CHARACTERIZATION OF SERRATIA-MARCESCENS NUCLEASE ISOFORMS BY PLASMA DESORPTION MASS-SPECTROMETRY

Isoforms of Serratia marcescens nuclease found in the natural nuclease produced by S. marcescens and in recombinant nuclease produced by Esc herichia coli were structurally characterized by peptide mapping using plasma desorption mass spectrometry. The nuclease isoforms produced a nd secreted from S. marcescens B10M1, which are present in much greate r amounts than in S. marcescens W225 nuclease produced by E. coli, wer e characterized completely and the information used to facilitate char acterization of the recombinant nuclease isoforms. After purification of the nuclease the isoforms were separated on a DEAE-cellulose anion- exchange column and then digested with endoproteinase Lys-C. The pepti des generated were isolated by reverse-phase HPLC and their molecular masses determined by plasma desorption mass spectrometry. Comparison o f the peptides from the native nuclease, Sm2, and the two isoforms, Sm 1 and Sm3, revealed that they differed only in the N-terminus, the lat ter being found to lack three amino acids in Sm1 and one amino acid in Sm3. No interior post-translational changes were found in either of t he three isoforms. Using this information we were able to confirm that Sm1, the isoform lacking three amino acids, was also present in very small amounts in recombinant S. marcescens W225 nuclease produced and excreted by E. coli.