) is usually dominated by the absorbances of the peptide bonds, which has permitted identification of spectra characteristic of various forms of secondary structure found in proteins. This attribute has made CD a standard tool for assessing the conformational integrity of proteins and peptides. Moreover, with its exquisite sensitivity to structural changes, CD is a logical first choice when examining stability and/or the effects of amino acid substitutions. In addition, CD bands above 260 nm originate from the aromatic residues and disulfide bonds, and interactions within their immediate environments. Spectra in this region can provide a fingerprint of the correctly folded conformation of a protein that is surpassed in sensitivity only by NMR spectroscopy. When characterizing the effects of mutations, CD is an ideal tool for ascertaining conformational stability and/or the kinetics of changes in response to temperature or upon additions to the solvent.
In the Aviv instrument, CD and absorbance spectra can be recorded simultaneously from 170 to 875 nm with temperature control for up to five samples from -5 to 110 ?C. Other accessories permit measurement of total fluorescence along with CD, automated titrations and stopped-flow kinetic studies. The programmability of the instrument and its accessories make sophisticated multidimensional experiments feasible.For a brief overview of spectroscopic tools for structural studies including CD, FTIR, Raman, and NMR, see: Pelton, J. T. and McLean, L. R. (2000) Spectroscopic methods for analysis of protein secondary structure. Anal. Biochem. 277, 167-176. 
QuantaMaster Model C-60/2000 Spectrofluorimeter (Photon Technologies International)
When a molecule absorbs a photon in the UV/Vis range, it enters an excited electronic state where, in a few instances, photochemistry can occur (see: photo-cross-linking). Most often the energy will simply and rapidly be dissipated to the solvent as heat. In some cases, however, only a portion of the absorbed energy is lost as heat with the rest being re-emitted as a photon of longer wavelength (i.e., lower energy) in a process known as fluorescence (or in some case phosphorescence). The fluorimeter permits both the identification of wavelengths resulting in excitation (excitation spectrum) and also characterization of the emitted wavelengths (emission spectrum). Fluorescence measurements are extremely sensitive, allowing measurements at very low concentrations and use for quantification. The emission spectrum of a particular substituent (e.g., tryptophan or an added fluorophore) is generally sensitive to its immediate environment providing some structural insights. Small non-fluorescent molecules can greatly reduce the fluorescence from a macromolecule through a process known as quenching, which can probe the accessibility of the fluorescent substituent. The modulation of emission intensity and/or wavelength by the addition of either fluorescent or non-fluorescent molecules can be exploited as a measure of interaction. A common example is the Forster (or fluorescence) resonance energy transfer (FRET) measurement, which monitors the impact of one fluorescent molecule on another providing insights into their proximity (e.g., bound or not bound). The excitation wavelength can be linearly polarized, and the degree to which this polarization changes upon emission is measured as anisotropy (polarization). Anisotropy measurements can provide information on the size of molecules that were excited, or changes in anisotropy can be exploited as another measure of interaction.
The Quantamaster is a photon-counting instrument providing high sensitivity. The current configuration has a single sample position with the temperature controlled by an external waterbath. Both excitation and emission wavelengths are controlled by monochrometers, which facilitates FRET measurements. Automated Glan-Thomson polarizers are available for both excitation and emission wavelengths, permitting automated anisotropy measurements.
Infinity AR60 Fourier-Transform Infrared Spectrophotometer (ATI Mattson Instruments)
Fourier-transform infrared (FTIR) spectroscopy focuses on the absorption of low energy photons in the infrared region of the spectrum. These photons promote transitions between various vibrational energy levels within molecules, and the specificity of the energies involved has long been used to identify functional groups within organic compounds. In proteins, the stretching vibrations of amide N-H and C=O bonds are extremely sensitive to hydrogen bonding patterns, i.e., beta-sheet, alpha-helix, beta-turns making FTIR an ideal complement to CD spectra (See: Pelton, J. T. and McLean, L. R. (2000) Spectroscopic methods for analysis of protein secondary structure. Anal. Biochem. 277, 167-176. ) Moreover, modern spectral deconvolution techniques coupled with isotopic labeling methods allow absorptions associated with individual functional groups to be resolved and assigned. Unlike most other spectroscopic tools, FTIR can readily be applied to optically turbid media and even the solid state. The time scale involved in FTIR transitions permit resolution of individual conformational isomers interconverting on the picosecond timescale.
The Infinity has an MCT detector for mid IR (400 to 4000 cm-1, where most protein studies are performed) and an InAs detector for near IR (2000 to 10000 cm-1) regions of the spectrum. Sampling accessories for liquids and solids are available and include demountable liquid cells, a 12-ton press for making solid pellets, and a Harrick Horizon ZnSe ATR (attenuated total reflectance) sampling system.
VP-Isothermal Titration Calorimeter (MicroCal)
Almost all reactions between molecules, whether involving the breaking and making of covalent bonds or non-covalent interactions, result in either the absorption or release of heat. Isothermal Titration Calorimetery (ITC) provides a direct measurement of these heats and is particularly well suited for the study of the noncovalent interactions involved in protein - protein and protein - ligand interactions. Data from a single experiment can often provide a complete thermodynamic characterization of the interaction, including the enthalpy (?H) and equilibrium constant (Ka) as well as the stoichiometry of the interaction, and these in turn can be used to derive the Gibbs free energy (?G) and the entropy (?S) for the reaction. If the reaction is measured at multiple temperatures, the change in constant pressure heat capacity (?Cp) can also be derived. Because heat is the quantity measured in ITC, the method requires no spectroscopic or isotopic labeling of any of the molecular constituents in the reaction. Measurements using ITC and BIAcore SPR technology overlap to some extent; however, unlike the BIAcore approach, ITC does not require immobilization of any of the species involved in the interaction. The near universal production/absorption of heat during chemical transformations, make it possible to use the ITC to measure the kinetics of some reactions as has been applied to the determination of Michaelis - Menten kinetic constants in some enzymatic reactions.
Advances in instrument design and sensitivity accompanied by the development of user friendly software have made reliable and accurate ITC measurements by non-specialists possible, thus expanding the range of biological interactions studied by calorimetric methods. The VP-ITC has a temperature range of 2 to 80 ?C. Affinity constants from 100 to ~109 M-1 can be directly measurable by ITC with even higher affinities possible through competition assays. In addition to the its normal mode of operation, the current instrument is capable of the single-injection method (SIM), which can facilitate higher throughput. Software is available for the measurement and interpretation of kinetic studies as well as binding measurements.
VP-Differential Scanning Calorimeter (MicroCal)
The impact of mutations on the stability of a protein, or changes in stability upon addition of ligands or excipients is often of interest. Changes in a spectroscopic observable as a function of temperature is often exploited to probe the "melting" behavior of a protein, which serves as a convenient probe of stability. Nonetheless, the use of spectroscopic tools (CD, UV/Vis, fluorescence, FTIR) requires that a unique temperature sensitive spectroscopic signature be identified, and it should be noted that different spectroscopic approaches or the selection of other wavelengths can yield different results (such differences can, however, provide further structural insights). Further complicating quantitative interpretation in these studies is the usual assumption that the transition involves only two-states (i.e., folded and unfolded), which yields a Tm and van't Hoff enthalpy (?HvH).
In Differential Scanning Calorimetry (DSC), the difference in the amount of heat absorbed by a protein solution and an equivalent amount of buffer is directly measured as the temperature of both solutions is simultaneously and slowly increased (or decreased). In the absence of thermally induced transitions, these data provide the constant pressure heat capacity (Cp) of the protein in solution, which in turn permits the calculation of the enthalpy change (?H) that accompanies any change in temperature. At a thermally induced transition (e.g., a "Tm"), the protein solution will require excess heat to keep its temperature the same as that of the buffer, this excess heat capacity determines the enthalpy associated with the transition (?Hmelt or more generally ?Hcal). Unlike the spectroscopic approaches no model is assumed in determining the enthalpy of a transition in DSC and a single run can reveal and characterize multiple transitions. Furthermore, disagreement between ?Hcal and ?HvH (whether derived from the calorimetric data itself or indirectly from spectroscopic data) provides evidence that a transition is not a simple two state reaction.
