Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements

Abstract

Binding of the fluorescent Ca2+ indicator dye fura-2 by intracellular constituents has been investigated by steady-state optical measurements. Fura-2's (a) fluorescence intensity, (b) fluorescence emission anisotropy, (c) fluorescence emission spectrum, and (d) absorbance spectra were measured in glass capillary tubes containing solutions of purified myoplasmic proteins; properties b and c were also measured in frog skeletal muscle fibers microinjected with fura-2. The results indicate that more than half, and possibly as much as 85%, of fura-2 molecules in myoplasm are in a protein-bound form, and that the binding changes many properties of the dye. For example, in vitro characterization of the Ca2+-dye reaction indicates that when fura-2 is bound to aldolase (a large and abundant myoplasmic protein), the dissociation constant of the dye for Ca2+ is three- to fourfold larger than that measured in the absence of protein. The problems raised by intracellular binding of fura-2 to cytoplasmic proteins may well apply to cells other than skeletal muscle fibers.

Resting cytoplasmic free Ca2+ concentration in frog skeletal muscle measured with fura-2 conjugated to high molecular weight dextran.

“…The spatially averaged fura dextran concentration in cytoplasm was estimated by a simple comparison of the F(360) measured in the muscle fibers and in quartz capillaries of the same diameter, as described previously (Konishi et al, 1993;also Klein et al, 1988). The indicator concentration may be somewhat overestimated, as a quantum efficiency of the indicator molecules is likely to be increased in the cytoplasm from that in salt solutions (Konishi et al, 1988). The cytoplasmic fura dextran concentration, thus estimated, was generally 30-100 ~M.…”

“…Two other runs with a spectrofluorometer (FP-770, JASCO) with 1 ~M fura dextran gave/~ values of 0.51 o.M and 0.56 ~M. The average value from these three runs, 0.52 (-+0.03) o~M, is 2-2.5 times larger than that of fura-2 at 1 mM [Mg 2+] (e.g., Konishi et al, 1988). Inclusion of ATP and creatine phosphate in the calibration solutions (solutions 5 and 6 of Table I) does not appreciably change the KD of fura dextran.…”

“…Fura dextran's dissociation constant (Ko) for Ca ~+ was estimated in vitro by measuring the fluorescence in the calibration solutions with various [Ca ~+]'s, which were prepared by mixing solutions 7 and 8 of Table I (18~ 1 mM [Mg2+]). The values of R at eight different [CaZ+]'s were least squares fitted with a theoretical 1:1 binding curve (for the details of the analysis, see Konishi et al, 1988). One run made in quartz capillaries on the microscope with 20 ~M indicator gave a best-fitted F~ value of 0.50 ~M (the left-most curve in Fig.…”

“…On the other hand, another very well controlled study, which used both Ca2+-sensitive microelectrodes and aequorin, reports that resting [Ca2+]i is below the detection limit of their methods, and gives [Ca2+]i values of 0.04-0.06 oLM as an upper limit. Major problems related to the use of these indicators appear to be the binding of the indicators to intracellular constituents, which alters Ca ~+ affinity, the optical signals of the indicators, or both (e.g., Konishi, Olson, Hollingworth, and Baylor, 1988;Blatter and Blinks, 1991;Baker, Brandes, Schreur, Camacho, and Weiner, 1994).…”

Calcium release and its voltage dependence in frog cut muscle fibers equilibrated with 20 mM EGTA.

“…B21 and B24 shows that EGTA is able to reduce the steady state value of free A [Ca] by scaling the term d#/4~rDcar by the factor exp (-r/hc~). Robinson and Stokes, 1959) and the assumption that the value of the diffusion coefficient in myoplasm is half of that in a dilute aqueous solution (Kushmerick and Podolsky, 1969;Konishi et al, 1988). The values of DEGTA and DC~GTA are both taken to be 1.7 • 10 -6 crn2/s (see first section of Results).…”