Spectral analysis of the conformation of polyadenosine diphosphoribose. Evidence indicating secondary structure.

Based on boronic acid chromatography, a rapid method was developed for the purification of polyadenosine diphosphoribose oligomers of various chain lengths. Polyadenosine diphosphoribose oligomers obtained by the new method were distinguished on the basis of spectral criteria. The purity of polyadenosine diphosphoribose was determined by high performance liquid chromatography, which is a highly sensitive method for the detection of contamination by nucleotides derived from other nucleic acids. The A280/A260 ratio, which has been used in the past as one of the criteria of purity of oligomers, was found to be an unsuitable index of purity because it significantly varied as a function of temperature or ionic strength, exhibiting characteristics of temperature- and ionic strength-dependent hypochromicity. Hypochromicity was highly significant at 260 nm but not at 280 nm. The A280/A260 ratio showed a correlation with the oligomer chain length, significantly diminishing below a chain length of 9 adenosine diphosphoribose units, with concomitant loss of hypochromicity. The increase in A280/A260 ratio above a chain length of nine was small and gradual. Circular dichroism of long, medium, and short oligomers was determined at varying temperatures (5-72 degrees C). For long chains, a temperature-dependent change of the major negative theta value and a red shift occurred from 249.5 to 267.5 nm. With medium chain length oligomers, there was no decrease in the theta value at 249.5 nm, only a red shift. In the case of short chain oligomers the major negative theta value was at 267.5 nm and its position was temperature-independent with only a small temperature-related decrease in size. At 72 degrees C, the different patterns of circular dichroism of the three groups of oligomers of differing chain lengths became indistinguishable. These results were interpreted to indicate a significant secondary structure of polyadenosine diphosphoribose of long chain length.