Gregory L. Baker and Mark E. Alden College of the Academy of the New Church Bryn Athyn, Pennsylvania 19009
Optical Rotation and the DNA Helix-To-Coil Transition
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An undergraduate project
A recent article in this Journnll described a n experil ment which measured the optical density of DNA using a simple ultraviolet photometer. A striking increase in optical density was observed in the 80-100'C temperature range where DNA passes through t h e cooperative helixto-coil transition.* Another method of observing this transition is t h a t of optical rotation. As plane polarized light is passed through the DNA solution, the plane of electric field vibration is rotated by the asymmetric character of .~ this molecular asymthe helical DNA s t ~ c t u r e Because metrv is drasticallv reduced a s t h e DNA undergoes the struc&al transition, optical rotation provides a possible method for estimating the transition temperature. While the experiment itself can b e carried out with a commercial photoelectric polarimeter, the construction of less costly apparatus for the experiment provides a useful laboratory -project for t h e undergraduate chemistry or nhvsics . " student. The e x ~ e r i m e n tto be described required relatively inexpensive equipment and was carried t o c o m leti ion by .a student (MEA) in a one term laboratory course. The Experiment Modern optical rotation experiments generally utilize the ultraviolet range because the effects to be observed are usually larger than in the visible range.' However, the available hardware in our laboratory dictated the use of visible light. A schematic diagram is shown in Figure 1. The intense light source was a projector lamp (750 W at 110 V) cooled by a small fan blade attached to a 3600 rpm motor. The draft from the fan was directed along a flexible dryer vent hose which channelled the cooling air onto the lamp but away from the main section of the experiment. Power for the lamp was obtained by a Variac adjusted to provide optimum intensity. The radiation was filtered to transmit yellowgreen light, thereby reducing the dispersive effects of white light from the lamp.s The main section of the apparatus consisted of the variable temperature polarimeter tube6 sandwiched between two large polarizing plates, one of whieh was free to rotate. The incident light beam was polarized by the first plate, then passed through the sample tube, and the second plate was rotated to achieve a minimum intensity transmission through it. Because of the large diPolarize
Figure 1. A schematic diagram of the apparatus. Except for a small aperture, the polarimeter tube, analyzer, and observer were isolated from any stray radiation.
Figure 2. A graph of the optical rotation as a function of temperature. The data was taken in the direction of increasing temperature.
ameter of the plates, it was possible to distinguish the null to within f0.3', without resorting to photoelectric equipment. The error in the temperature was about f2'C. The sample solution consisted of DNA from salmon testes dissolved in a 0.14 M NaCl solution of pH 7.' While the amount of rotation increases with higher DNA concentration so also does the opacity of the solution. Therefore it is important to achieve a compromise between high concentration and transparency of the DNA solution. In our case the optimum concentration was found to be about 4 g/l. A clear solution was obtained after several days. The Results Data were recorded from 30 to about 105°C in order of ascending temperature; a graph is shown in Figure 2 and may be compared to the results of footnote 1, which are
'Steinert, R., and Hudson, B., J. CHEM. EDUC., 50, 129 119731. ,. ...,.
?Steiner, R. F., "The Chemical Foundations of Molecular Biology," D. Van Nostrand Co., Ine., New York, 1965, p 280.
3Bush, C. A,, "Physical Techniques in Biological Research," 2nd Ed., val 1, (Editor: Oster, G.), Academic Press,' Ine., New York, 1971. p 351. &Adler,A. J., and Fasman, G. D., in "Methods in Enzymalogy," (General Editors: Colowick, S. P., and Kaplan, N. 0.1, val 12. Nucleic Acids. Part B (Editors:Grossman, L., and Moldave, K.), Academic Press. Inc., New York, 1968, p 269. >Thefilter system consisted of No. 4 and 7 Spectro-Radioscopic Filters which provided a yellow-green passband. A sodium source might have been preferable. The filters were originally purchased from Cambridge Botanical Supply Co., Waverly, Mass. 6The polarimeter tube (200 mm in length) was purchased from Arthur H. Thomas Co. of Philadelphia. Polarizing material may be obtained from Edmund Scientific Co., Barringon, N. J. 'The transition temperature has some dependence upon the pH of the containing solution. Cf. Marmur, J., Sehildkraut, C. L., and Daty, P., in "The Molecular Basis of Neaplasia," (Edited by the staff of M. D. Anderson Hospital and Tumor Institute), University of Texas Press. Houston. 1967, p 16. The DNA was purchased from Worthington Biochemical Cow, Freehold, N. J. Volume 57, Number 9, September 1974
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sketched in Figure 3. The optical rotation is relatively constant u p to 7 5 T , then decreases smoothly to essentially zero rotation a t about 100QC,indicating completion of the helix-to-coil transition. If further data are taken using descending temperatures, hysteresis effects are observed which seem to depend on a variety of parameters. These effects are discussed e l s e ~ h e r e . ~ In conclusion, the general trend of the data in Figure 2 bears out the results in footnote 1, thereby indicating that optical rotation can provide an alternative and complementary method for the study of molecular transitions in a student laboratory experiment. 40
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Acknowledgment
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TEMPERATURE
100
1°C)
Fioure 3. A schematic summaw of the optical density results in footnote " 1. The transition mcurs over approximately the same temperature range as is shown in Figure2.
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/ Journal of Chemical Education
It is a pleasure to acknowledge the interest and advice of Dr. Grant R. Doering.
#Cf.footnote 2.
