To better understand the function of YphP, we determined its reduction potential and analyzed its isomerase activity by using a continuous assay that measures the rate of isomerization of non-native to native disulfide bonds in tachyplesin I (10).
Given that SAMHD1 both hydrolyzes and is regulated by dNTPs, we developed a continuous assay for SAMHD1 activity in order to determine its quantitative kinetic parameters and to assess if nucleotides, nucleotide analogues, and Nt-AIs are substrates, activators, or inhibitors.
The reaction was initiated by addition of substrate (300 µM MeOSuc-AAPV-AMC) and enzyme substrate hydrolysis was monitored in a spectrofluorimeter in a continuous assay, by measuring the release of fluorescence (excitation 380 nm, emission at 460 nm).
Each is a continuous assay, free from interferences caused by other components present in crude enzyme preparations, and allows meaningful and reproducible kinetic parameters to be determined.
For substrates 63 and 64, whose turnover was substantially faster than 12 and 13, we were able to employ a continuous assay that monitors consumption of NADPH by a decrease in absorbance at 340 nm.
The next logical development was a continuous assay for Dam activity in a format suitable for high throughput analysis.
Hydroxypyruvate reductase activity was determined at 70°C using a continuous assay.
Methylsuccinyl-CoA dehydrogenase was assayed by a continuous photometric assay using ferrocenium hexafluorophosphate as an artificial electron acceptor; alternatively, a discontinuous assay with HPLC analysis was used as reported in [15].
The activity of the supported enzymes was determined by a continuous electrochemical assay involving the hydrolysis of N-acetyl-l-tyrosine ethyl ester.
The DPP-IV-like enzymatic activity in the heparinized blood plasma and in BMNC was measured by a continuous rate fluorimetric assay with 7- glycyl-prolylamido -4-methylcoumarin (Bachem, Switzerland, final concentration 50 μmol/L), as a substrate at pH 7- glycyl-prolylamido -4-methylcoumarin
