Continuous UV monitoring of fluorogenic substrates. I. Kinetic analysis of N-acetyl-beta-D-hexosaminidases

Determination of the uv absorption spectra of 4-MUF-GlcNAc and 4-MUF showed them to differ significantly at 350 nm. This finding was applied to the enzymatic hydrolysis of fluorogenic 4-MUF-O-substituted substrates with a continuous, spectrophotometric assay procedure. With the use of this automated technique, selected kinetic properties, i.e., Ki, Km, and V, of “purified” liver N-acetyl-β-d-hexosaminidases A and B and of crystalline Jack bean meal hexosaminidase were determined and found to be in close agreement with previousy published data obtained by conventional single point assay methods. The described technique is fast, accurate, and permits instantaneous measurements of the kinetic properties of certain enzymes implicated in a number of genetic disorders.