Measurement of Betaine‐Homocysteine Methyltransferase Activity via Fluorescent Detection of Free Thiols

High concentrations of homocysteine correlate with early onset heart disease and other disorders. The enzyme betaine-homocysteine methyltransferase (BHMT) consumes homocysteine by transferring a methyl group from betaine to the free thiol group of homocysteine to form methionine. BHMT activity in turn can cause an imbalance of betaine, leading to other health issues like fatty liver and digestive disorders. Clearly, then, understanding the activity and regulation of BHMT can lead to insights into several key health problems. Unfortunately, currently available in vitro BHMT enzyme assays include the use of NMR or HPLC and thus are either slow or not readily scalable. Our goals are to create a new high throughput in vitro assay to measure BHMT activity and to implement this assay to better understand the enzyme's regulation. This new assay utilizes the dye monobromobimane (mBBr), which becomes fluorescent upon conjugation to a free thiol group. The free thiol of BHMT substate homocysteine is consumed upon methylation; mBBr fluorescence, then, serves as a quantitative reporter of the progress of the BHMT reaction. We have confirmed that mBBr detects the model thiol, L-cysteine, in a 96-well plate format, and we can detect sub-nanomolar amounts of homocysteine deprotected from commercially available DL-homocysteine thiolactone. This assay has been used to follow the progress of BHMT activity and to investigate the effects of several potential inhibitors. The development of a simple fluorescent assay will enhance the speed of study of this and related enzymes and could spur high throughput searches for small molecule modulators.