A quantitative method for the assessment of homocysteine thiolactonase enzyme activity

This assay elucidates an accurate, simple, and precise protocol to quantify the activity of homocysteine thiolactonase (HTase). To establish HTase activity, the enzyme samples were incubated with a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, which contained suitable concentrations of the homocysteine thiolactone as a substrate. To stop the enzyme's reaction, the CUPRAC reagent (Cu(Nc)₂²⁺) was added after a suitable incubation time. The reduction of Cu(II)-neocuproine complex (Cu(Nc)₂²⁺) to highly coloured Cu(I)-neocuproine complex (Cu(Nc)₂⁺) by the produced homocysteine was quantified spectrophotometrically at 450 nm (CUPRAC method). The increase in the absorbance of the coloured Cu(I)-neocuproine complex (Cu(Nc)₂⁺) was correlated directly to the activity of HTase. ANOVA analysis was utilised to validate the new method against homocysteine thiolactonase activity using the H⁺ ions liberating method in matched samples. In conclusion, according to the obtained correlation coefficient (0.9995) from the comparison of the current method with the reference method, the current method is effective in assay HTase activity with high reliability.