Harkins A B, Kurebayashi N, Baylor S M
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6085.
Biophys J. 1993 Aug;65(2):865-81. doi: 10.1016/S0006-3495(93)81112-3.
Fluo-3 is an unusual tetracarboxylate Ca2+ indicator. For recent lots supplied by Molecular Probes Inc. (Eugene, OR), FMAX, the fluorescence intensity of the indicator in its Ca(2+)-bound form, is approximately 200 times that of FMIN, the fluorescence intensity of the indicator in its Ca(2+)-free form. (For earlier lots, impurities may account for the smaller reported values of FMAX/FMIN, 36-40). We have injected fluo-3 from a high-purity lot into intact single fibers from frog muscle and measured the indicator's absorbance and fluorescence signals at rest (A and F, respectively) and changes in absorbance and fluorescence following action potential stimulation (delta A and delta F signals substantially lagged behind that of the myoplasmic free Ca2+ transient. Our analysis of fluo-3's signals from myoplasm therefore focused on information about the level of resting myoplasmic free [Ca2+] ([Ca2+]r). From A, delta A, and in vitro estimates of fluo-3's molar extinction coefficients, the change in the fraction of fluo-3 in the Ca(2+)-bound form during activity (delta f) was estimated. From delta f, delta F, and F, the fraction of the indicator in the Ca(2+)-bound form in the resting fiber (fr) was estimated by fr = (delta f x F/delta F) + (1-FMAX/FMIN)-1. Since FMAX/FMIN is large, the contribution of the second term to the estimate of fr is small. At 16 degrees C, the mean value (mean +/- S.E.) of fr was 0.086 +/- 0.004 (N = 15). From two estimates of the apparent dissociation constant of fluo-3 for Ca2+ in the myoplasm, 1.09 and 2.57 microM, the average value of [Ca2+]r is calculated to be 0.10 and 0.24 microM, respectively. The smaller of these estimates lies near the upper end of the range of values for [Ca2+]r in frog fibers (0.02-0.12 microM) estimated by others with aequorin and Ca(2+)-selective electrodes. The larger of the estimates lies within the range of values (0.2-0.3 microM) previously estimated in this laboratory with fura red. We conclude that [Ca2+]r in frog fibers is at least 0.1 microM and possibly as large as 0.3 microM.
Fluo-3是一种不同寻常的四羧酸盐Ca2+指示剂。对于Molecular Probes公司（俄勒冈州尤金市）近期提供的批次产品，FMAX（指示剂与Ca(2+)结合形式的荧光强度）约为FMIN（指示剂无Ca(2+)形式的荧光强度）的200倍。（对于早期批次产品，杂质可能是报道的FMAX/FMIN值较小，为36 - 40的原因）。我们已将来自高纯度批次的Fluo-3注入青蛙肌肉的完整单纤维中，并测量了静息时指示剂的吸光度和荧光信号（分别为A和F）以及动作电位刺激后吸光度和荧光的变化（ΔA和ΔF信号显著滞后于肌质游离Ca2+瞬变信号）。因此，我们对来自肌质的Fluo-3信号的分析集中在关于静息肌质游离[Ca2+]（[Ca2+]r）水平的信息上。根据A、ΔA以及Fluo-3摩尔消光系数的体外估计值，估算了活动期间与Ca(2+)结合形式的Fluo-3比例的变化（Δf）。根据Δf、ΔF和F，通过fr = (Δf × F/ΔF) + (1 - FMAX/FMIN)^-1估算静息纤维中与Ca(2+)结合形式的指示剂比例（fr）。由于FMAX/FMIN很大，第二项对fr估计值的贡献很小。在16摄氏度时，fr的平均值（平均值±标准误）为0.086 ± 0.004（N = 15）。根据肌质中Fluo-3对Ca2+的表观解离常数的两个估计值，1.09和2.57微摩尔，计算得出[Ca2+]r的平均值分别为0.10和0.24微摩尔。这些估计值中较小的一个接近其他研究人员使用水母发光蛋白和Ca(2+)选择性电极估计的青蛙纤维中[Ca2+]r值范围的上限（0.02 - 0.12微摩尔）。较大的估计值在本实验室先前使用呋喃红估计的值范围（0.2 - 0.3微摩尔）内。我们得出结论，青蛙纤维中的[Ca2+]r至少为0.1微摩尔，可能高达0.3微摩尔。
