Spin-label oximetry: kinetic study of cell respiration using a rapid-passage T1-sensitive electron spin resonance display.

Abstract

An unusual ESR display has been developed that exhibits sensitivity to bimolecular collisions of dissolved oxygen in water with nitroxide radical spin probes at oxygen concentrations as low as 0.1 guM, requiring only 1 dul of sample. The method involves observation of the ESR rapid-passage signal when tuned to the dispersion using a loop-gap resonator. The bimolecular collision rate determines the phase of the signal. The method has been used in a closed-chamber geometry to study respiration of asynchronous populations of Chinese hamster ovary (CHO) cells. An integral of the Michaelis-Menten equation permits direct comparison with experiment and is shown to be incompatible with the data. The theory of diffusion limitation also is developed and shown to be inconsistent with experiment. The average oxygen concentration is found to decrease as V1,,t, where t is the time after sealing the chamber, to a critical oxygen concentration of 5.2 juM. Below 5.2 IAM, the concentration can be fitted to an exponential form, exp(-t/T), where X = 15 sec for 4000 cells per Il. It is believed that this experimental behavior is determined by complex enzyme kinetics.In a series of recent papers (1-7), we have developed the methodology of spin-label oximetry and have applied it to various cellular and liposomal membrane systems. The physical basis of the method depends on Heisenberg exchange between nitroxide radical spin labels and molecular oxygen, which occurs at the bimolecular collision rate. This rate can be estimated by using the Smoluchowski equation,where R is the interaction distance, which is generally assumed to be 4.5 A, and D is the diffusion constant of 02, which is assumed to be much greater than the diffusion constant of the spin label. These exchange events shorten both the spin-lattice relaxation time (T1) and the spin-spin relaxation time (T2) of the spin label.The T2 oximetric method was introduced by Backer et al. Heisenberg exchange between a fast-relaxing species (e.g., molecular oxygen) and a slow-relaxing species (e.g., a spin label) provides a coupling of the spin label to the lattice, as pointed out by Hyde and Sarna (10). This is the basis of the spin-label T1 method (1, 2, 5). One advantage of this method is that the spin label can be undergoing slow motion with no proton structure evident.One of the rationales for spin-label oximetry is that a molecular probe is used. It is a simple matter to vary the concentration of the probe to verify that it does not itself interfere with the kinetics of oxygen uptake (3). Polarographic probes are known to distort the distribution of oxygen in the sample under investigation (11, 12). Another rationale is that both T1 and T2 of spin labels happen to be rather close to the bimolecular collision rate of oxygen dissolved in water under standard conditions, which gives the method good sensitivity. Fluorescence probes are sometimes used for oximetry, where quenching of fluorescence by oxygen is the observed process, but there is a poor match between collision rate...

Activation of Hsp90/NOS and increased NO generation does not impair mitochondrial respiratory chain by competitive binding at cytochrome C Oxidase in low oxygen concentrations

“…The advantage of using this method is that only a micro-volume (~20-40 µl) of sample is necessary to acquire measurements. Moreover, EPR oximetry has been proven to be a technique that is accurate and highly sensitive (Froncisz et al 1985;O'Hara et al 2005;Presley et al 2006;Swartz 2004). From these measurements, we have found that the inhibitory role of NO is predominant at high pO 2 values; however, during endogenous NO generation, it is reduced or ineffective at lower pO 2 ranges (<10 mmHg).…”

In vivo Spectroscopy and Imaging of Nitroxide Probes

“…An advantage of EPR oximetry is that it is based on pure physical interaction, Heisenberg spin exchange, between paramagnetic molecules of probe and oxygen, and does not interfere with oxygen metabolism, therefore providing a basis for noninvasive oxygen measurements in biological systems. Initially, the paramagnetic probes for EPR oximetry were almost exclusively nitroxides (Backer et al, 1977;Lai et al, 1982;Froncisz et al, 1985;Bacic et al, 1989;Chan et al, 1989;Hyde & Subszynski, 1989). Both the longitudinal (T1) and transverse (T2) relaxation times of the NRs can be affected by collisions of the nitroxide with dissolved oxygen.…”

“…Two years later the T2 oximetry method was proposed and applied to follow mitochondrial respiration in samples containing about 100 liver cells (Backer et al, 1977). T1-sensitive EPR oximetry was developed by Hyde et al (Froncisz et al, 1985;Hyde et al, 1990). T2 oximetric applications using NRs are preferred for biological applications in low viscosity solutions because T2 is close to the collision rate resulting in optimal EPR sensitivity.…”

Spin-Labeled Small Unilamellar Vesicles with the T 1-Sensitive Saturation-Recovery EPR Display as an Oxygen-Sensitive Analyte for Measurement of Cellular Respiration

“…T 1 -sensitive oximetry methods were introduced and developed by Hyde and co-workers including saturation-recovery (the absolute T 1 method [4-6]), CW saturation [7, 8], passage display [9], and the multiquantum approach [10, 11]. Heisenberg exchange between a fast-relaxing species (e.g., molecular oxygen) and a slow-relaxing species (e.g., a spin label) leads to faster spin-lattice relaxation of the nitroxide.…”