Assessing the impact of product inhibition in a chromogenic assay.
Anal Biochem
; 580: 62-71, 2019 09 01.
Article
en En
| MEDLINE
| ID: mdl-31091429
ABSTRACT
Chromogenic substrates (CS) are synthetic substrates used to monitor the activity of a target enzyme. It has been reported that some CSs display competitive product inhibition with their target enzyme. Thus, in assays where enzyme activity is continuously monitored over long periods of time, the product inhibition may significantly interfere with the reactions being monitored. Despite this knowledge, it is rare for CSs to be directly incorporated into mathematical models that simulate these assays. This devalues the predictive power of the models. In this study, we examined the interactions between a single enzyme, coagulation factor Xa, and its chromogenic substrate. We developed, and experimentally validated, a mathematical model of a chromogenic assay for factor Xa that explicitly included product inhibition from the CS. We employed Bayesian inference, in the form of Markov-Chain Monte Carlo, to estimate the strength of the product inhibition and other sources of uncertainty such as pipetting error and kinetic rate constants. Our model, together with carefully calibrated biochemistry experiments, allowed for full characterization of the strength and impact of product inhibition in the assay. The effect of CS product inhibition in more complex reaction mixtures was further explored using mathematical models.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Factor Xa
/
Compuestos Cromogénicos
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Anal Biochem
Año:
2019
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
EEUU
/
ESTADOS UNIDOS
/
ESTADOS UNIDOS DA AMERICA
/
EUA
/
UNITED STATES
/
UNITED STATES OF AMERICA
/
US
/
USA