Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements.
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.
Konishi
M
M
Department of Physiology, University of Pennsylvania, Philadelphia 19104-6085.
Olson
A
A
Hollingworth
S
S
Baylor
S M
SM
D000200:Action Potentials; D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D003402:Creatine Kinase / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism; D016257:Fura-2; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008962:Models, Theoretical; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000378:metabolism; D009132:Muscles / Q000502:physiology*; D009994:Osmolar Concentration; D011896:Rana temporaria; D013050:Spectrometry, Fluorescence / Q000379:methods
D001572:Benzofurans; D009124:Muscle Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003402:Creatine Kinase; D005634:Fructose-Bisphosphate Aldolase; D002118:Calcium; D016257:Fura-2