As with ITC commercialization accompanied by advances in instrument design and sensitivity along with the development of user friendly software, now permits non-specialists to make reliable and accurate DSC measurements. The VP-DSC can scan both up and down in temperature from -10 to 130 ?C, at variable scan rates. The sample size is <1 mL and reliable results can be obtained at protein concentrations well under 1 mg/mL. The isoscan mode of operation is suited to the study of long term stability at a fixed temperature as might be required in pharmaceutical testing. The influence of very tight binding ligands on protein stability permits the determination of the associated affinity constant, Ka up to ~1020 M-1. The thermal properties of lipids and membranes can also be examined with this instrument.
BIAcore 2000 (Biacore)
The BIAcore 2000 exploits the optical phenomenon of surface plasmon resonance (SPR) to monitor interactions between molecules. Using one of several methods, a molecule (the ligand) is immobilized on a gold surface, which interacts with light to generate the surface plasmon. The instrument then flows the other molecule (the analyte) whether pure or in a complex mixture (e.g., a cell homogenate) over the surface where an interaction with the ligand will increase the mass of material at the surface. Because the method involves directly measuring changes in mass at the surface, neither ligand nor analyte need be labeled with radioactivity or spectral probes so that aside from the immobilization, minimal perturbations to the molecules involved are required. The increase in mass at the surface changes the optical properties of the surface plasmon, and these changes are monitored in real time. For a pure analyte, the kinetics of association and dissociation are directly observed. Equilibrium binding constants can be determined directly from measurements of saturation binding or through the kinetic parameters, and the specificity of an interaction can be probed. Although ITC (which requires neither immobilization nor labeling) also allows the determination of equilibrium binding constants, it is not generally amenable to kinetic studies of binding interactions, requires more material and is not as well suited to an analyte that is heterogeneous.
The BIAcore approach can be quite versatile and generally requires less material than most other non-spectroscopic/non-radioactive approaches. It is suited for screening the specificity of analytes towards a common ligand where relative rankings can be examined in terms of their kinetic and/or equilibrium constants to identify controlling features for specificity. The identification of a ligand's interaction partner from a cell or tissue homogenate is possible, and the instrument can be used for small scale affinity purification. The BIAcore can be used for analyte quantitation. Measurements are possible from 4 to 40 ?C. Typical Ka values for equilibrium measurements range from 104 to 1011 M-1. In kinetic measurements on rates of 103 to 106 M-1s-1 and off rates from 10-5 to 0.1 s-1 are routinely measured.
For additional information on using the BIAcore 2000, see: BioResources February 1998.
Model IK3102R-G Helium-Cadmium Laser (Kimmon Electric)
Photo-cross-linking can be a powerful method for revealing atomic contacts. It has found application in the study of protein - nucleic acid complexes and has the potential to identify an unknown nucleic acid-binding protein in a complex mixture. Modern biophysical photo-cross-linking in these systems exploits advances in nucleic acid chemistry to incorporate one or more 5-iodouridine nucleosides into a nucleic acid using standard chemical or enzymatic protocols. When a labeled molecule is briefly exposed to 325 nm light, the iodine-carbon bond in the 5-iodouridine undergoes homolysis. The resulting carbon radical is of low energy and forms covalent adducts with aromatic amino acid residues in its immediate vicinity. The yield of covalently cross-linked product can approach 100%.
The helium-cadmium laser in BIF produces a wavelength of 325 nm with an intensity >70 mW. The high flux makes this laser suitable even where the extinction coefficient of the photolyzable chromophore is small at 325 nm.
For additional information, see: Willis, M.C., Hicke, B.J., Uhlenbeck, T.R., Cech, T.R., and Koch, T.H. (1993) Photocrosslinking of 5-iodouracil-substituted RNA and DNA to proteins. Science 262, 1255-1257. Norris, C. L., Meisenheimer, P. L., and Koch, T. H. (1996) Mechanistic studies of the 5-iodouracil chromophore relevant to its use in nucleoprotein photo-cross-linking. J. Am. Chem. Soc. 118, 5796-5803.
