Kinetics of proflavin binding to bacteriophages T2L and T4D

We have measured the kinetics of proflavin binding to T‐even bacteriophages—the 700 S and 1000 S forms of T2L, T4D, and T4D os41—by difference spectroscopy at 430 nm. Measurements were carried out from 22° to 37°C. Binding is very slow to encapsulated DNA compared to free DNA, requiring hours to reach equilibrium. The kinetic data are compatible with the two‐step mechanism\documentclass{article}\pagestyle{empty}\begin{document}$$ P + N\mathop{{\buildrel\textstyle\rightarrow\over {\smash{\leftarrow}\vphantom{_{\vbox to.5ex{\vss_{k_{ - 1} }^{k_1 } PN_1 \mathop{{\buildrel\textstyle\rightarrow\over {\smash{\leftarrow}\vphantom{_{\vbox to.5ex{\vss_{k_{ - 2} }^{k_2 } PN_2 $$\end{document} where P is proflavin, N is nucleotide, and PN 1 and PN 2 are complexes. Computer integration of the rate equations allows evaluation of the rate constants; previous equilibrium measurements gave thermodynamic parameters. For all phage studied, the bimolecular step is endothermic with high positive entropy; the second, unimolecular step is highly exothermic with small negative entropy change. Both forms of T2L bind proflavin with essentially the same rate, as do T4D and the osmotic shock resistant mutant T4D os41. This suggests that the encapsulated DNA is equally accessible to proflavin in both forms of each phage. However, T4D binds dye appreciably faster than T2L, indicating that capsid permeability or DNA environment (glucosylation or packing) is different in the two species.