The role of polymeric chains as a protective environment for improving the stability and efficiency of fluorogenic peptide substrates.
Ana Arnaiz, Marta Guembe-García, Estefanía Delgado-Pinar, Artur J M Valente, Saturnino Ibeas, José M García, Saúl Vallejos
Author Information
Ana Arnaiz: Departamento de Química, Facultad de Ciencias, Universidad de Burgos, Plaza de Misael Bañuelos s/n, 09001, Burgos, Spain.
Marta Guembe-García: Departamento de Química, Facultad de Ciencias, Universidad de Burgos, Plaza de Misael Bañuelos s/n, 09001, Burgos, Spain.
Estefanía Delgado-Pinar: CQC, Department of Chemistry, University of Coimbra, Rua Larga, 3004-535, Coimbra, Portugal.
Artur J M Valente: CQC, Department of Chemistry, University of Coimbra, Rua Larga, 3004-535, Coimbra, Portugal.
Saturnino Ibeas: Departamento de Química, Facultad de Ciencias, Universidad de Burgos, Plaza de Misael Bañuelos s/n, 09001, Burgos, Spain.
José M García: Departamento de Química, Facultad de Ciencias, Universidad de Burgos, Plaza de Misael Bañuelos s/n, 09001, Burgos, Spain.
Saúl Vallejos: Departamento de Química, Facultad de Ciencias, Universidad de Burgos, Plaza de Misael Bañuelos s/n, 09001, Burgos, Spain. svallejos@ubu.es.
We have faced the preparation of fully water-soluble fluorescent peptide substrate with long-term environmental stability (in solution more than 35 weeks) and, accordingly, with stable results in the use of this probe in determining the activity of enzymes. We have achieved this goal by preparing a co-polymer of the commercial N-vinyl-2-pyrrolidone (99.5% mol) and a fluorescent substrate for trypsin activity determination having a vinylic group (0.5%). The activity of trypsin has been measured in water solutions of this polymer over time, contrasted against the activity of both the commercial substrate Z-L-Arg-7-amido-4-methylcoumarin hydrochloride and its monomeric derivative, prepared ad-hoc. Initially, the activity of the sensory polymer was 74.53 ± 1.72 nmol/min/mg of enzyme, while that of the commercial substrate was 20.44 ± 0.65 nmol/min/mg of enzyme, the former maintained stable along weeks and the latter with a deep decay to zero in three weeks. The 'protection' effect exerted by the polymer chain has been studied by solvation studies by UV-Vis spectroscopy, steady-state & time resolved fluorescence, thermogravimetry and isothermal titration calorimetry.
