Pure And Simple: Chromatography A Vital Tool In Biological Research

Biological Research Author: Holly Ahern It's the type of problem that crops up in cell biology, molecular biology, and neuroscience laboratories constantly. Hidden amid a compendium of compounds in a crude cell extract is a protein that researchers have sought since its gene was cloned months before. Perhaps the protein is an enzyme for which the catalytic mechanism has not yet been determined. Or maybe the compound is a recombinant protein that shows promise as a pharmaceutical agent. In either case, before further experiments can be performed, the biomolecule must be separated from the other substances in the cell extract and purified. Chromatography techniques provide scientists with the required tools for separating and purifying biological molecules, such as proteins and nucleic acids, from solutions that may contain hundreds or thousands of different substances. "Chromatography has been utilized for decades to perform biological separations," says Richard P. Cunningham, a professor of biology with the center for biochemistry and biophysics at the State University of New York, Albany. Cunningham, who investigates the catalytic mechanisms of enzymes involved in the repair of DNA damaged by oxidizing agents, is one of a growing number of investigators for whom chromatography has become a vital tool. They are taking advantage of the increasing selection of chromatography products and technology improvements that have greatly simplified the steps involved in biological separations. Researchers in Cunningham's laboratory use a variety of chromatography methods to obtain large amounts of purified enzyme from huge quantities of lysed bacterial cells. Once purified, the proteins are shipped to collaborator John A. Tainer, an X-ray crystallographer at the Scripps Research Institute in La Jolla, Calif. From crystals, Tainer reconstructs the protein's three-dimensional structure, using molecular modeling software. Once the structure is known, Cunningham and Tainer consider possible active sites and catalytic mechanisms, first on the computer and then in the laboratory, where Cunningham creates mutant forms of the enzymes to test his catalytic theories. "Crystallographers rely on highly purified preparations of protein and often require large amounts," explains Cunningham. "Thus, batch-to-batch variations in protein preparations need to be minimized. The recent improvements in biochromatography technology, such as the development of new support matrices that consist of uniformly sized, non-deformable particles, allow us to purify the enzymes rapidly and in a highly reproducible fashion." The new chromatographic supports (in columns) are combined with instruments that can be operated manually or automatically in chromatography systems. This enables researchers like Cunningham to purify biological substances, from relatively small amounts (milligram quantities) all the way up to the process-scale separations achieved by industrial laboratories. Biochromatography, the separation of biological molecules, traces back to the turn of the 20th century, when M. Tswett coined the term "chromatography" to describe his process of separating colorful leaf pigments in a column made of chalk (M. Tswett, Proceedings of the Warsaw Society for Natural Sciences, Section 14, Minute 6, 1903). Liquid chromatography techniques-in which substances in a moving liquid (the mobile phase) are separated or "partitioned" between the mobile phase and a stationary phase (usually linked to an inert matrix in a column)-were under development by the early 1940s and during the decades that followed (A.J.P. Martin, R.L.M. Synge, Journal of Biochemistry, 35:1358-68, 1941; L.R. Snyder, J.J. Kirkland, Introduction to Modern Liquid Chromatography, 1st and 2nd eds. New York, Wiley-Interscience, 1974 and 1979). The early liquid chromatography techniques were performed in large columns with "soft" (gels of silica or cellulose) supports under atmospheric conditions. As a result, separations were achieved slowly and were not always well resolved. Currently, commercially available media for liquid chromatography applications provide faster, higher-resolution separations for a wide range of applications, including several for chromatography of such biological molecules as proteins, amino acids, and DNA. For purifying proteins, life scientists can use a number of different chromatography techniques. These include size-exclusion chromatography (in which molecules are separated according to their size), ion-exchange chromatography (for charged molecules), and reverse-phase and hydrophobic-interaction chromatography (which dis- tinguish between molecules based on hydrophobic interactions between the sample and the stationary phase). In choosing which type of chromatography method to use, a researcher must consider the volume and complexity of the starting material, the substance or substances to be separated, and the level of purification that he or she hopes to achieve. Starting with a crude cell extract, for example, may require several chromatography steps before the desired substance is purified. "Devising a purification scheme for a particular protein may involve more than one chromatography step," explains Cunningham of SUNY-Albany. "For each step, we need to consider the chemistry of the molecule being separated and the nature of the chromatography medium employed in the separation." An investigator's choice of chromatography medium is central to the success of the separation, and there are several products on the market from which to choose. J.T. Baker Chemical Co. of Phillipsburgh, N.J., carries the BAKERBOND line of biochromatography media that are specifically designed for purifying and analyzing biomolecules. BAKERBOND media combine the chromatographic properties of silica beads (the support matrix) with a proprietary surface chemistry suitable for a