Analytical measurement of discrete hydrogen sulfide pools in biological specimens

Abstract

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.

Fig.1

Biological Pools of Labile Sulfur.

Fig.2

Optimization of the measurement techniques for the sulfide pools. (A) Effect of trapping time on hydrogen sulfide recovery. After 50 μL of 40 μM sodium sulfide was incubated with 450 μL of 0.1 M pH 2.6 phosphate buffer for 30 min, hydrogen sulfide gas was re-trapped by 500 μL of 0.1 M Tris-HCl buffer (pH 9.5, 0.1 mM DTPA) with a peak recovery time of 30 minutes. (B) Effect of sodium sulfide release and re-trapping on hydrogen sulfide recovery. The hydrogen sulfide detected from 40 μM of sulfide was compared before release and after releasing and re-trapping. Both releasing and trapping times were 30 minutes. (C) Effect of plasma on release of sulfide. 50 μL of plasma was incubated with 450 μL of 0.1M pH 2.6 phosphate buffer for different lengths of time and then re-trapped for 30 min demonstrating optimal release at 30 minutes.

Fig.3

Effect of plasma proteins on trapping hydrogen sulfide gas. (A) Comparison of sulfide level between samples trapped with plasma remaining in reaction vessel and samples trapped after removal of plasma. After 50 μL of plasma was incubated with 450 μL of 0.1M pH 2.6 phosphate buffer for 30 min, in the first group ∼25 μL of 3 M NaOH solution was added directly to plasma to adjust pH to 9.5 and then trapped for 30 min. In the other group, the plasma was removed from the reaction vessel and the released hydrogen sulfide was trapped by 500 μL of 0.1 M Tris-HCl buffer for 30min. (B) Effect of sulfide on protein persulfide formation. 0, 30, 300 μM of sodium sulfide was incubated with plasma and and the resulting generation of persulfide was measured at various time points. (C) Effect of sulfide on protein persulfide formation. Samples trapped with plasma had KCN added and then absorbance at 335 nm was measured.

Fig.4

Effect of TCEP on the derivatization of hydrogen sulfide with MBB. 40 μM sodium sulfide was reacted with MBB solution in the presence of 50mM TCEP (A) or 1 mM TCEP (B). (C) Effect of TCEP on diallyl trisulfide (DATS) reduction. 50 μL of 25 μM DATs was incubated with 450 μL of 0.1M phosphate buffer (pH 2.6, 1mM TCEP) at different time points, and the resulting hydrogen sulfide was trapped with 0.1M Tris-HCl buffer for 30min. (D) Efficiency of the protocol including reducing, releasing and trapping. 5 μM Na2S and 25 μM Dats were used with and without TCEP. (E) Effect of TCEP on stability of SDB across pH range.

Fig.5

Levels of free hydrogen sulfide, acid-labile sulfur and bound sulfane-sulfur in murine and human plasma. (A) C57BI/6J mice plasma in comparison with CSE-/- mice. (B) Human plasma from healthy male human controls.

Fig.6

Method overview illustration.