Quantified Maximum Entropy and Biological EPR Spectra

S.M. Glidewell and B.A. Goodman
Scottish Crop Research Institute
Dundee DD2 5DA

J. Skilling
Cavendish Laboratory
Madingley Road
Cambridge CB3 0HE


Free radicals are of importance in the functioning of all biological systems. Many biologically important free radicals have very short lifetimes and do not accumulate in tissues to levels which are readily detectable directly. In consequence, their presence is either determined indirectly by assay of stable end-products of radical reactions or by EPR spectroscopic detection of adducts with spin traps. This technique of spin-trapping has proved valuable in the study of reactions involving biological fluids and tissues, but its usefulnes is often limited by the low levels of adducts which are formed and the requirement for small amounts of aqueous material in the cavity of the spectrometer. Improvements in the signal to noise ratio of spectra can sometimes be effected by accumulation of the spectrum over time, but this is not an option in dynamic systems. In such situations, numerical methods are the only way of optimising the information which can be derived from the spectral data.

This work describes the use of a quantified maximum entropy method for the optimisation of analytical information from EPR spectra. EPR spectra are normally recorded as first derivatives of absorptions and, like all experimental data, are distorted from the ideal lineshape by a combination of systematic and instrumental factors. The first and crucial step in the analysis of a spectrum by maximum entropy methods is the definition of a function --- the point spread function (PSF) which represents the spread of the data around a point, or the distortion from ideality. For EPR spectra, the PSF is selected by differentiating a lineshape comprising Gaussian, Lorentzian and square wave contributions. Using this inital estimate of the PSF, the most probable result is calculated as the frequency disitribution of intensities which, convolved with the PSF, gives the best fit to the experimental data. Discrepancies between calculated and experimental data are used to guide the calculation by an iterative procedure and exploration around the optimum result allows the determination of the error associated with each line's frequency and intensity. The ability to use composite PSFs lends further refinement, either for the disentangling of multiline spectra or for the determination of the intensity of weak spectra of known radicals, a problem often encountered in biological EPR spectroscopy.

Quantified maximum entropy reconstruction of the complex multiline EPR spectrum of paraquat allows the accurate determination of the 4 hyperfine splittings. The approach is then used on a system involving unknown radical species formed by crushing lettuce in the presence of a spin trap. This reconstruction reveals the presence of at least 2 radicals and the parameters derived suggest that one of them is a hydroxyl radical adduct --- a result not obtainable by inspection.

MaxEnt 94 Abstracts / mas@mrao.cam.ac.uk