Multiple forms of medicinal leech destabilase-lysozyme

Earlier, three genes Ds1, Ds2, and Ds3 encoding corresponding destabilase-lysozyme isoforms were identified. However only one form of the enzyme encoded by Ds3 gene coincided with the protein CNBr fragments [Mol. Gen. Genet. 253 (1996) 20]. In this work we found by ESI-TOF mass spectrometry that the enzyme preparation consists of at least three forms with molecular masses of 12677.6, 12839.7, and 12938.2Da, each of which contains seven disulfide bridges. Only one mass (12839.7Da) fits to the calculated mass for the protein encoded by Ds3 gene. Further analysis of the CNBr fragments of the enzyme showed the heterogeneity of large 5.5 kDa peptide at positions 64 (threonine or arginine) and 67 (histidine or arginine) in the wild-type amino acid sequence. One CNBr peptide, with Arg and His at positions 64 and 67, respectively, correlates in the molecular mass with the protein encoded by Ds3. In addition, we have found a new acid form of destabilase-lysozyme, P-Ac, which differs from all known destabilase-lysozyme structures by its N-terminal amino acid sequence.