Chemical Education Today
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Research Advances by Angela G. King
Researchers worldwide are working to advance the broad field of chemistry. Their efforts range from extending the viable storage period of transplant-destined human organs to understanding and improving the quality of what we eat and drink. Organ Preservation Improved by Snow Flea Protein? Scientists in Illinois and Pennsylvania have developed a chemical way to make the antifreeze protein that enables Canadian snow fleas to survive frigid winter temperatures. They have recently described laboratory-produced proteins that could have practical uses in extending the storage life of donor organs and tissues for human transplantation. The so-called “snow flea antifreeze protein (sfAFP)” was discovered in 2005 by Peter Davies and colleagues in Toronto in Hypogastrura harveyi, the Canadian snow flea; they credited it with preventing ice crystals from forming within the snow flea’s body during the harsh arctic winter. sfAFP was shown to be a unique protein, with 45% of its amino acids being glycine. In the recent study, Stephen B. H. Kent and colleagues point out that scientists have tried for years without success to decipher the molecular structure of sfAFP. They were able to use chemical synthesis to make larger amounts of the protein, which exists naturally in only minute quantities in snow fleas. The larger synthetic quantities of the protein, which contains 81 amino-acid residues, were used for further research, including elucidation of the protein structure. The researchers made synthetic sfAFP, and showed that it has the same activity as the natural protein. They also produced variants, including the enantiomeric (mirror image, Figure 1) form (d-sfAFP) of natural sfAFP (l-sfAFP). Both forms of the protein were prepared from four synthetic peptide segments by sequential native chemical ligation. After purification by reverse-phase HPLC, the protein was folded in buffer at 4 °C, and two disulfide bonds formed, as indicated by a mass loss of 4 Da in LCMS data. The d-sfAFP and l-sfAFP proteins displayed an identical but oppositesign CD spectrum (Figure 2). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. “Our most significant advance was the use of the two mirror image forms of the protein to determine the previously unknown crystal structure of this unique protein,” said Kent. “That is a first in the history of protein X-ray crystallography.” The native protein and its enantiomer had identical antifreeze properties due to the achiral binding surface presented by ice. Antifreeze properties were measured using an established ice recrystallization inhibition activity assay (Figure 3). sfAFP has potential application for prolonging the storage of organs for transplantation. In particular, d-sfAFP appears less likely to trigger harmful antibodies and more resistant to destruction by natural enzymes, making it potentially more effective than the native form for use in organ and tissue preservation, the scientists note. 12
More Information 1. Pentelute, Brad L.; Gates, Zachary P.; Dashnau, Jennifer L.; Vanderkooi, Jane M.; Kent, Stephen B. H. Mirror Image Forms of Snow Flea Antifreeze Protein Prepared by Total Chemical Synthesis Have Identical Antifreeze Activities. J. Am. Chem. Soc. 2008, 130, 9702–9707. 2. Pentelute, Brad L.; Gates, Zachary P.; Tereshko, Valentina, Dashnau, Jennifer L.; Vanderkooi, Jane M.; Kossiakoff, Anthony A.; Kent; Stephen B. H. X-ray Structure of Snow Flea Antifreeze Protein Determined by Racemic Crystallization of Synthetic Protein Enantiomers. J. Am. Chem. Soc. 2008, 130, 9695–9701. 3. Research Advances previously covered research involving antifreeze glycoproteins found in arctic fish. See J. Chem. Educ. 2007, 84, 1402. 4. http://chemistry.uchicago.edu/fac/kent.shtml (accessed Oct 2008) provides much information on research in Kent’s laboratories. 5. More details of this research can be found online at http://
Figure 1. By creating an antifreeze protein found in the Canadian snow flea, scientists are reporting a development that could extend the storage life of donor organs. The figure shows representations of the structure of the protein in its mirror image forms prepared by total chemical synthesis. Image courtesy of Brad Pentelute.
