Internal Hydrolysis Indicator for Sample Specific Monitoring of ß-Glucuronidase Activity.

Metabolized forms of benzodiazepines (benzos) can cause issues with mass spectrometry identification. Benzodiazepines undergo a process called glucuronidation during metabolism that attaches a glucuronic acid for increased solubility. Often in clinical testing an enzymatic hydrolysis step is implemented to increase the sensitivity of benzodiazepines by hydrolyzing ß-D-glucuronic acid from benzodiazepine-glucuronide conjugates in urine samples using the ß-Glucuronidase enzyme. In this study resorufin ß-D-glucuronide, a substrate of the ß-Glucuronidase enzyme, was added to patient samples to determine if proper hydrolysis had occurred. The presence of resorufin as an Internal Hydrolysis Indicator (IHI) shows the activity and efficiency of the enzyme in each patient sample. Synthetic/patient urine samples were obtained and mixed with hydrolysis buffer containing resorufin ß-D-glucuronide. The ß-Glucuronidase enzyme was used to hydrolyze the benzodiazepine analytes as well as resorufin ß-D-glucuronide. The enzymatic hydrolysis addition increased the positivity rate of benzodiazepines by 42.5%. The ß-Glucuronidase substrate resorufin (IHI) displayed variability in area counts between patient samples. Comparative studies with internal standards and resorufin (IHI) showed no correlation between recovery and analyte variability. Hydrolysis reactions greatly improved the sensitivity of benzodiazepines by liquid chromatography time-of-flight mass spectrometry analysis. The large variation in resorufin (IHI) area counts amongst patient samples indicates possible variability in enzymatic hydrolysis activity. The enzymatic hydrolysis step is a part of the extraction procedure and should be controlled for in each patient sample.

The Mtr4 ratchet helix and arch domain both function to promote RNA unwinding.

Mtr4 is a conserved Ski2-like RNA helicase and a subunit of the TRAMP complex that activates exosome-mediated 3'-5' turnover in nuclear RNA surveillance and processing pathways. Prominent features of the Mtr4 structure include a four-domain ring-like helicase core and a large arch domain that spans the core. The 'ratchet helix' is positioned to interact with RNA substrates as they move through the helicase. However, the contribution of the ratchet helix in Mtr4 activity is poorly understood. Here we show that strict conservation along the ratchet helix is particularly extensive for Ski2-like RNA helicases compared to related helicases. Mutation of residues along the ratchet helix alters in vitro activity in Mtr4 and TRAMP and causes slow growth phenotypes in vivo. We also identify a residue on the ratchet helix that influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain has minimal effect on Mtr4 unwinding activity. We now show that combining the arch deletion with ratchet helix mutations abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates.