I am facing a problem to get a positive enzymatic reaction of ACE as a catalyst to hydrolyze a substrate (HHL) which liberates Hippuric acid (HA) and Hip-Leu (HL).
To be clear on the research progress, I had prepared 5 mM HHL (100 uL), 50 mU/ml & 100 mU/mL ACE (25 uL), inhibitor (food sample) (50 uL). For every sample (Inhibitor), a set of assay including sample (with inhibitor), control (w/o inhibitor), and blank (terminated enzyme reaction) was carried out.
The HHL and sample (Inhibitor) were combined and maintained at 37C for 10 min. ACE was also maintained at 37C for 10 min before the 2 solutions were combined and incubated at 37C. The reaction was terminated with 1M HCL and the solution filter through 0,2 um.
The determination of ACE-inhibitory activity was performed using UPLC. A good chromatogram profiling of standard peaks (HA and HHL) were obtained.
A positive result should yield a higher HA in control treatment, and lower HA in sample treatment containing inhibitor. Blank value must be much lower than those mentioned treatments.
However, the result from chromatogram profiling turned out to be negative (quite often) since the production of HA in sample treatment was relatively higher than control.
In addition, the result seemed unconvincing since the produced HA was too little to rely on as a positive data over 30 minutes incubation duration. Was the prepared concentration of enzyme insufficient or was this particular enzyme reaction definitely a slow reaction?
I suspect a false orientation of enzymatic reaction was implemented. Which step of pre-treatment of enzymatic reaction involving enzymatic inhibitor is correct?
Incubation of substrate with inhibitor (sample) prior addition of enzyme (ACE) or
incubation of enzyme with inhibitor (sample) prior addition of substrate (HHL)?
Comments and guidance on this queries are highly appreciated..
Incubate the enzyme with inhibitor before addition of substrate thats the correct sequence. What do you mean when you say you are facing problems getting a positive enzymatic reaction??
Ok didn"t read the rest of your question, Im also facing a similar problem, Im using Captopril as a positive control for my assays but Im getting even higher activity in comparison to my control,did you figure out why this was happening??
Well i can't answer ur question as I'm facing the same problem..the results seem inconsistent (as always)..sometimes my blank got even higher than sample n control..which assay technique u r using?
Ive been ussing the same protocol except I am detecting His-Leu and not HA using 1% OPA reagent and then measuring flourescence at an excitation of 365nm and emission of 495nm,
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