A highly sensitive single-enzyme homocysteine assay

A protocol for measuring the total plasma homocysteine (tHCY) concentration in very small samples using a selective, recombinant homocysteine alpha, gamma-lyase ( rHCYase) and a small portable fluorescence reader is described. The rHCYase produces hydrogen sulfide (H2S) from tHCY without interference from physiological concentrations of Cys or other plasma components. H2S is determined through reactivity with N,N-dibutyl phenylene diamine (DBPDA), which leads to the formation of a fluorescent chromophore. Only 5 mu l of plasma/serum sample is required, which can be obtained from a finger prick, suggesting great potential for mass screening. The assay takes similar to 20 min.