Linear Dichroism and Enzyme kinetics

Kinetics can be measured using linear dichroism (LD) by detecting changes in DNA structure due to enzyme action.

One example of this is restriction enzymes. They cut DNA at specific locations determined by a short palandromic base sequence (eg GAATTC for the enzyme EcoRI). We started with super-coiled plasmid DNA which has low alignment in the LD cells that we use (see LD section).

The graph below shows how LD changes when DNA is cut twice. Note that DNA gives a negative LD signal, so a minimum has the largest amplitude signal. The pictures are illustrative of the DNA structure in each phase of the reaction.

 

 

Several different enzymes have been looked at using this technique and the reaction has been modelled to attempt to obtain rate constants. We are also using modified plasmids to work out if the location of the restriction site affects the reaction rates.