Optimization of an Assay for High Throughput Measurement of Lysyl Oxidase Activity

Activity assays quantitatively measure enzyme activity and investigate the effectiveness of drugs. Reagents that react specifically with functional groups created by the enzyme allow for direct measurements, unlike many commercially available byproduct-based assays. Lysyl Oxidase (LOX) family of proteins are extracellular matrix crosslinking proteins. Particularly, Lysyl Oxidase Like 2 (LOXL2) has been identified as a target for treating vascular stiffening in isolated systolic hypertension. This hypertension frequently occurs with aging and results from central artery stiffening, a process mediated by LOXL2. Hence, there is interest in developing drugs targeting LOXL2. Recently, an assay has been developed which uses hydrazide reagents to directly label semi-aldehydes produced by LOX family enzymes. The study optimizes assay performance regarding 1) LOXL2 substrate content, 2) hydrazide concentration, and 3) incubation time for efficient screening of LOXL2 inhibitors. This versatile approach of labeling catalysis byproducts can be applied to studying enzyme activity involved in other diseases.