56 LIGHT ABSORPTION EFFECTS 



r* 



DNA solution heated 

 to some temperature 

 lower than denaturation 

 temperature 



DNA solution heated 

 above denaturation 

 temperature to produce 

 strand separation. This 

 is called melted DNA 



Melted DNA after 

 sudden chilling 



Fig. 23. A schematic representation of the partial and complete separation 

 of DNA strands by breaking some or all of the hydrogen bonds. If the com- 

 pletely separated strands are suddenly chilled, the strands form intra-strand 

 bonds before any inter-strand bonds can be formed. The opposite would have 

 been the case if the solution had been chilled slowly. 



It is worth pointing out that machines which measure light absorption 

 — colorimeters, spectrophotometers, etc. — express the result in terms of 

 what is called the optical density, which is defined as 



O.D. = log I /I x , 



since this equals E m cx and is therefore directly proportional to the con- 

 centration of molecules which absorb, which is the quantity usually being 

 sought experimentally. 



This expression has been obtained for a single wavelength; the optical 

 density will vary with wavelength if E or E m varies with wavelength. 

 By measuring the O.D. at various wavelengths for a given setup (keeping 

 c and x constant), we can determine the dependence of E on wavelength. 

 The plot of E versus wavelength is the intrinsic absorption spectrum of 

 the molecule. 



The absorption and scattering of light by a substance can depend very 

 strongly on its state of chemical combination and on the light-scattering 

 properties of the solvent in which it is suspended. We can see this from 

 an examination of two experimental situations which have proved of 

 interest and significance to biology. 



(a) The hyperchromia effect. DNA is a long double-stranded molecule, 

 held together by hydrogen bonds along the length of the molecule. As a 

 solution of DNA is heated, the thermal agitation becomes great enough 

 to break more and more of these hydrogen bonds. At any temperature, 

 there will be equilibrium between the breakage of these bonds and their 

 re-formation, so that at any time when there are still some intact hy- 



