Chemical Education Today
Reports from Other Journals
Research Advances by Angela G. King
New Way To Detect Destructive Enzyme Activity Scientists at the Pacific Northwest National Laboratory in Richland, Washington are reporting discovery of a muchneeded new method to identify the activity of destructive enzymes. These enzymes have been linked to a range of diseases caused by their degradation of extracellular matrix components and that serve as biomarkers for infection and inflammation. The enzymes belong to the matrix metalloproteinase (MMP) family. MMPs break down protein and have a metal such as zinc or calcium in their structure. “MMPs have been implicated in a variety of disease states, including arthritis, periodontal disease, and tumor cell invasion and metastasis,” Yuehe Lin and colleagues note. With MMPs offering an opportunity for diagnosis and monitoring the effectiveness of treatment, several tests for the enzymes have been developed including enzyme-linked immunoassay, Western blotting, gel electrophoresis, and radiolabeling. Those tests, however, have drawbacks, including inability to identify specific MMPs that are active in specific diseases or require complicated detection schemes. In a recent advance, researchers describe the new test as simple and sensitive. It involves use of an electrochemical proteolytic beacon (EPB) that signals “on” or “off ” when MMPs are active in a sample of tissue or body fluid. The beacons can be configured to signal when specific MMPs are active, including enzymes associated with certain infectious diseases. The test is based on previously documented electrontransfer properties of helix peptides. For the new test, ferrocene (FC) is used as the cleavage-sensing element or electroactive reporter used to label a specific sequence in an electrode-attached helix peptide. If the molecular assay senses cleavage in the presence of MMP, the EPB separates from the electrode surface, significantly decreasing the signal. To assemble the molecular device, FC is attached to the peptide through the terminal amino group of the helix peptide and researchers can synthesize helix peptides known to be active with a specific MMP, such as MMP-7. The EPB is prepared by self-assembly of a FC-peptide monolayer on a gold electrode surface. The research team found that the designed EPB is both specific for the designated MMP (for example MMP7 and not MMP-2 or MMP-3) as indicated by a drop in signal amplitude. The percentage decrease in signal is dependent on the log of [MMP] injected. There was no decrease in signal if the system was treated with an inert protein (bovine serum albumin) in place of the MMP. This breakthrough presents a simple and sensitive method for detecting MMP activity by converting peptide cleavage events into an electronic signal. By exploring multiple helix peptides and electroactive reporters, it may be possible to test for multiple MMP activities simultaneously. This basic research may soon make the identification of infectious agents and quantification of enzyme activity much easier.
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Figure 1. Schematic of EPB for detection of matrix metalloproteinase. (A) self-assembling electrochemical FC-peptide conjugate on the gold substrate; (B) cleavage of EPB in the presence of MMP-7. Reprinted with permission from J. Am. Chem. Soc. 2006, 128, 12382–12383. Copyright 2006 American Chemical Society.
More Information 1. Liu, Guodong; Wang, Jun; Wunschel, David S.; Lin, Yuehe. Electrochemical Proteolytic Beacon for Detection of Matrix Metalloproteinase Activities. J. Am. Chem. Soc. 2006, 128, 12382– 12383. 2. An undergraduate experiment involving cyclic voltammetry and ferrocene has been published in this Journal. See Gomez, Marielle E.; Kaifer, Angel E. Voltammetric Behavior of a Ferrocene Derivative: A Comparison Using Surface-Confined and DiffusionControlled Species. J. Chem. Educ. 1992, 69, 502. 3. Additional information on Lin’s research can be found online at http://emslbios.pnl.gov/id/lin_y (accessed Jan 2007).
Ancient Hair Dye Based on Modern Nanotechnology A hair dye developed 2,000 years ago relied on nanotechnology to change the graying hair of people in ancient Greece and Rome into a youthful black color, scientists in France report. Philippe Walter and colleagues studied a hair-dyeing recipe first described in Greco-Roman times, which is the basis of modern hair dyes that gradually darken gray or white hair. The recipe, which is amazingly similar to those used by medieval Arabian authors and throughout the Renaissance, uses only three ingredients. Lead oxide (PbO) and slaked lime (Ca(OH)2) are mixed with water to form a paste that is applied to hair strands. Over time the paste will darken hair to a black color. Scientists have previously determined that the hair darkens because sulfur atoms in hair keratin proteins react with the lead oxide to form a dark PbS precipitate known
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Chemical Education Today
Reports from Other Journals This is hypothesized that cystine degradation releases sulfur, which is then used to form the galena precipitate. The lead sulfide crystals look much like the lead sulfide quantum dots synthesized recently using techniques from materials science, they state. “In contrast to modern nanotechnology, the dyeing process is characterized by basic chemistry methods and has been developed more than 2,000 years ago with low-cost natural products,” the scientists report.
