Biochim. ttiophys. Acta, 194 (I969) 486-497 86 BIOCH1MICA ET BIOPHYSICA . BBA 35479 SPECTRAL CHANGES ACCOMPANYING THE POLYMERIZATION OF THE DINITROPHENYL DERIVATIVE OF G-ACTIN KATSUHISA TAWADA,a, * HIROSHI ASAI b AND BERNARD R. GERBERo, ** Departments of Physics, a Nagoya University, Nagoya (Japan), ~ Waseda University, Tokyo (Japan) and e Department of Biochemistry, State University of New York Downstate Medical Center, Brooklyn, N.Y. (U.S.A.) (Received July 28th, 1969) SUMMARY I. Dinitrophenyl (DNP-) derivatives of F-actin, one of the nmscle proteins, were prepared which retain their capacity to depolymerize and repolymerize (self- assemble) repeatedly, when the ionic strength of their solutions was respectively decreased or increased. In parallel with the self-assembly of the DNP-G-actin, a hyperchromic change in the near ultraviolet absorption spectrum occurred near 36o m/x, a spectral region attributable to absorbance by the dinitrophenyl chromo- phore covalently bound to the protein. 2. The spectral change near 36o m# could be correlated with substitution of one or more (up to three) of three rapidly reacting amino acid residues, probably lysyl residues, within the protein. 3. Subjecting the actin derivative in its monomeric form to either large hydro- static compression or to the action of 6 M urea, agents which unfold actin, produced the same end result, namely, a loss in polymerizability of the protein. 4. Remarkably, the effect of high pressure on DNP-G-actin was to displace its absorption band with maximum at 2 36o m# to shorter wavelengths, whereas the effect of 6 M urea was to shift this band to longer wavelengths. These two agents apparently bring about different local structures of the DNP- sites. Moreover, since the spectrum of the protein derivative obeys Beer's Law (through 1.2%, w/v) we conclude for the present that the hyperchromic change reflects a conformational change of the protein and not simply the close contact of actin monomers on polymerizing. The DNP- group reflects changes in its microenvironment (such as alterations of the secondary, tertiary or quaternary structure of the protein) by shifts in its absorption spectrum similar to those demonstrated with other molecular probes. 5. Measurements of solvent perturbation and temperature difference spectra on a model compound, e-N-DNP-lysine, confirmed the finding that this group's spectrum Present address: Department of Biophysical Engineering, Osaka University, Osaka, Japan. • ~ Present address: Department of Biology, University of Pennsylvania, Philadelphia, Pa. 191o 4.