Fluorescence of actin-bound hydrophobic molecules.
2-(N-methylanilino)naphthalene-6-sulfonic acid (MANS) binds to G-actin at a single high affinity hydrophobic site (Kd = 41 microM). Salt-induced polymerization of MANS-G-actin results in a general enhancement of sample emission intensities at all wavelengths. At 430 nm, KCl-induced polymerization yields a 2,3-fold enhancement, while MgCl2-induced polymerization gives a 2.0-fold increase. Polymerization of MANS-G-actin in the absence of agitation produces MANS-F-actin samples that have fluorescence polarization values at 430 nm of 0.33. Subsequent mixing or sonication of such MANS-F-actin samples results in a dramatic drop in fluorescence polarization values to 0.14. After cessation of mixing, the polarization values do not recover to their initial levels. Circular polarization of luminescence studies on MANS-actin demonstrate that agitation of MANS-F-actin samples drastically alters emission anisotropy values. 9-Anthroyl choline (9AC) binds to G-actin at a single hydrophobic site (Kd = 68 microM). The fluorescence of 9AC-actin is sensitive to salt-induced polymerization and depends upon the identity of the salt employed. KCl causes a drop in the fluorescence intensity at 490 nm to 70% of the value for 9AC-G-actin, while MgCl2 produces a 30% increase in intensity. Polarization experiments with 9AC-actin produced qualitatively the same results as did those with MANS-actin. Differences in the behaviours of MANS-actin and 9AC-actin in response to polymerization by KCl and MgCl2 and in response to the binding of deoxyribonuclease I suggest that the binding sites on actin for MANS and 9AC do not overlap completely.(ABSTRACT TRUNCATED AT 250 WORDS)
D000199:Actins; D000817:Anilino Naphthalenesulfonates; D002794:Choline / Q000031:analogs & derivatives*; D005453:Fluorescence; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D011108:Polymers
D000199:Actins; D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011108:Polymers; C019057:2,6-mansyl chloride; C035321:9-anthroylcholine; D002794:Choline