Journal of Chemical Education • Vol. 86 No. 1 January 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
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Figure 3. Ice recrystallization inhibition assays at (a) 0 and (b) 4 h. Samples from left to right are (1) 10 mM sodium phosphate, pH 7.5, with 100 mM NaCl; (2) approximately 25 mg/mL AFP I; (3) approximately 25 mg/mL sfAFP, reduced form; and (4) approximately 25 mg/mL sfAFP, oxidized form. Reprinted with permission J. Am. Chem. Soc. 2008, 130, 9702–9707. Copyright 2008 American Chemical Society. Figure 2. CD spectra of the protein enantiomers d- and l-sfAFP. CD spectra were recorded on an Aviv model 202 instrument at room temperature by dissolving 0.03 mg (prepared from a stock solution) of d- or l-sfAFP protein in 300 μL of 50 mM phosphate buffer, pH = 6.9. A 1-mm path length cell was used. Reprinted with permission from J. Am. Chem. Soc. 2008,130, 9702–9707. Copyright 2008 American Chemical Society.
www.spectroscopynow.com/coi/cda/detail.cda?id=19219&type=Featur e&chId=8&page=1 (accessed Oct 2008).
Edible “Antifreeze“ Prevents Unwanted Ice Crystals A scientist in Wisconsin reports development of an edible and tasteless “antifreeze” that prevents the formation of ice crystals that can spoil the smooth, silky texture of ice cream and interfere with the palatability of other frozen foods. In a recent report, Srinivasan Damodaran explains that preventing the formation of large ice crystals is a major challenge for frozen food manufacturers and consumers who store packages in home freezers. Since water is a major component of food products, removing water is not a viable option for preventing the formation of ice crystals. Although several different substances, such as hydrocolloid gums and polysaccharides, have been added to frozen foods to prevent ice crystal growth, none is really effective, the researcher says. Damodaran’s solution is gelatin hydrolysate, a protein known to act as a natural antifreeze. The researcher measured the ability of low molecular weight (2–5 kDa) peptides derived from the papain-mediated hydrolysis of gelatin of the inhibition of ice crystal growth in ice cream mix. In a controlled study using batches of ice cream prepared with and without the non-toxic compound, ice cream containing the antifreeze developed sig-
nificantly smaller and fewer ice crystals than batches prepared without the gelatin peptides or with a higher molecular weight fraction of the hydrolysate added (Figure 4). Molecular modeling of gelatin peptides revealed that the compounds form an oxygen triad plane at the C-terminus that has oxygen–oxygen distances similar to those found in ice crystal nuclei. Damodaran suggests that the peptides inhibit ice crystal growth by the binding of their oxygen triad to the prism face of ice nuclei through hydrogen bonding. More complete understanding of the molecular interactions that inhibit ice crystal growth may aid the design of more effective peptide cryoprotectants. The researchers are also now striving to determine the amino acid sequence of two different peptide fragments from gelatin hydrolysate that have amazing ice crystal inhibiting activity and could potentially be used for organ and tissue preservation. More Information 1. Damodaran, Srinivasan. Inhibition of Ice Crystal Growth in Ice Cream Mix by Gelatin Hydrolysate. J. Agric. Food Chem. 2007, 55, 10918–10923. 2. Ice cream has been used to stimulate student interest in thermodynamics, colligative properties, and bonding. See J. Chem. Educ. 2000, 77, 1392; 1992, 69, 658; 1989, 66, 669 and 1983, 60, 1004. 3. This research is also described online at http://www.sciencedaily.com/videos/2008/0702-edible_antifreeze_saves_ice_cream.htm (accessed Oct 2008).
Thujone: Not the Active Ingredient in Absinthe A new study may end the century-old controversy over what ingredient in absinthe caused the exotic green aperitif ’s supposed mind-altering effects and toxic side-effects when consumed to excess (Figure 5). In the most comprehensive analysis
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Control ice cream mix quick frozen at ź40 pC
Control Ice cream mix after 7 cycles at ź14 to ź12 pC
Ice cream mix with 2% gelatin peptide after 7 cycles at ź1 to ź12 pC
Figure 4. The effect of gelatin hydrolysate peptide fragment on ice crystal growth in ice cream mix. Figure courtesy A. Damodaran.
