Department of Pathology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA.
Free Radic Biol Med. 2012;52(11-12):2276-83. doi: 10.1016/j.freeradbiomed.2012.04.007. Epub 2012 Apr 19.
Hydrogen sulfide (H₂S) is a ubiquitous gaseous signaling molecule that plays a vital role in numerous cellular functions and has become the focus of many research endeavors, including pharmacotherapeutic manipulation. Among the challenges facing the field is the accurate measurement of biologically active H₂S. We have recently reported that the typically used methylene blue method and its associated results are invalid and do not measure bona fide H₂S. The complexity of analytical H₂S measurement reflects the fact that hydrogen sulfide is a volatile gas and exists in the body in various forms, including a free form, an acid-labile pool, and bound as sulfane sulfur. Here we describe a new protocol to discretely measure specific H₂S pools using the monobromobimane method coupled with RP-HPLC. This new protocol involves selective liberation, trapping, and derivatization of H₂S. Acid-labile H₂S is released by incubating the sample in an acidic solution (pH 2.6) of 100 mM phosphate buffer with 0.1mM diethylenetriaminepentaacetic acid (DTPA), in an enclosed system to contain volatilized H₂S. Volatilized H₂S is then trapped in 100 mM Tris-HCl (pH 9.5, 0.1 mM DTPA) and then reacted with excess monobromobimane. In a separate aliquot, the contribution of the bound sulfane sulfur pool was measured by incubating the sample with 1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride), a reducing agent, to reduce disulfide bonds, in 100 mM phosphate buffer (pH 2.6, 0.1 mM DTPA), and H₂S measurement was performed in a manner analogous to the one described above. The acid-labile pool was determined by subtracting the free hydrogen sulfide value from the value obtained by the acid-liberation protocol. The bound sulfane sulfur pool was determined by subtracting the H₂S measurement from the acid-liberation protocol alone compared to that of TCEP plus acidic conditions. In summary, our new method allows very sensitive and accurate measurement of the three primary biological pools of H₂S, including free, acid-labile, and bound sulfane sulfur, in various biological specimens.
硫化氢（H₂S）是一种普遍存在的气态信号分子，在许多细胞功能中起着至关重要的作用，并且已成为许多研究努力的焦点，包括药物治疗干预。该领域面临的挑战之一是准确测量具有生物活性的 H₂S。我们最近报道，通常使用的亚甲基蓝法及其相关结果是无效的，并且不能测量真正的 H₂S。分析 H₂S 测量的复杂性反映了这样一个事实，即硫化氢是一种挥发性气体，以各种形式存在于体内，包括游离形式、酸不稳定池和作为硫烷硫结合。在这里，我们描述了一种使用单溴代丁二酰亚胺法结合反相高效液相色谱法离散测量特定 H₂S 池的新方案。该新方案涉及 H₂S 的选择性释放、捕获和衍生化。通过在封闭系统中用 100mM 磷酸盐缓冲液（pH 2.6）与 0.1mM 二亚乙基三胺五乙酸（DTPA）孵育样品来释放酸不稳定的 H₂S。然后将挥发的 H₂S 捕获在 100mM Tris-HCl（pH 9.5，0.1mM DTPA）中，并与过量的单溴代丁二酰亚胺反应。在另一份样品中，通过用 1mM TCEP（三（2-羧乙基）膦盐酸盐）孵育样品来测量结合的硫烷硫池的贡献，TCEP 是一种还原剂，用于还原二硫键，在 100mM 磷酸盐缓冲液（pH 2.6，0.1mM DTPA）中，并且 H₂S 测量的方式类似于上述描述的方式。酸不稳定池的值通过从酸释放方案获得的值中减去游离硫化氢的值来确定。通过从单独的酸释放方案的 H₂S 测量值中减去 TCEP 加酸性条件下的值来确定结合的硫烷硫池的值。总之，我们的新方法允许非常灵敏和准确地测量各种生物标本中 H₂S 的三个主要生物池，包括游离、酸不稳定和结合的硫烷硫。
