W. Gale Rhodes,' William H. Flurkey, and Ruth M. Shipley Maryville College Maryville, Tennessee 37801
Thermal Proteinoids A project in molecular evolution for the undergraduate biochemistry laboratory
Biochemistry of the Cell, a n introductory undergraduate biochemistry course a t Maryville College is teamtaught by members of the biology and chemistry departments.2 Emphasis is placed upon the understanding of concepts and the development of hypothetical models in cell chemistry rather than upon memorization of structures, pathways, and processes. Discussion of biochemical phenomena includes a n in-depth view of cell and organelle morphology as it is related to chemical structure and function. Tests are 24-hour, open-book, requiring the ahility to discuss concepts, solve problems, and use the texts and reading materials as effective reference sources. We have included in our course a section on molecular evolution. Progress in this area is reviewed in the final chapter of A. L. Lehninger's excellent textbook ( I ) and other assigned readings (2-4). Among the topics discussed are abiotic production of amino acids, carbohydrates, purines, pyrimidines, and porphyrins; pmteinoids and pmteinoid microspheres; and the construction of phylogenetic trees via sequence comparison of proteins from various species. his-is an excelrent concluding section because it ties together a large number of topics from the earlier portions of the course. We also considered i t desirable to offer the opportunity for some related experimental work in the laboratory set. tion of the course, which operates in the following way earh three-week period of the course, each studenl chooses an experimental project, rarries i t out, and suhmits n laboratory report A numher of open-ended projects are offered by the instructors: furthermore, students may propose their own projects by consulting the literature. Projects are designed so that, in addition to providing exposure to laboratory work in a given area of biochemistry, they also provide experience in a wide range of common biochemical techniques. 2) A student may elect to extend a project to six or nine weeks for two or three lab credits by conceiving, proposing, and carrying out additional work related to the original project. 1 ) Within
The project that we have developed involves the preparation of thermal polyanhydroaminoacids (proteinoids) according to the methods of Fox and Harada (5) and the measurement of esterase activity towards p-nitrophenyl acetate (NPA), according to Rohlfing and Fox (6). To simplify the handling of kinetic data;the rate of proteinoid-catalyzed release of p-nitrophenol is compared to the rate of release of p-nitrophenol catalyzed by a n equal weight of chymotrypsin. Experimental Preparation of proteinoids Proteinoids are prepared from reactants consisting of 2 Parts by weight, each, of aspartic and glutamic acids, 1 Part of an equimolar mixture of the 16 common neutral and hasic amino acids (51, and varying proportions of histidine hydrochloride hydrate. In a typical experiment, 10 g glutamic acid in a 250-ml filter at 170-1800C until flask is heated in an oil I To whom inquiries should be addressed. ZDr. RObert Ramger, Department of Biology; Dr. Gale Rhodes, Department of Chemistry.
molten. Ten grams of aspartic acid, 5 g of equimolar mixture of neutral and basic amino acids, and the appropriate amount of histidine are added. The resulting mixture is stirred with a glass rcd and maintained at 170"C, under nitrogen, for 3 hr. After cooling, the bmwn product is dissolved in 10% aqueous sodium hicarbonate (foaming occurs, so the bicarbonate must be added slowly). Aliquots of this mixture ere subjected to the ninhydrin and biuret tests (7). The mixture is then dialyzed against distilled water 3-5 da, changing the water every4-6 hr. The liquid is then removed from the dialysis tubes, avoiding any insoluble material. Biuret and ninhydrin tests are repeated. The resulting solution of soluble, nandiffusible proteinoids is evaporated to dryness at 50°C in uocuo. Dry proteinoids are removed and ground to a fine powder with mortar and pestle. Assay of Esterase Activity (6) Assays are carried out in 0.067 M phosphate buffer, pH 6.2. The buffer is made 2% in acetone and 2 X IW3 M in p-nitrophenyl acetate (Eastman) just prior to the assay by dissolving the required amount of NPA in an appropriate quantity of acetone and adding the mixture to the buffer (NPA dissolves rapidly in acetone but very slowly in the buffer). To a 10-ml aliquot of buffered NPA solution is added 4 mg of proteinoid and the increase in absorbance at 4W nm is measured with