Lecture Demonstrations in Kinetics
Dennis M. Pederson Colifornio Stote College Son Bernordino, 92407
Relevant to the Biology Student
A large number of students taking chemistry courses today have as their goal a career in biologically related fields. It is thus not surprising that one often hears the question: Why do I need to know this? This is especially common in an area like chemical kinetics, where the reactions examined usually involve simple gaseous molecules while those of much more interest to the biolom -- student are often ignored or only given cursory treatment. Examination of the kinetics of enzvme-catalvzed reactions allows one to illustrate several basic kinetic principles while injecting some relevancy for the biology student. For this purpose I have developed a set of convenient and rapid lecture demonstrations using the enzyme 8-galactosidase and o-nitrophenyl-8-D-galactopyranoside (ONPG).
ONPC
Gslactoae
Examples of the use of enzyme kinetics in chemistry have appeared previously in this Journal but these seem to be much better suited for use as laboratory experiments.l.2.3 The system described here is ideal for a lecture demonstration. It is easily set up, one of the reaction products is colored, and all of the usual kinetic principles can he illustrated in a very short period of time. Experimental The following are required: a standard pH 7.0 buffer, 0.1 M soM solution of dium phosphate and M MgCI.; a 2.5 x ONPG in the standard buffer; a solution of 8-galactosidase in the standard buffer that yields a change in absorbance of 0.2-0.4 when assayed at 25'C; a Bausch and Lomb Spectronie 20 or similar soectmobotometer: and Dewar flasks for temoerature control.' Enzyme activity is assayed by pipetting 5.0 ml of the standard buffer into a test tube, allowing the solution to equilibrate at the Effect of Temperature and Mercury Compounds on the Enzyme Catalyzed Hydrolysis of ONPG .. . -. .-
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desired temperature for 2-3 min, and adding 0.1 ml of the enzyme solution followed by 1.0 ml of the ONPG solution. If several assaw are to be run. the ONPG solution mav be added to each tube at 30-s intervals. After exactly 4 min have elapsed following addition of the ONPG, 2.0 ml of 1 M NazCOa is added. The absorbance of the solution is then measured at 420 nm. If a suitable spectrophotometer is unavailable, most of the concepts may be illustrated qualitatively by comparison of the color intensities. Results Some typical results illustrating the effect of temperature and mercury compounds are given in the table. Since at these concentrations the absorbance change is directly proportional to the rate constant, a plot of log A420 versus 1 / T will yield the activation enerm for this reaction, apprbximately 10 kcal/mole. As is the-case for a number of other reactions, an increase of 10°C leads to an approximate doubling of the rate. The data also illustrate that unlike most reactions, a continued increase in temperature does not result in a continued increase in rate. This effect is due to denaturation of the enzyme-a process that begins a t approximately 45°C. The results obtained by adding mercury compounds illustrate very nicely an effect of mercury pollutants on biological systems. Other ideas easily demonstrated with this system include: the rate of a reaction with and without a catalyst (enzyme) present; the order of the reaction with respect to an enzyme (add 0.05, 0.1, 0.2 ml of enzyme solution); stereospecificity of a catalyst (use o-nitrophenyl-a-Dgalactopyranoside4); saturation of the active sites of a catM and 5 X M alvst bv substrate (use 2.5 X O N P G ~ ;and the dependence of enzyme activity (add 2 ml of 1 M HCI or 1M NalCOx . .before addine These demonstrations have been perf&med in our freshman chemistry class and for visiting high school science students with a very favorable response from both groups. 'Miller, J. F., and Cory, J. G., J . CHEM. EDUC., 48, 475 ,L"..,. ZFried,R., and Howse, M., J. CHEM. EDUC., 48,847 (1971). 3Hurlbut, J. A., Ball, T. N., Pound, H. C., and Graves, J. L., d. CHEM. EDUC.. 60.149 11973). 11071b
Mo. 5Data for these demonstrations is available fmm the author an retruest.
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Journal of Chemical Education
