Purification of Brussels sprout isoperoxidases

Extracts of Brussels sprouts contain a complex mixture of isoperoxidases. Purification of the anionic and cationic groups of isoenzymes by ion-exchange chromatography has resulted in identification of four distinct isoperoxidases (A1; A2, C1 and C2) of different molecular size. Each preparation showed single staining bands for isoperoxidase activity and by the more sensitive silver staining technique for protein. However, only the C2 isoperoxidase preparation showed a single band by Western blotting against polyclonal horseradish antibody, whereas the other isoenzymes still showed cross contamination with other isoperoxidases although minor in the instances for the a1 and A2 isoperoxidases. Also these results show that, even after extensive chromatographic purification of peroxidases, it is still necessary for assessment of homogeneity, prior to structural studies, to use the more sensitive Western antibody technique. Amino acid sequencing yielded a short identical sequence for A2 and C1 which was also 95% identical to some previously detected drought/salt stress proteins.