buffered NPA solution (without proteinoid) as a reference. An identical assay is carried out using 4 mg of a-chymotrypsin (39 crystalline, Nutritional Biochemicals Corp.) instead of the proteinoid. Comparison is made of the initial rate of ahsorhance change in the two assays. Catalvtic activitv measured bv our students has been in generalggreement'with the work of Rohlfing and Fox (6); with highest activities found when reactant mixtures contained 8.0 wt % histidine, expressed a s the free base. Activity ievels for dialyzed proteinoids are in the range of 3-5% of that of an equal weight of chymotrypsin. This project fits well into our laboratory scheme and can he readily adapted to the ground rules for other lahoratories. First, in addition to providing experimental exposure to the subject of molecular evolution, the project demands utilization of a variety of methods, including the ninhydrin and hiuret tests, dialysis, preparation and use of phosphate buffers, and spectrophotometric measurement of reaction rates. Second, the project offers numerous possibilities for extended studv. Students can carrv out estimation of proteinoid mole'ular weight bv use (if 2,a-dinitrofluumbenzene 61;frartionarion of proreinoids by Sephadex chromatography, and isolation of active fract i o n ( ~ ) preparation ; and photography of proteinoid microspheres (9); determination of dependence of catalvtic activity on tertiary structure by denaturation of proieinoids (10); detection of other catalytic activities such as hydrolysis of phosphates, decarhoxylation, and deamination (11); more sophisticated analysis of the kinetics of the esterase activity; preparation and study of proteinoids from a wide variety of starting mixtures and many other exper. iments (12, 13). In preparation of the lab report, the student is encouraged to search the literature for the contrasting points of view concerning possible roles of proteinoids in prehiological evolution. Fox (14) has presented a n excellent summary of the proteinoid theory. Relevant papers plus many other discussions of subjects related to Volume 52. Number 3, March 1975
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molecular evolution can be found in references (I, 12-13, 15, 16). Finally, in a paper which describes proteinoid experiments for a n undermaduate biolom course. Veeotskv bas pointed out that such experiments-are relevantto ;wide variety of courses in the undergraduate biology and chemistry curricula, including general biology, genetics, microbiology, cell biology, physiology, organic chemistry, and biochemistry. As he states The subject of the prebiological or ahiotic synthesis of living matter has a great appeal to all questioning minds. At the same time, this topic is a central theme of biology, essential for comprehension of the basic nature of life and its early evolution. Thus, it is not surprising that students find life's origin an absorbing subject and that authors of textbooks in many areas of biology frequently include at least some consideration of this matter (12). This effort was supported by the Division of Chemical Educatiou-DuPont Small Grants Program.
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LIerature Cited (1) lahniwer. A. L., '"Biochemiafry," Worth Publishers, Ine., New Yark, 1910. p. 768. (21 Dickarsan, R.E., Scientific American. 216, (41.58 (1972). Noturmissemchaftm, 59.1(1969). (3) Fax, S. W., (41 Wald, G..R o c . Nan. Amd. Sci, 52.595lI9Ml. (51 For. S. W.,and Harads. K..J Amer Chem. Soe.. 82.3747 (1960). (61 Rohlfing, D.L.,and For, S. W.,Areh. Bioehem. Biophys., 118.122 (1967). (7) Clark. J. M.. 'lwerimenfal Biochcmistw." W. H. Freeman and Comrrenv. San ~
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'(81 Hands. K.,and$& S.W.,Amh. Biaekm. Blophya., 86,274 (1960). (91 Hsu, L. L., Bmke, S., andFoi, S. W., Cvr~nrsinModernBlolog..4, 12(1971l. (101 Roh1fing.D. L.,and Fm,S.W.,ArehBioehm. Biophys, 118, ln(19671. (111 Dose, K.,and Zaki, L., in "Molecular Evolution: Chemical Evalution and the Ou. sin of Life." IEdifora: Ponnamaruma. C.. and Buvet., R.1. ., American Elwvicr hbliahing ~ o ~ p mhe.. y . New Vmk, 1971. p. 263. 112) Vs%nsky, Allen, in "Molecular Evolution: F'rebiolmical and Biolorieal." (Editors:
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(141 For, S.W.,N o t u r m i s s a & h ~ l s ~60.339 (1973). 1151 For, S. W. (Editor), "The Origin of Ptebiologiesl Systems and of their Molcculru Matzices."Acadomic PIDPB,New York, 1965. 1161 Calvin. Melvin. "Chemical Evolution: Molecular Evolutbn Towuda the Origin of Living Syatoms on the Earth and Elsewhere." Oxford Uniueraity Pres. New York, 1969.
