Changes in OD at 235 nm do not correspond to the polymerization step of actin.

Discrepancies were observed when the polymerization of rabbit muscle actin was monitored by delta OD235 and viscometry (eta). For example, in the presence of (beta,gamma)-methyleno ATP, the delta OD signal was as large as with ATP although polymerization was very poor (eta 1.1, compared with eta = 1.7 in the presence of ATP). Furthermore, when monomeric actin, kept for 1 h in the presence of a stoichiometric equivalent of ADP, was exposed to conditions favoring polymerization (addition of MgCl2), a considerable delta OD235 signal appeared, although the actin had completely lost its polymerizability (eta = 1.0). We conclude that the observed changes in OD235 cannot reflect polymerization itself, but must be caused by another reaction preceding the assembly. Under normal conditions, this reaction is supposed to be the slowest step of filament formation and so to determine the velocity of the whole process. In conclusion, monitoring of actin polymerization by delta OD235 is a valid method only when polymerization has been assessed by another, independent method.