Conclusions

The investigation of cleavage patterns generated by the hydolysis of a well defined substrate by a polymer cleaving enzyme provides a deeper insight in the mechanisms lying underneath the enzymatical reaction. The time dependant occurence of cleavage products reveals preference of the enzyme with regard to possible points of attack. Using a well-designed substrate with explicite difference between the different cleavage positions leads to a large yield in information. However, CPA can not provide reaction parameters as it is difficult to perform quantification on MALDI-TOF-MS data. Furthermore, MALDI-MS hardly can be used to prove the absence of a certain compound.

CPA with RNase T1 and RNase T1 RV showed the capabilities of this technique. Using a substrate with to different cleavage positions - one at each terminus of the eight-nucleotide-long oligonucleotide - it has been possible to reveal a preference of RNase T1 for a cleavage position embedded into a longer oligonucleotide chain. This may be either due to a general preference of RNase T1 towards longer substrates or due to binding site beyond $N1^B_S$.