Mechanistic basis for 2-aminopurine quenching by morpholine- and piperazine-based Good’s buffers

2-Aminopurine (2AP) is the most widely used fluorescent nucleobase analog in DNA and RNA research. While quenching of 2AP by DNA bases has been extensively characterized, the effect of extrinsic quenchers has received far less attention. This study examines the fluorescence quenching mechanisms of 2AP by commonly used buffers in biochemical research. We systematically investigated four Good's buffers-MES, MOPS, HEPES, and PIPES-along with their parent compounds morpholine and piperazine across a range of pH conditions and concentrations. For morpholine-containing buffers (MES and MOPS), quenching occurs predominantly at pH values at or above their respective pKa values and is negligible at more than two pH units below the pKa. In contrast, piperazine-containing buffers (HEPES and PIPES) exhibit substantial quenching even below their pKa values due to the presence of two basic nitrogen atoms in the piperazine ring, one of which remains unprotonated and reactive across the investigated pH range. Time-resolved fluorescence measurements demonstrate that quenching is primarily dynamic for MES, MOPS, and HEPES, while PIPES shows significant static quenching contributions. Results are consistent with a mechanism involving photoinduced electron transfer from unprotonated tertiary amines to excited-state 2AP. The thermodynamic feasibility of this mechanism is supported by the low oxidation potentials of these tertiary amines compared to primary amine-containing buffers such as TRIS, which does not quench 2AP fluorescence. These results have significant practical implications for fluorescence-based studies using 2AP as a structural or dynamic probe in nucleic acid research. Buffer selection can substantially alter both quantum yields and fluorescence lifetimes of 2AP, potentially leading to misinterpretation of experimental data if these effects are not properly accounted for.