| Popis: |
In order to develop a new detection technique for a DNA probe method, subtilisin, a protease with favorable thermal stability for the method, was examined for its ability to digest a substrate carrying electron spin. To prepare the substrate for subtilisin, a spin label, 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl, was bound on carboxyl groups of hydrophilic polymer beads by N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide through glycyl- l -phenylalanine linkage. Before the subtilisin treatment, only weak broadened ESR signals were given from a suspension of this substrate in aqueous media. After the subtilisin digestion, however, sharp signals with high intensity were obtained, indicating that proteases can be used as labeling enzymes in a DNA probe method coupled with ESR detection upon usage of such a specially designed substrate. |