DAWN Multi-angle Laser Light Scattering (Wyatt Technology)
Light scattering can be a useful, nondestructive approach for characterizing macromolecular mass and sometimes shape. As a laser beam passes through a sample, a small amount of the light is scattered in all directions with the scattering intensities and angular dependence a function of the molecular weight and shape of the molecule. The DAWN multi-angle laser light scattering (MALLS) instrument simultaneously measures the average intensity of light scattered at various angles from the beam. The result is a nearly instantaneous measurement of the weight-average molecular weight (Mw) of all macromolecular species present in the sample. Mw is the same thermodynamically rigorous molecular weight as measured in sedimentation equilibrium experiments. MALLS provides rapid information on Mw at a single concentration, but sedimentation equilibrium on a single sample can reveal the concentration dependence of Mw (e.g., resulting from self-association). MALLS measurements as a function of concentration, leads to another thermodynamic parameter the second virial coefficient, which is a measure of the interactions between the macromolecule and its solvent. If the size (i.e., radius) of the molecule in solution is >~1/10 of the wavelength of the laser, the angular distribution of scattering measures the radius of gyration of the molecule. The radius of gyration is rigorously defined and can lead to structural insights by comparison of the measured value with those calculated for various shapes. The hydrodynamic radius (Stokes radius) as measured by sedimentation velocity or dynamic light scattering is that of an equivalent sphere and contains contributions from mass, shape and bound solvent the latter making shape inferences more difficult.
MALLS provides a rapid approach to the measurement of Mw over a wide range of molecular weights. The second virial coefficient is easily obtained, and in some instances a direct measure of molecular shape is possible. MALLS generally requires more material than do sedimentation methods. MALLS can be useful as a relatively rapid approach for characterizing proteins and for investigating macromolecular associations.
N4-Plus Dynamic Light Scattering (Beckman Coulter)
In dynamic light scattering (DLS) a detector is focussed on a small volume in a sample that is illuminated by a laser beam. The detector measures fluctuations in the intensity of light scattered from this volume (in MALLS the detector measures the time-averaged intensity scattered from the same volume). The fluctuations are the result of macromolecules diffusing into and out of the small volume that the detector is monitoring. From the data the translational diffusion constant of the macromolecule is determined, which in turn can be related to the radius that a sphere with the same diffusion constant would have. This is the Stokes radius (also measured by sedimentation velocity) and contains contributions from the mass, shape and bound solvent components. A measure of the polydispersity of the sample is also derived during the analysis with higher polydispersity suggesting aggregation in the case of proteins. In some cases populations of species of differing radii and polydispersity can be resolved. Protein preparations exhibiting low polydispersity have generally proven more suitable for crystallization trials than those with high polydispersity. The method is well suited to the characterization of large polymers and liposomes.
The N4-Plus can measure at six angles with Peltier based temperature control from 0 to 90 ?C. For measurements at the 90? angle samples under 100 µL can be measured. Particles diameters from 3 to 3000 nm are accessible.
AVS 310 Capillary Viscometer (Schott Gerate GmbH)
The viscosity of a solution is a measure of its resistance to flow and dissolved macromolecules (e.g., synthetic polymers, high molecular weight polysaccharides) can have pronounced effects. Viscosity measurements of synthetic polymer solutions are often part of their routine characterization, and have been used to deduce molecular weights of the polymer and their degree of branching. Most measurements of the hydrodynamic radius of a protein (e.g., with sedimentation velocity or DLS) require knowledge of the solvent viscosity.
From the time that a fixed volume of solvent takes to flow through a capillary viscometer, the solvent viscosity can be determined. By measuring viscosities of solutions at varying concentrations of macromolecule, the intrinsic viscosity of the macromolecule can be deduced. The intrinsic viscosity reflects properties of the macromolecule itself, i.e., its shape, volume, and any bound solvent. The technique can be used to measure solution viscosities in the range of 0.35 to 30,000 centipoise for a solution density of ~1 g/mL.
DMA5000 Density Meter (Anton Paar USA)
The the solvent density impacts the macromolecular mass and size that is deduced from many solution measurements (e.g., sedimentation equilibrium), and an accurate density measurement can reduce the uncertainty of the final calculated value. The partial specific volume (psv) of a macromolecule also impacts such results; while the psv is usually computed using additivity rules, it can be deduced from the change in density that accompanies changes in macromolecular concentration. Density measurements have also been exploited in the study of the "binding" of small molecules (e.g., salts and sugars) to macromolecules to understand the molecular basis of their stabilizing/destabilizing effects.
The DMA5000 permits a highly accurate measurements of densities from 0 to 3 g/cm3 on ~1 mL of sample over the temperature range of 0 to 90 ?C.