Variation of the permeability of bacteriophage T4: analysis by use of a protein-specific probe for the T4 interior.

The permeability of bacteriophage T4 and the change in T4 permeability caused by mutation to osmotic shock resistance are investigated here by quantification of the kinetics with which both a DNA-specific probe (ethidium) and a protein-specific probe [1,1'-bi(4-anilino)naphthalene-5,5'-di-sulfonic acid, or bis-ANS)] bind to T4. In the case of an osmotic shock-resistant mutant, T40s41, both ethidium and bis-ANS bind with first order kinetics. The first-order rate constant (k*) for both bis-ANS and ethidium is a function of anion type and concentration. Adenosine triphosphate, phosphate, bisulfite, sulfate, and acetate anions all reduce k* below the k* observed when chloride is the only anion. When chloride is the only anion at 25 degrees C, k* values for binding to T40s41 are orders of magnitude above k* values for binding to wild-type T4 (T4wt). At 25 degrees C, k* for T4wt is too small to measure but k* for T4wt increases at 50-55 degrees C to values approaching those measured for T40s41, without inactivating T4wt, when chloride is the only anion; during heating, T4wt is stabilized by both ethidium and bis-ANS. Binding to T4wt is reversible at 50-55 degrees C, but not at 25 degrees C. Equilibrium binding of bis-ANS to T40s41 reveals 112 +/- 24 sites per T4 capsid. Equilibrium binding of ethidium to T40s41 reveals both high- and low-affinity sites previously observed in the packaged DNA of other bacteriophages. The ATP-induced decrease in k* is not accompanied by a decrease in equilibrium binding. The following hypotheses are presented to explain the above data: (a) All detected bis-ANS binding sites on T4 are interior to the outer surface of T4. (b) The value of k* for both bis-ANS and ethidium is controlled at the port(s) of passage through the outer shell of the T4 capsid. (c) The anions present control k* values at the port(s) of entry, probably by controlling the size of this port. The effects on k* of phosphate explain the otherwise paradoxical observation [P. J. McCall and V. A. Bloomfield (1976) Biopolymers 15, 2323-2336] that in a phosphate buffer the permeabilities of T4wt and T40s41 are the same.