Kinetic investigations of RNase T1

To characterise RNase T1 variants, enzyme kinetic investigations were performed to obtain reaction parameters. The standard assay uses dinucleoside monophosphates (normally 5',3'-GpC) as substrate and follows the first reaction step (the transesterification) spectrophotometrically by the change of extinction at 280 nm[26] due to unstacking effects. However, 5',3'-GpC itself absorbs light at 280 nm, thereby interfering with the detection and limiting the maximal substrate concentration. Using more native substrates like tri- or tetranucleotides is almost impossible. Furthermore, this method only allows investigations of the first reaction step, as it is not possible to detect the hydrolysis of the cyclic 2',3'-phosphodiester (the intermediate) simultaneously. Chromatographic assays with TLC or HPLC are also used, but are limited, too. TLC does not allow a reliable quantification of the reaction products without further techniques like $^{32}$P labelling, and both chromatographic methods require a long separation time which limits the time resolution. Furthermore, the detection and elucidation of unknown products is difficult or impossible using the techniques described above.