FLUORESCENCE LIFETIME QUENCHING STUDIES ON THE ACCESSIBILITIES OF ACTIN SULFHYDRYL SITES

The fluorescence decay of N-iodoacetyl-N-(5-sulfo-1 -naphthyl)ethylenediamine( 1, 5-IAEDANS) labeled G-actin exhibited two decay components, a major component of lifetime 17.3 ns that accounted for 96% of the fluorescence, and a minor component of lifetime 33.3 ns. Lifetime quenching studies by the addition of acrylamide yielded quenching rate constants, kq, of 2.31 × 108 M-1 s-1 and 6.5 × 107 M-1 s-1 for the major and minor components, respectively. Upon polymerization of the labeled actin, only one lifetime of 19.3 ns was detectable. The presence of a minor component was revealed when acrylamide was added. The values of kq for labeled F-actin decreased from those of G-actin to 1.35 × 108 M-1 s-1 and 1.52 × 107 M-1 s-1 for the major and minor components, respectively. Pretreatment of the G-actin with N-ethylmaleimide (which is known to react primarily with cysteine-373 in actin) substantially blocks the subsequent incorporation of 1, 5-IAEDANS. These data were interpreted as follows: The major decay component for labeled G-actin is associated with a major labeling site, probably located at cysteine-373. The minor decay component corresponds to a minor labeling site (or class of sites) of unknown location. The major labeling site is more polar and more accessible than the minor labeling site. Upon polymerization, both sites become less accessible. Intensity quenching studies on labeled F-actin in the presence and absence of tropomyosin revealed that tropomyosin further decreased the accessibility of the major labeling site (kq decreased by about 30%). This finding can be interpreted to indicate that tropomyosin binds to actin at a site near cysteine-373. © 1979, American Chemical Society. All rights reserved.