LIGHT ABSORPTION 



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Temperature 



Fig. 24. A sketch of the optical density (O.D.) of a DNA solution as the 

 temperature is slowly raised from room temperature to about 100°C. T m is the 

 melting temperature, defined as the temperature at which half the O.D. change 

 is found. The increase in O.D. is known as the hyperchromic effect. 



drogen bonds, the strands will be held together. It is possible to raise the 

 temperature so high that all the bonds will be broken at once. If, at this 

 temperature, the solution is suddenly chilled (by placing the tube of 

 DNA solution in an ice bath), the chances are greater that the individual 

 strands will fold up and make internal hydrogen bonds than that they 

 will have time to find the complementary strands to re-form the DNA. 

 This is sketched in Fig. 23. 



If the light absorption depends on the chemical state of the substance, 

 it would be possible for the solution to absorb different amounts of light 

 in each of the situations shown in the figure. In fact, when DNA is 

 "melted" there is a 40% increase in light absorption at the peak absorp- 

 tion wavelength (260 mfi) for nucleic acids. Figure 24 shows the light 

 absorption of a DNA solution at various temperatures. The DNA is said 

 to have a melting temperature T m at the temperature halfway between 

 the native and melted (or denatured) states. 



It turns out that the melting temperature T m is quite characteristic of 

 the DNA of various organisms and that related organisms have melting 

 temperatures which are very close to each other, so that this light absorp- 

 tion measurement has phylogenetic significance. The increase of light 

 absorption is known as the hyperchromic effect, and is useful in a number 

 of experimental situations. Since RNA and single-stranded DNA have 

 very small hyperchromic effects, it is possible to deduce their presence or 

 absence by light absorption measurements. 



(b) Matching of refractive indices. Light is bent in direction when it 

 goes from one medium to another of different optical properties (you 

 know this from the classical case of long objects appearing broken when 

 half in and half out of water). The light-bending property of a substance 

 is known technically as its refractive index. The contents of a cell will 



