Structural Biochemistry/The Hypochromic Effect

The course of denaturation of DNA can be followed spectrophotometrically because the relative absorbance of UV by DNA solutions at 260 nm increases as much as 40% as the bases unstack. This absorbance increase is called hyperchromic shift

Definition

Difference in absorbance of ultraviolet light between single stranded and double stranded DNA

Hypochromicity describes a material’s decreasing ability to absorb light. Hyperchromicity is the material’s increasing ability to absorb light.

The Hypochromic Effect describes the decrease in the absorbance of ultraviolet light in a double stranded DNA compared to its single stranded counterpart. Compared to a single stranded DNA, a double stranded DNA consists of stacked bases that contribute to the stability and the hypochromicity of the DNA.

When a double stranded DNA is denatured, the stacked bases break apart and thus becomes less stable. It also absorbs more ultraviolet light since the bases no longer forms hydrogens bonds and therefore are free to absorb light. Ways to denature DNA include high temperature, addition of denaturant, and increasing the pH level.

Importance of Hypochromic Effect

Nucleic acid melting curve showing hyperchromicity as a function of temperature

The measurement of absorption of light is important in monitoring the melting and annealing of DNA. At the melting temperature (Tm), the DNA is half denatured and half double stranded. By lowering the temperature below the Tm, the denatured DNA strands would anneal back into a double stranded DNA. When temperature is above the Tm, the DNA is denatured.

Because melting occurs almost instantly at a certain temperature, monitoring the absorbance of the DNA at various temperature would indicate the melting temperature. By being able to find the temperature at which DNA melted and annealed, scientists are able to separate DNA strands and anneal them with other DNA strands. This is important in creating hybrid DNAs, which consists of two DNA strands from different sources. Since DNA strands can only anneal if they are similar, the creation of hybrid DNAs can indicate similarities between genomes of different organisms.

References

• Berg, Jeremy & Tymoczko, John. (2006). Biochemistry 6th edition. W.H. Freeman and Company.