Departamento de Química, Facultad de Ciencias, Universidad de Burgos, Plaza de Misael Bañuelos s/n, 09001, Burgos, Spain.
CQC, Department of Chemistry, University of Coimbra, Rua Larga, 3004-535, Coimbra, Portugal.
Sci Rep. 2022 May 25;12(1):8818. doi: 10.1038/s41598-022-12848-4.
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.
我们已经成功制备了具有长期环境稳定性（在溶液中超过 35 周）的完全水溶性荧光肽底物，并且在使用该探针测定酶活性时得到了稳定的结果。我们通过制备商业 N-乙烯基-2-吡咯烷酮（99.5%摩尔）和荧光基质的共聚物来实现这一目标，该基质用于测定胰蛋白酶活性，具有乙烯基基团（0.5%摩尔）。我们已经在水相中测量了这种聚合物的胰蛋白酶活性，与商业底物 Z-L-Arg-7-氨基-4-甲基香豆素盐酸盐及其专为此制备的单体衍生物的活性进行了对比。最初，感应聚合物的活性为 74.53 ± 1.72 nmol/min/mg 酶，而商业底物的活性为 20.44 ± 0.65 nmol/min/mg 酶，前者在数周内保持稳定，而后者在三周内迅速衰减至零。通过 UV-Vis 光谱、稳态和时间分辨荧光、热重分析和等温滴定量热法研究了聚合物链的“保护”作用。
