Open AccessSpin-label oximetry: kinetic study of cell respiration using a rapid-passage T1-sensitive electron spin resonance display.
Author(s) -
Wojciech Froncisz,
ChingSan Lai,
James S. Hyde
Publication year - 1985
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.82.2.411
Subject(s) - oxygen , chemistry , analytical chemistry (journal) , electron paramagnetic resonance , spin (aerodynamics) , limiting oxygen concentration , kinetics , diffusion , chinese hamster ovary cell , nitroxide mediated radical polymerization , nuclear magnetic resonance , respiration , kinetic energy , thermodynamics , chromatography , physics , biochemistry , receptor , radical polymerization , organic chemistry , quantum mechanics , copolymer , polymer , botany , biology
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 microM, requiring only 1 microliter 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 Vmaxt, where t is the time after sealing the chamber, to a critical oxygen concentration of 5.2 microM. Below 5.2 microM, the concentration can be fitted to an exponential form, exp(-t/tau), where tau = 15 sec for 4000 cells per microliter. It is believed that this experimental behavior is determined by complex enzyme kinetics.