Lai C S, Hopwood L E, Hyde J S, Lukiewicz S
Proc Natl Acad Sci U S A. 1982 Feb;79(4):1166-70. doi: 10.1073/pnas.79.4.1166.
Magnetic interactions between dissolved oxygen and nitroxide radical spin probes lead to broadening of the ESR lines. We have used a closed-chamber method based on this property to determine the maximum rate of O2 uptake per cell (Vmax per cell) in cultured mammalian cells. A suitable spin probe and a cell suspension are mixed in an aerated medium, and the rate of disappearance of dissolved O2 is measured. The effects of temperature, pH, and microwave power on the determination of dissolved oxygen in solution were studied. For asynchronous Chinese hamster ovary cells, oxygen uptake is 3.8 X 10(7) oxygen molecules per cell per sec and appears to be enzymatically limited at oxygen concentrations greater than 10 microM. About 5-10 X 10(5) cells were used for each measurement, making it possible to study mitotically synchronized cells. Using this method, we have found that Vmax per unit cell volume changes during the cell cycle of Chinese hamster ovary cells from a minimum in mitosis to maxima in both G1 and late S phases. Advantages and limitations of spin probes for studying the O2 uptake of intact cells are discussed.
溶解氧与氮氧化物自由基自旋探针之间的磁相互作用会导致电子自旋共振（ESR）谱线变宽。我们基于这一特性采用封闭腔室法来测定培养的哺乳动物细胞中每个细胞的最大耗氧率（每个细胞的Vmax）。将合适的自旋探针与细胞悬液在通气培养基中混合，然后测量溶解氧的消失速率。研究了温度、pH值和微波功率对溶液中溶解氧测定的影响。对于异步培养的中国仓鼠卵巢细胞，每个细胞每秒摄取3.8×10⁷个氧分子，并且在氧浓度大于10微摩尔时似乎受到酶的限制。每次测量大约使用5 - 10×10⁵个细胞，这使得研究有丝分裂同步化的细胞成为可能。使用这种方法，我们发现中国仓鼠卵巢细胞在细胞周期中单位细胞体积的Vmax从有丝分裂期的最小值变化到G1期和S期后期的最大值。讨论了自旋探针在研究完整细胞耗氧方面的优缺点。
