ABSTRACT
The influence of pH and state of polymerization on the site of attachment of the fluorophore 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) to actin was examined. Possible interference of buffer constituents which react with NBD-Cl was also observed. F-actin is primarily labeled at cysteine below pH 6.5 and at lysine at values of pH above neutrality. Time dependences of the excitation spectra support the contention that NBD-Cl may react first with cysteine and then undergo transfer to a neighboring lysine. The quantum yield enhancement of NBD bound to cysteine upon polymerization is markedly less than the 2-fold increase observed when it is bound to lysine. Labeling reactions carried out with G-actin give predominantly the lysine derivative regardless of pH. NBD-Cl was found to react with a number of the constituents of buffer systems normally employed with actin. The spectra of these reaction products are sufficiently similar to those of the NBD-amino acid derivatives to require care when interpreting reaction rate data by spectrophotometric or fluorometric methods.
