The close-packed nature of enzymes and enzyme-substrate

Dec 1, 1976 - ... nature of enzymes and enzyme-substrate complexes. Andrew Williams. J. Chem. Educ. , 1976, 53 (12), p 778. DOI: 10.1021/ed053p778.3...
0 downloads 0 Views 2MB Size
TLC Monitoring of Triglyceride Saponification R. A. Heller Wilfrid Laurier University Waterloo, Ontario, Canada N2L 3C5 The saponification of triglycerides is a common student experiment which may be used to achieve a variety of lahoratory teaching goals. A modification which we have found verv successful is to monitor the reaction usine a tlcmethod adapted from an Eastman Chromatogram procedure.' By taking samples from the saponification mixture a t appropriate times and analyzing them by tlc, the student is able to observe the formation of mono- and dielvceride intermediates in the conversion of triglycerides to fatty acids. Ten grams of suitable fat or oil (butter, olive oil, etc.) are added to 40 ml of refluxing 7% methanolic KOH. Samples (2 ml each) are removed at 5,10, and 30 min. Each of the samples is acidified and extracted with ether. The ether extracts are washed with saturated NaCl solution and dried over anhydrous MgSOa. Small portions of the resulting solution are applied to the base line of an oven-dried Silica Gel tlc plate, together with a sample of the starting material and appropriate standards. The tlc is then developed in hexanelethyl etherlacetic acid, 80:20:1, visualized by spraying with 0.2% ethanolic 2',7"-dichlorofluoroscein, and examined under uv light. With care, the results clearly show the expected pattern. The fatloil sample shows a single spot corresponding to the triglyceride standard (Rf = 0.55). The five-minute sample shows triglyceride, appreciable amounts of 13-and 1,3-diglycerides (R, = 0.20.0.25) free fatty acid (Rl = 0.30) plus small amounts of monoglyceride (Rf = 0.05). At 10 min, little or no triglyceride is seen, while the monoglyceride, diglyceride, and fatty acid quantities are large. At 30 min, only fatty acid is present. '"Separation of Glycerides", Analytical Procedure, Eastman Chromatogram Systems, Eastman Kodak Co., Rochester, N.Y. 14603.

Linear Combinations of Ligand Orbitals for a Tetrahedral Molecule Thomas C. W. Mak and Wai-Kee Li The Chinese Uniuersity of Hong Kong Shatin, N.T., Hong Kong The construction of symmetry-adapted linear combinations (SALC's) of the ligand orbitals for AL, molecules is by now a standard topic in a physical or inorganic chemistry course which includes molecular orbital theory in its syllabus. When 778 /

Journal of Chemical Education

the system is relatively simple, e.g., small n value or only a bonding is considered, the pictorial approach described in Gray's book* is often preferred. On the other hand, for the more complicated cases, the technique of projection operatom2 is kmployed instead. Among the various symmetries for cases with n Q 6, the construction of the SALC's for a tetrahedral molecule, including T bonding, is the only one that poses any serious technical problems. Indeed, even though the forms of the SALC's are commonly found in the l i t e r a t ~ r ethe , ~ way of obtaining them is not readily available. In a supplement tothis note, the construction of the SALC's for a molecule with Td symmetry is shown in a step-by-step manner. This is not only a problem on molecular orbital theory, but it is also an exercise on rudimentary group theory and vector algebra as well. The supplement may he obtained from either one of the authors. Gray, H. B., "Electrons and Chemical Bonding," Benjamin, New Yark, 1965. Cotton,F.A,, "Chemical Applications of Group Theory," 2nd ed., WiIey-Interscience, New York, 1911. See, for example, Ballhausen, C. J., and Gray, H. B., "Molecular Orbital Theory," Benjamin, New York, 1965.

The Close-Packed Nature of Enzymes and Enzyme-Substrate Complexes Andrew Williams University Chemical Laboratories Canterbury, Kent, England The best way of illustrating the close-packed rigid nature of enzymes and globular proteins is to view a space-filling model constructed using X-ray crystallographic coordinates. Failing this, the use of stereoscopic illustrations is helpful; non-stereoscopic pictures have some merit and have been reported from time to time. The close packing of globular proteins can he demonstrated without recourse to photographs or to the construction of expensive space-filling models. Sufficient Styrofoam balls (costing only a few pennies) are stacked in a face-centered close-packing arrangement and glued to provide a surface capable of supporting four or five connected vacancies. The cleft thus produced is suitable to accommodate a substrate made from similar but colored balls linked bv metal oins. Rotational isomerization of the substrate is possible about the a ins hut in the cleft the molecule is held in a defined conformation so that reactive groups are in appropriate locations to

react with complementary centers on the substrate. The model will also demonstrate the termolecular complex and, if vacancies are two layers thick, the compulsory order often found in the formation of a ternary complex; it is clearly seen how the binding of the "second" substrate will block that of the "first". The model also provides a simple demonstration of the conversion of binding energy into "strain" energy. When the active-site cleft has a shape or size not precisely complementary to that of the substrate the non-directional forces may caused the substrate to bind but in so doing become strained by the steric hindrance in the cleft and activated. Provided the model is composed of sufficient Styrofoam balls i t mav easilv be seen how the close-packing prevents entry of substrate molecules into the inteGor of inenzyme; the reason for active-sites as clefts a t the surface of enzymes then becomes self evident. The model is rigid as is the enzyme by virtue of its close-packing and the interatom bonds: in the former case glue and in the latter disulfide and peptide links. A detailed account of close-packing in enzymes and its consequences is available from the author.

Acetic Acld In Toluene: A Safer Conductivity Experiment John W. Hill University of Wisconsin River Falls, Wisconsin 54022 Many beginning laboratory courses involve the testing of a solution of dry hydrogen chloride in benzene for electrical conductivity. Distilled water is added, and the solution is tested again. The experiment can be faulted on two counts: (1)The benzene-HC1solution is difficult to prepare, store, and handle, and (2) benzene is quite toxic. A safer system involves glacial acetic acid and toluene. Neither reagent conducts electricity when dry, nor does amixture of the two. Addition of distilled water gives a two-layer solution. The lower (aqueous) layer readily conducts electricity. This svstem illustrates the same principles as the benz e n e - ~ ~ l s o l u t i oItn has . the advantages thatareticacid in lens irritatinz and easier u, handle than gaseous hydrogen chloride and that toluene is considerably leis toxic than benzene.

Volum 53, Number 12, December 1976 / 779