Elsevier

Analytical Biochemistry

Volume 421, Issue 1, 1 February 2012, Pages 203-207
Analytical Biochemistry

Passive loss of hydrogen sulfide in biological experiments

https://doi.org/10.1016/j.ab.2011.10.016Get rights and content

Abstract

Hydrogen sulfide (H2S) is a volatile gas of considerable interest as a physiologically relevant signaling molecule, but this volatility has typically been overlooked in the context of biological experiments. We examined volatility of 10 and 100 μM H2S (Na2S·9H2O) in real time with polarographic electrodes in three commonly employed experimental apparatuses: 24-well tissue culture plates (WP), muscle myograph baths (MB), and the Langendorff perfused heart apparatus (LPH). H2S loss from all apparatuses was rapid and exponential, with half-times (t1/2) of 5 min (WP), less than 4 min (MB), and less than 0.5 min (LPH). The t1/2 for H2S loss from MB bubbled with 100% oxygen was slightly longer than that for MB bubbled with 100% nitrogen; both were significantly shorter than stirred but unbubbled MB (>9 min). Therefore, even without tissue, H2S rapidly disappears from buffer under a variety of experimental conditions, and this is due to volatilization, not oxidation. The inability to maintain H2S concentration, even briefly, questions the accuracy of dose–response studies and the relevance of long-term (>10 min) exposure to a single treatment of H2S. These results also help to explain the discrepancy between low H2S concentrations in blood and tissues versus high concentrations of exogenous H2S required to produce physiological responses.

Section snippets

H2S measurement

An amperometric H2S sensor was built in-house [11] or purchased (model HS-700, Innovative Instruments, Tampa, FL, USA) and used in conjunction with an Apollo free radical analyzer (World Precision Instruments, Sarasota, FL, USA). These sensors measure H2S gas that is in the equilibrium: H2S  HS  S2−, with pKa1 = 6.9 and pKa2 > 11 [24]. At physiological pH (∼7.4 blood and ∼6.9 intracellular), the concentration of H2S ranges from 25% to 50% of the total dissolved sulfide. However, the equilibrium can

Formation of H2S on solvation

H2S gas is formed nearly instantaneously on solvation in buffer (Fig. 1). The reaction is nearly 80% complete within the first 10 s. By 20 s, H2S gas concentration reaches a maximum and falls exponentially thereafter. The t1/2 for H2S loss varied with the stirring rate but was not characterized further. Physical dissolution of H2S appears to be the rate-limiting step in formation of H2S gas within the time resolution of this method.

Muzaffar and coworkers [25] showed that when NaHS is placed in

Summary

Over the past 25 years, our concept of H2S has gone from a toxic gas to a signaling molecule with potential clinical relevance. Our knowledge of the effects of exogenous H2S on biological systems has outpaced our understanding of how these systems affect H2S. The development of H2S gas sensors capable of measuring H2S in real time and on unadulterated tissues has downgraded our estimates of endogenous H2S production and tissue concentrations. In this study, we have shown that even the residence

Acknowledgments

The authors thank K. Hurley for technical assistance. This work was supported in part by National Science Foundation Grants IOS 0641436 and IOS 1051627.

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