Spin-Label No-Metry In Lipid Bilayer Membranes

Abstract

Electron paramagnetic resonance (EPR) detection of free nitric oxide (NO) by spin-trapping is now widely applied in model and biological systems (Archer, 1993; Henry et al., 1993). This method involves chemical interaction of NO with hemoglobin, nitroso-compounds, or other spin-traps during which a stable visible EPR adduct is formed, but NO is “trapped” and removed from the system. The other method involving EPR spectroscopy is called spin-label NO-metry and can be defined as the use of nitroxide radical spin labels to monitor the NO diffusion-concentration product. Here during bimolecular collisions of NO (fast relaxing, unseen paramagnetic species) with nitroxide radical spin labels (slow relaxing, visible EPR species), physical interaction between molecules occurs that involves Heisenberg exchange and/or dipole-dipole interaction (Molin et al., 1980). This method does not disturb the concentrations of colliding species. As was shown qualitatively by Singh et al. (1994) during collisions of NO with spin labels located in water or in membranes, both the linewidth and the spin-lattice relaxation time of spin labels are altered. In our previous paper (Lomnicka & Subczynski, 1996), we demonstrated that spin-label NO-metry is also a quantitative method because every collision of NO with spin label leads to an observable event — EPR line broadening. These results allow us to connect the Smoluchowski equation for colliding molecules, with NO-induced line broadening of the EPR spin label spectrum, expressed in frequency units. Here R is an interaction distance for a collision (4.5 A as was shown by Lomnicka and Subczynski, 1996, and p is the probability that a spectroscopically observable event occurs when a collision occurs. It is also assumed that the diffusion coefficient of NO, D _NO, is much higher than the diffusion coefficient of the spin label. This assumption should always be considered critically, but if it can be made, then an experimental observable that depends on w yields the so-called diffusion-concentration product of NO, D _NO [NO]. Equation (5.2) is appropriate in principle if the line shape in the absence of NO is Lorentzian. Here DH _pp is the NO-induced peak-to-peak line broadening, and g is the magnetogyric ratio of the electron.