Chemical Education Today
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Research Advances by Angela G. King
New Metal Detector To Study Human Disease Zinc may be a familiar dietary supplement to millions of health-conscious people, but it remains a mystery metal to scientists who study zinc’s role in Alzheimer’s disease, stroke, and other health problems. While zinc is the second most abundant trace element in humans, just 2–3 grams of zinc are present in the entire human body. The metal is a key building block in enzymes and other substances involved in functioning of the nervous system, the immune response, and the reproductive system. Scientists are just beginning to fathom how the body keeps levels of zinc under the precise control that spells the difference between health and disease. Until now scientists have not had a grasp on how zinc is distributed within the human body or incorporated into metalloproteins. Due to the presence of many zinc ligands in cells, free (or rapidly exchanging) zinc concentrations within cells has been estimated in the femtomolar range. If the concentration of free zinc is so low as to make its insertion into transcription factors and other proteins following their synthesis unlikely, then it is postulated that it must be carried there and inserted by another molecule, as is the case for copper. Thus, precisely measuring cellular concentrations of free zinc is critical to
understanding the biological mechanism of zinc ions. A collaborative team of scientists from the University of Maryland School of Medicine and the University of Michigan, Ann Arbor, have now developed a biochemical metal detector to help crack the mystery. It is a biosensor that has yielded the first measurements of the tiny amounts of free zinc ordinarily present inside living cells. “The question of how much zinc is available in a cell has emerged at the forefront of chemical biology”, Amy R. Barrios of the University of Southern California, Los Angeles, wrote in an accompanying Point of View in ACS Chemical Biology. Barrios described the new research as “a critical step forward” and predicted “many more exciting breakthroughs” in measuring levels of metals in human cells. In the past, scientists could only measure the relatively high levels of zinc in sick cells. The collaborative team, led by biochemist Richard Thompson, developed a fluorescent indicator system utilizing apocarbonic anhydrase as a sensor transducer. Human carbonic anhydrase II (CA) exhibits very high affinity for zinc at pH 7.5 and is unaffected by the presence of Ca2⫹ or Mg2⫹ at concentrations higher than their in vivo levels. To visualize and quantify the binding of CA to free zinc, the research team employed Dapoxyl sulfonamide (Figure 1). Dapoxyl sulfonamide does not bind to
apo-CA in the absence of zinc and displays a weak emission at 617 nm under those conditions. However, Dapoxyl sulfonamide coordinates to the zinc of holo-CA, and the result is an enhanced fluorescent emission at 617 nm with UV excitation. The new sensing technology enables low free zinc levels to be measured by reporting the fractional occupancy of the protein (which is controlled by the free zinc concentration) as a change in fluorescence polarization, lifetime, or a ratio of fluorescence intensities; the last is ideal for studying cells in the fluorescence microscopes widely used for studying cellular calcium levels (Figure 2). The team circumvented the difficulty of introducing a protein-based biosensor into cells by attaching a transactivator of transcription (TAT) peptide to the CA-based biosensor. TAT induces cultured cells to take up the protein. Uptake of the TAT-fused fluorescent labeled CA had no apparent ill effects on cells at concentrations required for fluorescent microscopy. It allowed intracellular zinc ions to be imaged and quantified in the picomolar range. Because proper zinc levels are so important in health and disease, scientists have been seeking ways of measuring zinc inside and outside of cells for more than a decade. “This is an important discovery”, said Sarah B. Tegen, managing editor of ACS Chemical
Figure 1. Schematic of ratiometric zinc determination with modified CA and Dapoxyl sulfonamide. In the absence of Zn, Dapoxyl sulfonamide does not bind to CA, therefore no FRET occurs to the label on the protein and only weak emission at 617 nm occurs. In the presence of Zn, Dapoxyl sulfonamide binds to Zn, is excited with UV light, and FRET occurs, exciting the fluorescent label causing emission to occur at 617 nm. Reprinted with permission from ACS Chem. Biol. 2006, 1, 103–111. Copyright 2006 American Chemical Society.
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Chemical Education Today
Reports from Other Journals
Figure 2. Fluorescence ratio image of free zinc inside PC-12 cells. a: brightfield; b: ratio image false color; c: excitation at 365 nm, emission at 617 nm, exposure time of 20 ms, pseudocolor red; d: excitation at 365 nm emission at 617 nm, exposure time of 500 ms, pseudocolor red. The false color calibration bar (at right) indicates the concentration of cytoplasmic rapidly exchangeable zinc ions. Reprinted with permission from ACS Chem. Biol. 2006, 1, 103–111. Copyright 2006 American Chemical Society.
