Reactivity of the alpha-putrescinylthymine amino groups in phi W-14 deoxyribonucleic acid

Bacteriophage phi W-14 DNA contains the hypermodified pyrimidine alpha-putrescinylthymine (putThy) [Kropinski, A. M. B., Bose, R. J., & Warren, R. A. J. (1973) Biochemistry 12, 151]. The primary amino groups of the putrescinyl side chains react with trinitrobenzenesulfonate (TNBS) but at a much slower rate than the reactive lysine side chains of proteins. Both primary and secondary amino groups in the putrescinyl side chains react with acetic anhydride, but the reaction does not go to completion under the conditions employed. Some of the secondary amino groups react faster with acetic anhydride than do some of the primary amino groups. Acetylation lowers the thermal transition temperature (Tm) of phi W-14 DNA, confirming that the unusually high Tm of this DNA is due to the charged putrescinyl groups, but it does not affect the buoyant density. The extent of lowering of the Tm is proportional to the degree of acetylation. Since some of the secondary amino groups are acetylated before some of the primary amino groups, the effect of acetylation on the Tm suggests that both the primary and secondary amino groups can neutralize negative charge repulsion in phi W-14 DNA. Only the putThy residues in the DNA are modified by TNBS and acetic anhydride.