|T H E N I H C A T A L Y S T||M A R C H A P R I L 1997|
H O T M E T H O D S
Anita E. Yu, Ph.D., NCI
Robert Fisher, Ph.D., NCI
William Stetler-Stevenson, M.D., Ph.D., NCI
A new category of biotechnologymolecular interaction analysis using surface-plasmon resonance is increasingly picking up steam and has been drawing mounting interest worldwide since its introduction in 1990. This class of techniques exploits changes in the behavior of light at boundaries of different refractive indices to detect the concentration and mass movement of biomolecules. Analytical instruments designed around the optical phenomenon detect surface-plasmon resonance (SPR) and yield sensitive, radioactive-label-free measurements of biospecific interactions in real time.
SPR signals in these instruments are generated by changes in the refractive index of a solution close to the surface of a specially coated sensor chip. The changing refraction of the boundary layer solution is directly related to the concentration of solute and molecular interactions taking place on the surface of the chip, where one of the system interactants is bound.
The new methodology is useful for studying a tempting variety of molecules, including proteins, peptides, nucleic acids, carbohydrates, lipids, and low-molecular-weight molecules, such as signaling ribonucleotides and therapeutic drugs. The specificity of the selected probe coated on the sensor permits direct analysis of biomeolecules in complex mixtures, such as serum, tissue-culture supernatant, and membrane extracts, even without purification.
In the pharmaceutical and biotechnology industries, SPR is replacing earlier techniques for interaction analysis and beginning to provide some new kinds of information. For example, Schuster and co-workers have used SPR to demonstrate the functional formation on the sensor-chip surface of a tetrameric complex involving four components of the chemotactic signaling sytem in E. coli (1). Other examples include identification of B61 as a ligand for the ECK (2), a member of a large orphan receptor proteintyrosine kinase family headed by EPH, and measurement of T-cell-receptor affinity and thymocyte-positive selection (3).
The first step in SPR analysis is immobilization of one of the interactants in a dextran matrix on a sensor chip, which forms one wall of a micro-flow cell. Immobilization can be achieved using a range of chemical techniques, such as direct amine coupling or ligand capturing. The machine then injects samples containing the other potential interactant(s) over the surface of the chip in either a controlled flow or stirred-cell-type system. Any changes in surface concentration resulting from the interaction between the immobilized interactant and a component of the bulk solvent sample generate an SPR signal, which is expressed in arbitrary units, called resonance units (RU). One RU is proportional to 1 pg of mass per square millimeter of surface area.The continuous display of RU as a function of time, referred to as a sensorgram, tracks the progress of interactants' association and dissociation. When analysis of one interaction cycle is completed, the sensor-chip surface can be regenerated by treatments that remove any bound analyte but don't affect the activity of the immobilized ligand.
Major advantages of SPR over other techniques for detecting and measuring interaction include label-free detection and real-time monitoring. Label-free detection means that practically any interactant can be studied, often without having to purify it in advance. Real-time measurement allows investigators to monitor the association and dissociation process extremely closelydown to a time resolution of 0.1 sthus providing a kinetic description of the interaction that is seldom possible with other existing techniques, such as colormetric, fluorometric, or Scatchard plot analyses.
Several companies have been active in developing SPR-based technology+. Biacore, Inc. (formerly Pharmacia Biosensor) first introduced a commercial instrument exploiting this technology in 1990 and currently has two instruments on the marketthe BIAcore 1000 and BIAcore 2000. These systems use a carboxymethylated dextran surface for the standard chip, to which ligand is then coupled, usually by amine coupling. This chip then forms one surface of a flow cell, and solute containing the second ligand(s) is allowed to flow across the chip. These systems offer an alternative to direct coupling known as ligand capture, in which the surface is first coated with an immobilized capturing molecule (such as a specific antibody or streptavidin) that selectively binds to the first ligand of interest. Recent adaptations of this approach include use of nickel ions to capture His-tagged recombinant proteins.
Amersham International, PLC, has been developing SPR-based immunotechnology that uses antibodies labeled with latex particles (beads). The beads amplify changes in refractive-index properties of the sensor surface-solution interface that occur when the antibodies bind to the immbolized antigen layer. This technology is promising, provided that nonspecific interaction between latex-labeled antibodies and the sensor surface is minimal.
Serono Diagnostics is now developing a fluorescence-based evanescent-wave immunosensor that incorporates a novel capillary-fill design. The system consists of two glass plates separated by a narrow capillary gap of ~ 100 mm. The lower plate acts as an optical waveguide and is coated with an immobilized layer of antibodies. Some fundamental drawbacks of this technology include low capillary flow for viscous samples, such as blood; the necessity of an incubation time of several minutes; and system-changeover costs. Unfortunately, each analyte to be tested required a dedicated sensorand this entails labeling and immobilization of antibodies on the plate prior to physical reassembly of the instrument.