Biology. “We need to know how the body controls levels of zinc inside cells. Too much zinc can kill nerve cells. With too little, nerve cells will not work properly. “Now we have a metal detector, technology that can measure tiny amounts of zinc in living cells. Understanding how zinc is stored and released in different cells throughout the body may help us understand some of the nerve damage that occurs during a stroke and other nerve injuries.” “We believe this new technique can help us understand how zinc is involved in plaque formation in Alzheimer’s disease, how prolonged seizures or stroke kill brain cells, and how the cell normally allocates zinc to different proteins,” said Thompson. Thompson explained that almost all zinc inside cells is incorporated into proteins, where it plays many vital roles, such as helping to read the genetic code of DNA. “We know that if there is much zinc in the cell that is not attached to protein or otherwise encapsulated—so-called ‘free zinc’—the cell is stressed or may be undergoing programmed cell death. This has been observed in animal models of epilepsy and stroke.”
More Information 1. Bozym, Rebecca A.; Thompson, Richard B.; Stoddard, Andrea K.; Fierke, Carol A. Measuring Picomolar Intracellular Exchangeable Zinc in PC-12 Cells Using a Ratiometric Fluorescence Biosensor. ACS Chem. Biol. 2006, 1, 103–111. 2. This Journal has published an undergraduate lab involving zinc and CA. See Williams, Kathryn R.; Adhyaru, Bhavin. Removal of Zinc from Carbonic Anhydrase. A Kinetics Experi-
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Salinized Fresh Water? Dangerous driving conditions aren’t the only threat brought on by winter storms. Recent research indicates that road salt, used liberally in some areas of the Northeast U.S. to help improve road conditions in times of snow and ice, is threatening the freshwater supply. Overuse of the existing freshwater supply has already diminished this important resource. To compound the issue, industrial pollution, agricultural practices, and automotive waste have been known to negatively impact the freshwater supply. Now a collaborative team led by Gene Likens of the Institute of Ecosystem Studies has determined that increases in the use of salt deicer are salinizing fresh water rapidly enough that within 100 years many surface water supplies in the Northeast may not be potable for human consumption and will be toxic to freshwater life. The scientists used long-term data from streams and rivers draining rural
watersheds in Baltimore County, MD, the Hudson River Valley, and the White Mountains to investigate the rate of salinization and baseline chloride concentration. These rural sites were selected in part due to small population growth changes and their low density of roads. In contrast, the team examined the same changes in the Baltimore metropolitan area, which had an approximate increase of impervious surface of approximately 39% between 1986 and 2000. Records show that the city of Baltimore utilized more than 82,000 metric tons of NaCl as deicing material during the study period. Their results show that in all areas of the study there were strong increases in baseline Cl⫺ concentration in the past three decades. In some areas, rural streams had chloride concentrations equal to that of 25% seawater. They also found that salinization is related to the extent of impervious surface coverage (Figure 3). In many suburban and urban areas, the chloride concentration already exceeds the 250 mg/L limit recommended for the protection of freshwater life. The team also noted a seasonal fluctuation, with chloride concentration peaking during late fall and winter, an indication that the contribution from sources such as septic effluent and water softeners is low compared to that contributed from deicing. Since substantially decreasing the accumulation of road salt in aquifers and groundwater will likely take decades, the freshwater supply is already damaged. The research team suggests that salinization associated with suburbanization and urbanization should gain attention as a significant threat to the Northeast U.S. freshwater ecosystems.
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ment for Upper-Level Chemistry Laboratories J. Chem. Educ. 2004, 81, 1045–1047. 3. More information on the structure of CA is available. See Ship, Noam J.; Zamble, Deborah B. Analyzing the 3D Structure of Human Carbonic Anhydrase II and Its Mutants Using Deep View and the Protein Data Bank. J. Chem. Educ. 2005, 82, 1805–1808. 4. This Journal has previously published an experiment investigating the binding of sulfonamides to CA. Manalang, Mary G.; Bundy, Hallie F. Azosulfonamides: Preparation and Binding to Carbonic Anhydrase: A Bioorganic Chemistry Experiment. J. Chem. Educ. 1989, 66, 609–610.
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More Information 1. Kaushal, Sujay S.; Groffman, Peter M.; Likens, Gene E.; Belt, Kenneth T.; Stack, William P.; Kelly, Victoria R.; Band, Lawrence E.; Fisher, Gary T. Increased Salinization of Fresh Water in the Northeastern United States. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 13517–13520. 2. Two environmental science courses have been described that measure salinity as part of the educational lab experience. See Adami, Gianpiero. A New Project-Based Lab for Undergraduate Environmental and Analytical Chemistry. J. Chem. Educ. 2006, 83, 253–256; and Lunsford, Suzanne K.; Slattery, William. An Interactive Environmental Science Course for Education Science Majors. J. Chem. Educ. 2006, 83, 233–236. 3. More information on Gene Likens and the Institute of Ecosystem Studies is available online at http://www.ecostudies.org (accessed Jul 2006).
