Preparation and characterization of a new molecular cytochemical probe: 5-iodoacetamidofluorescein-labeled actin

Abstract

The new technique of molecular cytochemitry (Taylor DL, Wang YL (1978): Proc Natl Acad Sci USA 75:857) requires the use of functional fluorescent analogs of cellular components with optimal fluorescence characteristics. An analog of actin suitable for this technique is prepared by reacting purified rabbit striated muscle actin with 5-iodoacetamidofluorescein (5-IAF). The conjugate is purified by DEAE-cellulose ion exchange chromatography and cycles of polymerization-depolymerization, yielding a relatively homogeneous product with the fluorescein group covalently attached to cystein 373. The fluorescently labeled actin maintains normal polymerizability and activates heavy meromyosin Mg2+ adenosine triphosphatase to the same extent as unlabeled actin. Furthermore, fluoresecent paracrystals are readily detectable in fluroescence microscope upon adding excess Mg2+ or Ni2+ ions. Spectrofluorimetric studies of the bound fluorescein indicate that the peak excitation and emission wavelengths, the shapes of the spectra, and the peak fluorescence intensities are somewhat sensitive to polymerization and heavy meromyosin binding. Possible causes of these spectral changes are analyzed and future applications of this fluorescently labeled actin in vitro as well as in vivo are discussed.