of old bottles of original absinthe—once quaffed by the likes of van Gogh, Degas, Toulouse-Lautrec, and Picasso—a team of scientists from Europe and the U.S. have concluded the culprit was plain and simple: high alcohol content, rather than thujone, the terpene compound widely believed responsible for absinthe’s effects. Although consumed diluted with water, absinthe contained about 70% alcohol, giving it a 140-proof wallop. Most gin, vodka, and whiskey are 80–100-proof and contain 40–50% alcohol or ethanol. Absinthe use and abuse was widespread in late 19th-century Paris among bohemian artists and writers. Reference to “the Green Fairy” or “the Green Muse” can be seen throughout artwork from this period. The drink’s popularity spread through Europe and to the U.S. However, illness and violent episodes among drinkers gave absinthe the reputation as a dangerous drug, and it was banned in Europe and elsewhere in 1915. In the new study, Dirk W. Lachenmeier and colleagues point out that scientists know very little about the composition of the original absinthe produced in France before that country banned the drink in 1915. Only a single study had analyzed one sample of pre-ban absinthe. Now researchers have analyzed 13 samples of pre-ban absinthe from sealed bottles—“the first time that such a wide ranging analysis of absinthe from the pre-ban era has been attempted,” they say. The analysis included thujone (Figure 6), widely regarded as the “active” ingredient in absinthe. “It is certainly at the root of absinthe’s reputation as being more drug than drink,” according to Lachenmeier. Thujone was blamed for “absinthe madness” and “absinthism”, a collection of symptoms including hallucinations, facial contractions, numbness, and dementia. However, the study found relatively small concentrations of thujone, amounts less than previously estimated and not sufficient to explain absinthism. Thujone levels in pre-ban absinthe actually were about the same as those in modern absinthe, produced since 1988, when the European Union (EU) lifted its ban on absinthe production (Figure 7). Laboratory tests found no other compound that could explain absinthe’s effects, including inconspicuous levels of fenchone, pinocamphone, copper, and antimony. Levels of individual terpenes were noted by use of gas chromatography–mass spectrometry, while atomic absorbance 14
Figure 5. 13 samples of authentic absinthe dating from the pre-ban era (i.e. prior to 1915) were analyzed for parameters that were hypothesized as contributing to the toxicity of the spirit. Adapted with permission from J. Agric. Food Chem. 2008, 56, 3073–3081. Copyright 2008 American Chemical Society.
spectrometry and inductively coupled plasma mass spectrometry were utilized for the determination of copper and antimony levels, respectively. “All things considered, nothing besides ethanol was found in the absinthes that was able to explain the syndrome of absinthism,” according to Lachenmeier. He says that thujone cannot explain pre-ban absinthe’s reputation as a psychotropic substance. Recent historical research on absinthism concluded that the condition probably was alcoholism, Lachenmeier indicates. “Today it seems a substantial minority of consumers want these myths to be true, even if there is no empirical evidence that they are,” says Lachenmeier. “It is hoped that this paper will go some way to refute at least the first of these myths, conclusively demonstrating that the thujone content of a representative selection of preban absinthe… fell within the modern EU limit.” More Information 1. Lachenmeier, Dirk W.; Nathan-Maister, David; Breaux, Theodore A.; Sohnius, EvaMaria; Schoeberl, Kerstin; Kuballa, Thomas. Chemical Composition of Vintage Preban Absinthe with Special Reference to Thujone, Fenchone, Pinocamphone, Methanol, Copper, and Antimony Concentrations. J. Agric. Food Chem. 2008, 56, 3073–3081. Available online (ACS Author Choice Free Access): http://pubs.
O
H Figure 6. Structure of the terpene thujone. Structure by A. King.
Journal of Chemical Education • Vol. 86 No. 1 January 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
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http://www.jce.divched.org/Journal/Issues/2009/Jan/abs12.html Abstract and keywords Full text (PDF) with links to cited URLs and JCE articles
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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Total thujone [mg/l]
acs.org/cgi-bin/sample.cgi/jafcau/2008/56/i09/pdf/jf703568f.pdf (accessed Oct 2008). 2. The Virtual Absinthe Museum, the most comprehensive site on absinthe available on the Internet, offers more information on all aspects of absinthe production and prohibition; it can be found online at http://www.oxygenee.com/ (accessed Oct 2008). 3. More chemistry and details of another disproven theory regarding santonin in absinthe can be found at J. Chem. Educ. 1991, 68, 27. 4. Interactive 3D modeling of thujone is available online at http:// www.3dchem.com/molecules.asp?ID=142# (accessed Oct 2008).
35 30 25 20 15 10 5 0
Preban absinthe (n=13)
Modern absinthe (n=9)
Figure 7. Comparison between total thujone content in pre-ban and modern absinthe. The columns show the average contents with standard deviation as error bars. Image courtesy D. Lachenmeier.
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