132P London, UK
Pharmacology 2017

 

 

Development of a SF-7 AM based fluorescence assay for detection of Hydrogen Sulphide in the Porcine Heart

Y. Y. AL-TAIE, R. E. Roberts, M. J. Garle, S. P. Alexander. School of Life Sciences/ Cardiovascular Research Group, School of Life Sciences/ Medical School, Nottingham, United Kingdom.

Introduction: Hydrogen sulphide (H2S) is mainly synthesised endogenously from L-cysteine through the enzymes Cystathionine β-Synthase (CBS) and Cystathionine γ-lyase (CSE) and from 3-mercaptopyruvate through 3-mercaptopyruvate sulphurtransferase (3-MST)1. There is an increasing evidence for generation and activity of H2S in the vasculature and there is much less information about its role in the heart. The aim of this study was to develop an assay to measure the H2S-production in the myocardium.

Methodology: Cytosolic fractions of porcine hearts (PHC) were prepared by homogenisation in Tris-EDTA buffer and centrifugation at 30,000 g for one hour. H2S production through the CBS/CSE pathway was determined using 10mM L-cysteine, whereas H2S production through the MST pathway was determined using 10 mM mercaptopyruvate as substrate. H2S generation from these substrates was measured using the methylene blue (MB)2 assay and compared to that detected using the fluorescent probe sulfidefluor-7 acetoxymethyl ester (SF7-AM) 3. Comparisons between more than two data group were made using ANOVA followed by Sidak’s post-hoc test. For comparisons between two data sets, a two-tailed unpaired/paired Student’s t-test was carried out. A P-value of less than 0.05 indicated a significant difference between the data set; n= number of animals.

Results: The optimal incubation time in the methylene blue assay was 60 minutes for CBS/CSE pathway and 30 minutes for MST pathway while it was 90 minutes for SF7-AM assay in both CBS/CSE and MST pathways. Increasing the pH of the incubation buffer from 7.4 to 9 resulted in an increase in the detection of H2S production in the CBS/CSE pathway measured by MB and SF7-AM assays. Increasing the pH to 9 caused an increase in H2S detected through the MST pathway by the MB assay, but not using the SF7-AM assay. Dialysis of the PHC (using semipermeable membrane) resulted in an increase in H2S detected through the CBS/CSE and MST assays using the SF7 assay (table 1).

Table 1: The effects of modifications on H2S detection:

Modification Control Modification n p-value
pH 9 (MB) 5.7±0.5 nmoles/ mg protein 7.6±0.7 nmoles/ mg protein 6 <0.05
pH 9 (SF7-AM/ CBS/CSE) 24.6±2.6 relative fluorescence unit /mg protein 36.63± 3.7 relative fluorescence unit/ mg protein 6 <0.05
pH 9 (SF7-AM/ MST) 32.4±3.3 relative fluorescence unit/ mg protein 37.9±4.5 relative fluorescence unit/ mg protein 6 NS
Dialysis 8.7±1.7 relative fluorescence unit/ mg protein 16.49±2.0 relative fluorescence unit/ mg protein 6 <0.05

Conclusion: In short, SF7-AM can be used to detect H2S production through both the CBS/CSE pathways and the MST pathway in the porcine heart. Dialysis seems to lead to an increase in H2S detected, which may be due to a removal of an endogenous inhibitor.

References:

(1) Dunn et al., (2016) Pharmacol Ther,158: 101-113.

(2) Rashid et al., (2013) British Pharm, 168, 1902-1910.

(3) Lin et al., (2013) PNAS, 110 (18), 7131-5.

(4) Yasir et al., (2016) BPS Winter Meeting.