Kim Danny H, Marbois Beth N, Faull Kym F, Eckhert Curtis D
Department of Environmental Health Sciences, University of California, 10833 Le Conte Avenue, Los Angeles, California 90095, USA.
J Mass Spectrom. 2003 Jun;38(6):632-40. doi: 10.1002/jms.476.
This paper describes for the first time the direct measurement of boric acid (B(OH)(3)) and borate (B(OH)(4) (-)) adduction to NAD(+) and NADH by electrospray ionization mass spectrometry (ESI-MS) and (11)B NMR spectroscopy. The analysis demonstrates that borate binds to both cis-2,3-ribose diols on NAD(+) forming borate monoesters (1 : 1 addition), borate diesters (1 : 2 addition) and diborate esters (2 : 1 addition), whereas, only borate monoesters were formed with NADH. MS in the negative ion mode showed borate was bound to a cis-2,3-ribose diol and not to the hydroxyl groups on the phosphate backbone of NAD(+), and MS/MS showed that the 1 : 1 addition monoester contained borate bound to the adenosine ribose. Boron shifts of borate monoesters and diesters with NAD(+) were observed at 7.80 and 12.56 ppm at pH 7.0 to 9.0. The esterifications of borate with NAD(+) and NADH were pH dependent with maximum formation occurring under alkaline conditions with significant formation occurring at pH 7.0. Using ESI-MS, the limit of detection was 50 micro M for NAD(+) and boric acid (1 : 1) to detect NAD(+)-borate monoester at pH 7.0. These results suggest esterification of borate with nicotinamide nucleotides could be of biological significance.
本文首次描述了通过电喷雾电离质谱（ESI-MS）和¹¹B核磁共振光谱法直接测量硼酸（B(OH)₃）和硼酸盐（B(OH)₄⁻）与NAD⁺和NADH的加合物。分析表明，硼酸盐与NAD⁺上的顺式-2,3-核糖二醇结合形成硼酸单酯（1:1加成）、硼酸二酯（1:2加成）和二硼酸酯（2:1加成），而与NADH仅形成硼酸单酯。负离子模式下的质谱显示硼酸盐与顺式-2,3-核糖二醇结合，而非与NAD⁺磷酸主链上的羟基结合，串联质谱显示1:1加成的单酯中硼酸盐与腺苷核糖结合。在pH 7.0至9.0时，观察到硼酸盐与NAD⁺形成的单酯和二酯的硼化学位移分别为7.80和12.56 ppm。硼酸盐与NAD⁺和NADH的酯化反应依赖于pH，在碱性条件下形成量最大，在pH 7.0时也有显著形成。使用ESI-MS，在pH 7.0时检测NAD⁺-硼酸单酯，NAD⁺和硼酸（1:1）的检测限为50 μM。这些结果表明硼酸盐与烟酰胺核苷酸的酯化反应可能具有生物学意义。
