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. Author manuscript; available in PMC: 2017 Sep 13.
Published in final edited form as: Methods Enzymol. 2014;549:189–219. doi: 10.1016/B978-0-12-801122-5.00009-X

Figure 5.

Figure 5

UV absorbance-based determination of pKa. A & B) Plot of absorbance at 240 nm (solid circles) and 290 nm (open circles) versus pH to determine pKa of cytosine in two unstructured nucleotides (A) UC, and (B) UUCUU. C) Plot of absorbance at 285 nm for a 19 mer oligonucleotide containing A+•C wobble pair (solid circles), and a controloligonucleotide containing an GC base base pair (hollow circles). Note: The shifted pKa due to the formation of the protonated A+ •C wobble pair could be detected even though the magnitude of hyperchromicity is only 4%. The pKa was determined by fitting the data to Eq. (4). (Adapted from Moody et al. 2005).