wide range of applications. Researchers interested in ion-exchange chromatography of proteins, for example, would consider BAKERBOND WP-PEI medium for anion (negative ion) exchange; or BAKERBOND WP-Sulfonic medium for cation (positive ion) exchange. Bulk media for ion exchange chromatography, such as the Series 50 (DE-52, DE-32, and DE-23) and the Series 90 (DE-92 and QA-92) anion exchange media, and a line of cation exchange (CM-52, CM-23, SE-53) products are available from Whatman Inc. of Fairfield, N.J. These media consist of microgranular cellulose material bonded with a charged functional group (positively charged for the anion exchangers, and negatively charged for the cation exchangers). The media are preswollen, which decreases column preparation time. These media are characterized by fast flow times and high binding affinities for proteins, hormones, polypeptides, and other biopolymers bearing positive or negative charges. Fisher Scientific of Pittsburgh and VWR Scientific of West Chester, Pa., both market an extensive selection of chromatography media and other supplies such as columns, flow adapters, and other fittings for liquid-column chromatography. Fisher distributes Whatman's line of ion-exchange chromatography media. The company also distributes purification matrices from Promega Corp. of Madison, Wis., such as Heparin Agarose for protein purifications and SoftLink Resin for affinity chromatography, in which biomolecules labeled with the ligand biotin are attracted to the avidin resin. VWR distributes a selection of chromatography media, such as Sterogene-derivatized resins from Sterogene Bioseparations in Arcadia, Calif., for affinity chromatography and BAKERBOND products from J.T. Baker. Sigma Chemical Co. of St. Louis offers a comprehensive line of products for all types of chromatographic techniques, including media for ion-exchange, size-exclusion, reverse-phase and hydrophobic-interaction chromatography; and chromatofocusing, in which compounds are separated on the basis of their isoelectric point (the pH at which molecules bear no charge). Sigma also carries an extensive selection of media for affinity chromatography of biological molecules, including resins bearing nucleotides (for protein or nucleic acid purification) and other substances, such as aminobenzamidine, polymyxin B, and vitamin B for more specialized applications. A chromatography technique that utilizes convective flow of the liquid phase through the particles of the chromatographic support is patented by PerSeptive Biosystems Inc. of Framingham, Mass., and its use is licensed with the purchase of products in PerSeptive's POROS chromatography line. "Since it was introduced, POROS has provided life science researchers a faster, easier, and more cost-effective way to obtain pure preparations of biomolecules for their studies," claims Daryl Vanderburgh, marketing manager for laboratory scale applications at PerSeptive. VERSATILE: Beckman's BioSys 500 series instruments can be operated with both low-pressure and high-pressure columns. During conventional liquid chromatography, molecules move through the column by convection of the molecules around and between the particles or beads that make up the solid support matrix. Transport through the particles' pores (to the inner surfaces) occurs relatively slowly by diffusion. POROS media consists of particles with considerable mechanical strength that are engineered with two classes of pores, which enhances the transport of molecules by providing convective flow through the beads as well as around them. "This allows chromatography to occur at flow rates 10 to 100 times faster than that of conventional chromatography media with high capacity and high resolution," says Vanderburgh. POROS media can be purchased from PerSeptive as bulk media, as Self Pack (a trademark technology) media ready for the researcher to pack his or her own high-performance columns, or as prepacked columns. Ion-exchange, hydrophic-interaction, affinity, and reverse-phase chromatography are all supported by the POROS line. Before the 1970s, samples were passed through a solid phase that was generally packed into a large-diameter glass column, to maximize the surface area of the stationary phase. Because the flow of material through the column was driven by gravity, separations were generally slow and poorly resolved. By shrinking the column size and applying pressure, biochromatographers achieved faster separations with better resolution. "Adding pressure prevents the sample from diffusing throughout the column as it passes through, and this im- proves the resolution of the separation," explains Cunningham. From the cumbersome liquid chromatography techniques of the 1960s evolved low-pressure (LP) liquid chromatography in the latter part of the decade, followed by high-performance liquid chromatography (HPLC) in the late 1970s. With these methods, pressure is applied to the column from a peristaltic pump (LP) or a more powerful pressure-delivery system such as a dual-piston pump (HPLC), which forces the mobile phase through the column at a much greater flow rate than simple diffusion alone. Because faster flow rates lead to quicker separations with better resolution, liquid chromatography performed under pressure is a powerful analytical and preparative tool for chemists and life scientists. In both LPC and HPLC, molecules are separated through chromatographic columns of uniformly sized microparticles packed into small columns or cartridges. "For HPLC, the limiting factor for many years was the chromatography media, because the support matrix must be rigid enough to withstand the applied pressure and still maintain the desired chromatographic qualities," notes Tom Pritchett, principal scientist for proteins at Beckman Instruments Inc. of Fullerton, Calif. Many of the media currently available for chromatography are suitable for both LPC and HPLC applications. Firms such as