Fuel Cell Enzyme Many microorganisms living under anaerobic or semianaerobic conditions utilize hydrogenases, enzymes with Fe, or FeNi active sites folded inside, as part of their metabolic mechanism. Like the microorganisms that contain them, most hydrogenases are extremely sensitive to the presence of oxygen. However, Ralstonia eutropha (Re) is an example of an aerobic bacterium that has circumvented the susceptibility of hydrogenase to oxygen gas and can gain energy from the oxidation of hydrogen in the presence of oxygen. Re hosts three physiologically distinct hydrogenases, one of which is membrane bound hydrogenase (MBH), which contains a Ni–Fe catalytic center. MBH enables Re to grow with hydrogen gas as its only source of energy, even in the presence of oxygen. Based on its ability to function despite the presence of O2, scientists were prompted to investigate the electrocatalytic potential of MBH, hoping to find a catalyst for use in hydrogen fuel cells that could tolerate both oxygen and carbon monoxide. An international team of researchers, led by Fraser Armstrong of the University of Oxford, obtained Re MBH through the cultivation of cells containing an MBH overproduction plasmid. Re MBH solution was pipetted onto electrode surfaces for protein film voltammetry. Polarizing the electrode at ⫹142 mV, researchers could measure the enzyme activity directly by observing the current generated by hydrogen oxidation. Excluding the background drop due to enzyme dissociation from the electrode, scientists found that the addition of CO to the gas mixture did not shut down the enzyme or even decrease its activity. Cyclic voltammetry confirmed that the introduction of CO at any potential has no effect on the ability of Re MBH to oxidize H2. Experiments in which the partial pressure of oxygen was increased in a stepwise manner demonstrated that O2 did decrease the activity of the enzyme, but activity was quickly regained when the O2 was removed from the fuel cell. It is also important to note that a significant current due to H2 oxidation was observed even at [O2] greater than found in air. This research represents a breakthrough in the search for new catalysts for use with hydrogen-cycle technologies. The tolerance Re MBH demonstrates for CO suggests it could www.JCE.DivCHED.org
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Figure 3. Relationship between impervious surface and mean actual concentration of chloride in streams of the Baltimore LTER site for a 5-year period (R2 = 0.81). Sites are located along a gradient of urbanization. Dashed lines indicate thresholds for damage to some land plants and for chronic toxicity to sensitive freshwater life. Reprinted with permission from Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 13517–13520. Copyright 2005 National Academy of Sciences, U.S.A.
be employed as a catalyst in fuel cells powered by synthesis gas, an abundant fuel containing both hydrogen and CO in copious amounts. Alternatively, Re MBH’s ability to tolerate oxygen suggests a catalyst for an H2/O2 fuel cell in which there is no need for a membrane between the anode and cathode. The research conducted preliminary investigations into developing the latter application, and constructed a simple fuel cell device by soaking graphite strips in dilute enzyme solution and dipping them in a beaker of buffer. Their results demonstrate the feasibility of a “membrane-free” H2/O2 fuel cell and encourage further exploration of this enzyme catalyst, or the development of a synthetic catalyst based on their new insight.
More Information 1. Vincent, Kylie A.; Cracknell, James A.; Lenz, Oliver; Zebger, Ingo; Friedrich, Barbel; Armstrong, Fraser A. Electrocatalytic Hydrogen Oxidation by an Enzyme at High Carbon Monoxide or Oxygen Levels. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 16951–16954. 2. More information on hydrogen fuel cells can be found in previous issues of this journal. See J. Chem. Educ. 1988, 65, 725– 726; J. Chem. Educ. 2004, 81, 1088–1089; J. Chem. Educ. 1988, 65, 272–273. 3. F. A. Armstrong’s research, including his work on bio-fuel cells, is described at http://www.chem.ox.ac.uk/researchguide/ faarmstrong.html (accessed May 2006). 4. The work of collaborator Bärbel Friedrich involving the structure and function of NiFe hydrogenases is online at http:// www.biologie.hu-berlin.de/microbio/ (accessed May 2006).
Angela G. King is Senior Lecturer in Chemistry at Wake Forest University, P.O. Box 7486, Winston-Salem, NC 27109;
[email protected].
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