pH and polymerization dependence of the site of labeling of actin by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.
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.
D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010069:Oxadiazoles / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism*; D011817:Rabbits; D013053:Spectrophotometry
D000199:Actins; D010069:Oxadiazoles; D011108:Polymers; D005969:Glutamate Dehydrogenase; D009327:4-Chloro-7-nitrobenzofurazan