DETERMINATION OF DISULFIDE BRIDGES IN NATURAL AND RECOMBINANT INSECT DEFENSIN-A

The primary-structure comparison of natural insect defensin A from Phormia terranovae and recombinant insect defensin A from Saccharomyces cerevisiae has been accomplished using a combination of Edman degradation and liquid secondary ion mass spectrometry. The natural and recombinant proteins have the same primary structure with identical disulfide-bond designations activative (Cys3 Cys30, Cys16 Cys36 and Cys20 Cys38) and determined from the peptide obtained after thermolysin digestion. The combined use of Edman degradation and mass spectrometry allowed the disulfide-bridge structure to be determined with a total of only 40-mu-g (9.9 nmol) natural peptide. Mass spectrometry provides a rapid means of disulfide-bridge verification, requiring not more than 20-mu-g recombinant insect defensin A, which is compatible with use in batch analysis.