Pierce Chemical Co. of Rockford, Ill., and Bio-Rad Laboratories in Hercules, Calif., sell chromatography media for HPLC prepacked in cartridges that are easy to install in a chromatography instrument. Pierce's Cartridge Column System allows researchers to select the size of the cartridge to optimize separation speed and resolution. Shorter cartridge columns reduce analysis time, while longer columns provide higher resolution (along with a longer separation time). Pierce's Aquapore Columns for reverse-phase and ion-exchange chromatography were designed specifically for the HPLC separation of proteins, peptides, and other polyfunctional macromolecules. Bio-Rad's prepacked Econo-Pac cartridge columns are available in 1 ml or 5 ml volumes and come filled with a variety of supports. Up to three cartridges can be connected in tandem to triple the system's sample capacity. For researchers in the first stages of a purification who need to optimize their separation schemes, Bio-Rad sells Protein Purification Sampler Packs. These contain five different media cartridges (included are three ion-exchange cartridges, a hydrophobic interaction cartridge, and a cartridge containing HTP hydroxyapatite). With the Sampler Pack, an investigator can perform five different test runs on a sample in a single day. The separating power of LPC and HPLC are enhanced with the addition of instruments that automate steps and control the chromatographic runs. Integrated systems consisting of a pressure-generating pump, the chromatography column (or columns), a friction collector and monitor to collect and analyze substances as they elute from the column, and a computerized control system can be purchased for separations by LPC and HPLC. The Econo System from Bio-Rad is an integrated system designed for the purification of milligram quantities of proteins and nucleic acids by low-pressure chromatography. The fully automated version of the Econo System is a totally hands-off system, with everything, including sample loading and fraction collection, under the control of the computer. HIGH RESOLUTION: Bio-Rad's BioLogic System, a high-pressure chromatography device. Bio-Rad's BioLogic System is a high-pressure chromatography instrument with Micosoft Corp.'s Windows-based software that features pull-down menus and intuitive icons that simplify programming and system operation. The BioLogic System supports a wide range of chem-istries for chromatography, including ion-exchange, hydro- phobic-interaction, size-exclusion, reverse-phase, and affinity chromatography. Bio-Rad's HPLC Series 5000 System is a complete HPLC unit. Pharmacia Biotech Inc. of Piscataway, N.J., manufactures the FPLC (Fast Protein Liquid Chromatography) System for both manual and automatic biochromatography applications. With this system, researchers can program multi-step purification schemes into the instrument to control parameters such as flow rate, gradient formation, and fraction collection. The automated FPLC System handles both small and large sample volumes and enables fast, high-resolution separations. FROM PHARMACIA: The SMART System, left and the FPLC (Fast Protein Liquid Chromatography System Pharmacia's SMART System is a micropurification chromatography system that recovers and purifies biologically active material present in a sample at very low concentrations (from micrograms to picograms). The system consists of specially designed instruments, software, and prepackaged columns to purify biomolecules for applications such as peptide sequencing and protein structure/function studies. Beckman Instruments Inc. has introduced the BioSys 500 and 510 protein-purification systems, which are the first in a series of workstations being developed by Beckman for bioseparation applications. Unlike the FPLC, the BioSys 500 series instruments can be operated with both low-pressure and high-pressure chromatography columns. Over the next year, according to the company, Beckman will be working on the ProScale program, a system that will consist of BioSys instruments, HyperD media (high-capacity chromatography media produced by BioSepra Inc. of Marlborough, Mass., and supplied by Beckman) and a series of technical information and application bulletins. With the ProScale program, Beckman hopes to simplify the protein-purification process for researchers. "The goal of ProScale is to provide systems, media, and technical information to researchers to make protein purification a rational process, as opposed to the 'hit and miss' method that novice protein purifiers often employ," says Beckman's Pritchett. PerSeptive Biosystems has developed a line of bioseparation instruments for applications ranging in scale from the research lab up through bioprocess scale in industrial labs. The BioCAD Workstation (for process development) and BioCAD/Sprint Workstation (for the research lab) are completely integrated products designed around PerSeptive's POROS media to allow researchers to take advantage of perfusion chromatography. PerSeptive also carries the ConSep system, a product acquired from Millipore Corp. of Bedford, Mass., for low-pressure chromatography. For many life scientists, biochromatography provides the means to an end. "In many cases, chromatography is viewed as a tool that researchers use to obtain a protein or other biomolecule, which is the actual target of their research," comments PerSeptive's Vanderburgh. Beckman's Pritchett agrees. But, he adds: "In some areas, however, chromatography has become a specialized field. The growth of the biotechnology industry has led to a large number of companies involved in the production of biopharmaceuticals. For the people involved in protein purification on the larger scale, chromatography is not just a tool, it's a discipline." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y.

Pure And Simple: Chromatography A Vital Tool In Biological Research

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