Cytochrome P450 enzymes are a super family of haem-containing mono-oxygenases which are found in all kingdoms of life. They show extraordinary diversity in their reaction chemistry and are involved in the biotransformation of a plethora of both exogenous and endogenous compounds. There has been a variety of approaches for the immobilisation of these biological redox systems and direct electrochemistry of these proteins can go towards providing biomimetic environments for fundamental studies together with a basis for designing devices without the need for electron transfer mediators. The incorporation of these proteins into mesoporous films or onto various electrode surfaces has generated interest due to the possibility of direct electron exchange between the proteins redox active sites and the host electrode. Here, two different P450 protein systems were investigated. The water soluble P450cam or Cyp101, from the soil dwelling bacteria Pseudomonas Putida and Cyp6g1, a microsomal protein form the fruit fly Drosophila Melanogaster. In this work, firstly the Cyp101 proteins were expressed and purified from a microbial culture starting material. The various steps in the purification process freed the protein from a confining matrix followed by the separation of the protein and non-protein parts of the mixture. In the latter system the enzyme is already embedded into a microsomal unit, thus more likely to mimic a biologically active environment. The utilisation of methoxy-resorufin ether (MRES) is described as an electrochemical probe for investigating the activity of the microsomal protein. This substrate also exhibits fluorescent properties which provided a dual detection system for the enzymes activity. The work then went on to investigate the absorption and reactivity of Cyp101 in porous nanoparticulateTiO2 film electrodes and on an edge plane graphite electrode.
|Date of Award||1 Jul 2007|
|Supervisor||Toby Jenkins (Supervisor)|
Studies of cytochrome P450 in biomimetic films
Dash, H. (Author). 1 Jul 2007
Student thesis: Doctoral Thesis › PhD