The NCI Extracellular Matrix Pathology Laboratory has successfully incorporated SPR technology into several studies. For example, one area where SPR technology has been helpful is in understanding the interactions between gelatinases and their endogenous tissue inhibitors of metalloproteinases (TIMPs). Gelatinases A and B are two members of the matrix metalloproteinase (MMP) family. The MMPs are collectively responsible for the degradation of most components of the extracellular matrix. Gelatinases A and B degrade elastin, fibronectin, gelatin, and collagen types IV, V, and VII, and they have been closely associated with invasive phenotype of many human tumors. Gelatinases and synthesized and secreted from cells as inactive precursors (progelatinases), and they have been shown to bind to TIMPs through the C-terminal domains of these two molecules. Because the activity and activation of gelatinases A and B are tightly regulated by TIMP-1 and TIMP-2, respectively, the exact mode of binding of TIMPs to gelatinases is of tremendous interest, as is their exact mechanism for inhibition of MMP activity.
We use the biosensor system to study systematically the kinetics of gelatinase-TIMP interactions. The interactions of surface-bound TIMPs with the progelatinases and gelatinases in solution is monitored in real time. Progelatinase A binds tightly to immobilized TIMP-2 with a rapid k-on rate and a very slow k-off rate. The k-on rate for the active enzyme is approximately the same as that for the proenzyme, whereas the k-off rates are different. The estimated associated equilibrium constant for activated gelatinase A is 6 x 109 M-1. As expected, TIMP-2 binds to activated gelatinase B with lower affinity and does not bind to progelatinase B. Unexpectedly, the association of progelatinase A with immobilized TIMP-2 was biphasic, and saturation binding is influenced by the free CA++ concentration. The kinetics of the binding of progelatinase A to TIMP-2 suggest that the enzyme possesses a single binding site with two binding states. This kinetic data from the SPR analysis suggest that the initial interactions between TIMP-2 and both progelatinase A and gelatinase A are identical, probably occurring by initial binding between the C-terminal domains of this inhibitor and enzyme pair. However, activation provides a binding site for the N-terminus of TIMP-2, resulting in tighter binding and a slower k-off rate (A.E. Yu, R.J. Fisher, D.E. Kleiner, U.M. Wallon, C.M. Overall, and W.G. Stetler-Stevenson, unpublished observations).
Robert Fisher at the NCI-Frederick Cancer Research and Development Center and members of his lab have pioneered the design, interpretation, mathematical modeling, and global data-fitting of SPR data and have used this technology to study and model severeal systems, including the interaction of transcription factors with duplex DNA (4). They find that by using SPR technology, appropriately designed experiments will yield information about stoichiometry of the components, association rate constants, and dissociation rate constantseven for very complex molecular interaction systems.
A serious drawback of the SPR technology-based instruments is the cost. For example, the approximate base price for BIA 1000 and 2000 are $155,000 and $235,000, respectively. (More information is available from the company's home page.) Affinity Sensors (a division of the Thermo BioAnalyses Corp.) manufactures biosensor instruments and has a home page. Due to the popularity of SPR technology, almost all biosensor instruments on the NIH campus are heavily used. Interested individuals are encouraged to talk to one of the contact people below to find out more about instrument availability and the process for obtaining service or scientific applications.
Despite the cost, SPR technology offers many benefits and potentially has a wide range of applications, providing researchers with an avenue to data not otherwise easily approachable. Scientists who turn to SPR as a way to reduce their reliance on radioactive tracers may ultimately find that the technology gives them more than they bargained for and opens some new doors.
1. S.C. Schuster, R.V. Swanson, L.A. Alex, R.B. Bourret, and M.I. Simon, "Assembly and function of a quaternary signal transduction complex monitored by surface plasmon resonance." Nature 365, 343-46 (1993).
2. T.D. Bartley, R.W. Hunt, A.A. Welcher, W.J. Boyle, V.P. Parker, R.A. Lindberg, S.L. Hsieng, A.M. Colombero, R.L. Elliott, B.A. Guthrie, P.L. Hoist, J.D. Skrine, R.J. Toso, M. Zhang, E. Fernandez, G. Trail, B. Varnum, Y. Yarden, T. Hunter, and G.M. Fox. "B61 is a ligand for the ECK receptor proteintyrosine kinase." Nature 368, 558-60 (1994).
3. S.M. Alam, P.J. Travers, J.L. Wung, W. Nasholds, S. Redpath, S.C. Jameson, and N.R. Gascoigne. "T-cell-receptor affinity and thymocyte positive selection." Nature 381, 616-20 (1996).
4. R.J. Fisher, M. Fivash, J. Casas Finet, J.W. Erickson, A. Kondoh, S.V. Bladen, C. Fisher, D.K. Watson, and T. Papas. "Real-time DNA binding measurements of the ETS1 recombinant onco proteins reveal significant kinetic differences between the p42 and p51 isoforms." Protein Science 3, 257-66 (1994).
Anita E. Yu, NCI, Laboratory of Pathology; phone: 402-1936, fax: 402-2628.
William G. Stetler-Stevenson, NCI, chief, Extracellular Matrix Pathology Section, Laboratory of Pathology; phone: 402-1521, fax: 402-2628.
Robert J. Fisher, NCI, Protein Chemistry Laboratory, SAIC-Frederick; phone: 846-1633, fax: 846-6164.
* In response to the recommendations of the NIH Committee on Alternatives to the Use of Radioactive Techniques, The NIH Catalyst will now use its "Hot Methods Clinic" as a forum also for methods that do not rely on radionuclides. This is the first in what we hope will be a series of articles.
+ Disclaimer: Mention of a specific product in this article does not constitute a commercial endorsement of that product, nor does it constitute a rejection of other techniques and products that may be equally effective but unknown to the authors and editors of this article.
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