Real-time detection of enzymatically formed hydrogen sulfide by pathogenic variants of cystathionine beta-synthase using hemoglobin I of Lucina pectinata as a biosensor.

Classical homocystinuria is a rare disease caused by mutations in cystathionine β-synthase (CBS) gene (OMIM 613381). CBS catalyzes the first step of the transsulfuration pathway that converts homocysteine (Hcy) into cystathionine (Cysta) via a number of co-substrates and mechanisms. Formation of Cysta by condensation of Hcy and cysteine (Cys) produces a molar equivalent of hydrogen sulfide (H 2 S). H 2 S plays important roles in cognitive and vascular functions. Clinically, patients with CBS deficiency present with vascular, ocular, neurological and skeletal impairments. Biochemically, CBS deficiency manifests with elevated Hcy and reduced concentration of Cysta in plasma and urine. A number of pathogenic variants of human CBS have been characterized by their residual enzymatic activity, but very few studies have examined H 2 S production by pathogenic CBS variants, possibly due to technical hurdles in H 2 S detection and quantification. We describe a method for the real-time, continuous quantification of H 2 S formed by wild-type and pathogenic variants of human recombinant CBS, as well as by fibroblast extracts from healthy controls and patients diagnosed with CBS deficiency. The method takes advantage of the specificity and high affinity of hemoglobin I of the clam Lucina pectinata toward H 2 S and is based on UV-visible spectrophotometry. Comparison with the gold-standard, end-point H 2 S quantification method employing monobromobimane, as well as correlations with CBS enzymatic activity determined by LC-MS/MS showed agreement and correlation, and permitted the direct, time-resolved determination of H 2 S production rates by purified human recombinant CBS and by CBS present in fibroblast extracts. Rates of H 2 S production were highest for wild-type CBS, and lower for pathogenic variants. This method enables the examination of structural determinants of CBS that are important for H 2 S production and its possible relevance to the clinical outcome of patients.