Immobilized enzyme electron spin resonance: A method for detecting enzymatically generated transient radicals

The study of enzymatically generated, transient radicals provides valuable information about radical reactivity as well as enzyme function. ESR methods to detect transient radicals are generally based on continuous flow and have the potential to consume large quantities of enzyme, substrate, and buffer. Experimental approaches have been pursued to minimize sample volumes, although none have made the continuous-flow ESR approach generally applicable for enzymes and substrates available in limited quantities. We have developed an alternative approach to the traditional continuous-flow ESR method that provides the same high-resolution ESR spectra, but does not consume large quantities of enzyme, substrate, or buffer. The method utilizes enzyme immobilized onto an inert substrate packed directly into an ESR flat cell. Flowing substrate solution over the immobilized enzyme generates in situ, transient radicals, which can then be observed on the submillisecond time scale. We have termed this method immobilized enzyme ESR, abbreviated IE-ESR. In this paper, we have described the details of the IE-ESR technique and have presented data collected using the IE-ESR technique for transient radicals from limited quantity enzymes, limited quantity substrates, and D2O buffers. An extension of this technique to ESR spin trapping has also been discussed.