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Figure 2. Optical macrophotographs of hair (A–C) and microphotographs (D–F) of the corresponding transverse cross sections, showing progressive blackening during treatment with lime and lead oxide in water (25 oC, pH = 12.5). (A and D) nontreated, (B and E) 6 h, (C and F) 72 h. (G) time dependence of total adsorbed lead concentration in the bulk sample measured by X-ray fluorescence spectroscopy. Pb maps of the respective treated samples are obtained by SEM-EDX, (H) 6 h, (I) 72 h, and show a progressive radial fixation from the cuticle to the center of the hair (concentration increasing from dark blue to green). Reprinted with permission from Nano Lett. 2006, 6, 2215–2219. Copyright 2006 American Chemical Society.
as galena. The French team of scientists recently found that the dye works by causing formation of nanocrystals of lead sulfide. That chemical compound forms inside hair shafts and colors hair black without damaging the hair. X-ray microdiffraction analysis of hair dyed for three days with a solution made with equal masses of Ca(OH)2 and PbO in ultrapure water showed that galena nanocrystals with an average size of 4.8 nm had formed. The analysis also revealed that the ␣-helical keratin structures were preserved during the dying process. The 3-D distribution of the Pb in hair was determined by scanning confocal electron microscopy (SCEM) of hair cross sections and high-resolution transmission electron microscopy (HRTEM) of longitudinal sections of hair. Pb accumulations are observed in the cuticle and cortex, in globular lipid aggregates (200 nm) and membrane complex located between macrofibrils. Researchers attributed this to interactions between lead ions with lipid components of the hair. On the contrary, distinct nanocrystals of PbS accumulated only within the macrofibril. The nanocrystals were linearly arranged along the axis of the hair about 8–10 nm apart. The researchers noted that when compared to hair samples treated with slaked lime only or PbO only, during the dying process, the amount of cystine decreases while the amount of lanthionine and lysino-alanine residues increase.
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1. Walter, Philippe; Welcomme, Eleonore; Hallegot, Philippe; Zaluzec, Nestor J.; Deeb, Christopher; Castaing, Jacques; Veyssiere, Patrick; Breniaux, Rene; Leveque, Jean-Luc; Tsoucaris, Georges. Early Use of PbS Nanotechnology for an Ancient Hair Dyeing Formula. Nano Lett. 2006, 6, 2215–2219. 2. An undergraduate lab using the mineral galena as a source of lead ions is available. See Nechamkin, Howard; Dumas, Philip. J. Chem. Educ. 1978, 55, 601. 3. Safety information on lead oxide is available in this Journal. See Young, Jay A. Lead(II) Oxide. J. Chem. Educ. 2006, 83, 1457. 4. Additional information on historical and ancient hair dye chemistry can be found at http://www.madehow.com/Volume-3/HairDye.html (accessed Jan 2007) and Food Chem Toxicol. 2004, 42, 517–543.
Squeezing More Shelf Life Out of Milk Putting the squeeze on milk may be a long-sought solution to the search for improved ways of killing harmful bacteria in milk and increasing its shelf life without introducing off-flavors into the beverage, researchers report. Most milk sold in the U.S. has been subjected to high-temperature– short-time (HTST) pasteurization to kill microorganisms that could be harmful. Milk treated in this manner can last for almost three weeks if refrigerated. For a long time a search has been on for a treatment method that would allow consumers to keep milk fresh at room temperature or extend the refrigerated shelf-life. An existing technique, ultrahigh-temperature pasteurization (UHT), does produce milk that stays fresh at room temperature for six months. However, some say that UHT leaves a “cooked” flavor in milk that has limited the popularity of UHT milk in the U.S. Previous work has attributed the “cooked” aroma to volatile sulfur compounds, aldehydes, and methyl ketones and scientists can quantify the levels of these compounds in milk samples. Through recent experiments, a team of scientists at Oregon State University led by Michael C. Qian and Antonio Torres learned how a new food processing technology affects the taste of milk. Called high hydrostatic pressure processing (HPP), it involves putting foods under pressures that crush and kill bacteria while leaving food with a fresh, uncooked taste. Qian’s team investigated the generation of volatile compounds under HPP and compared the results to volatile generation under atmospheric pressure. Headspace solid-phase microextraction and gas chromatography were used to identify components of milk samples.
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Chemical Education Today
Reports from Other Journals Photo: Michael Qian
Results demonstrated that HPP generates minimal change in the volatile profile of milk samples; however the high pressure does favor the formation of aldehydes over methyl ketones and hydrogen sulfide over methanethiol. “Milk processed at a pressure of about 85,000 pounds per square inch for five minutes, and lower temperatures than used in commercial pasteurization, causes minimal production of chemical compounds responsible for the cooked flavor. HPP gives milk a shelf life at refrigerated temperature of at least 45 days,” they note. HPP does affect some properties of milk. It can reduce the size of casein micelles, which increases milk viscosity while decreasing its whiteness. It can also shift the phase transition temperature, which in turn affects the crystallization properties of milk fat. There’s still more work to be done before scientists can fully understand the potential of HPP. Scientists are currently trying to unravel how reaction kinetics impact the development of volatile compounds during different processing methods.
Figure 3. This HPP unit in a research pilot plant uses high pressure to give milk a refrigerated shelf life of 45 days without much impact on taste.
More Information 1. Vazquez-Landaverde, Pedro A.; Torres, J. Antonio; Qian, Michael C. Effect of High-Pressure-Moderate-Temperature Processing on the Volatile Profile of Milk. J. Agric. and Food Chem. 2006, 54, 9184–9192. 2. This Journal has published numerous articles on milk as it relates to teaching chemistry. See J. Chem. Educ. 1993, 70, 480; 2003, 80, 762; 1998, 75, 1618; 1996, 73, 848; and 1973, 50, 136 for examples. 3. Michael Qian’s research Web page is available at http:// oregonstate.edu/dept/foodsci/faculty/mcq.htm and an online report of this
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research project can be found at http://extension.oregonstate.edu/news/ story.php?S_No=428&storyType=news (both sites accessed Jan 2007). 4. Qian gives an interview relating interesting aspects of working on food chemistry at http://curdnerds.com/node/ 181?PHPSESSID=2dc5d0abc629fcf8a4930701f368a44c (accessed Jan 2007).
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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