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Biophys J. 1988 Dec;54(6):1089-104.

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

Author information

1
Department of Physiology, University of Pennsylvania, Philadelphia 19104-6085.

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.

PMID:
3266079
PMCID:
PMC1330420
DOI:
10.1016/S0006-3495(88)83045-5
[Indexed for MEDLINE]
Free PMC Article

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