A fluorometric method for measuring homocysteine thiolactonase activity

Abstract

The enzyme homocysteine thiolactonase (HTase) is involved in the metabolism of homocysteine, an amino acid. As a byproduct of the metabolism of methionine, a secondary amino acid, homocysteine (Hcy), a sulfur-containing amino acid, is produced. Hcy levels in the blood are linked to higher risks of heart disease, stroke, and other disorders. To create Hcy, HTase catalyzes the hydrolysis of homocysteine thiolactone (HTL). This process helps control the body's homocysteine levels and prevents the accumulation of homocysteine in the blood.This essay describes a precise, easy-to-follow procedure for measuring homocysteine thiolactonase (HTase) activity. This protocol (NAM-HTase) determines HTase activity via incubation with γ-thiobutyrolactone, an enzyme substrate, in a pH-appropriate solution at 37 °C for five minutes. It then uses thiol fluorometry to monitor the produced thiol groups. The newly formed thiol groups are reacted with the fluorescent probe N-(9-acridinyl)maleimide (NAM) and quantified using fluorometric intensity at an Ex/Em of 360/432 nm to evaluate HTase activity. The method is linear for 4-mercaptobutyric acid and homocysteine, from 0.005 ± 0.001 to 6 ± 0.04 µM. By comparing the HTase activity in matched samples to that of a standard method, this new protocol was found suitable for clinical applications. Its accuracy compared to other methodologies was shown by a correlation of 0.9995 between its values and those of a standard method. Using the H+ ion releasing method in matched samples, analysis of variance was used to verify the novel method against HTase activity. In conclusion, the proposed NAM-HTase method successfully measured HTase activity with high reliability.