TRANSITION-METAL COMPLEXES AS ALTERNATIVES TO PROTEOLYTIC-ENZYMES - REGIOSELECTIVE CLEAVAGE OF MYOGLOBIN BY PALLADIUM(II) AQUA COMPLEXES

The palladium(II) aqua complexes [Pd(H2O)(4)](2+), cis-[Pd(en)(H2O)(2) ](2+), and cis-[Pd(dtco-OH)(H2O)(2)](2+) effect hydrolytic cleavage of horse myoglobin in aqueous solution. The conditions were optimized wi th the third complex. Its structure was determined by X-ray crystallog raphic analysis of its precursor, the square-planar complex cis-[Pd(dt co-OH)Cl-2], in which the chelating ligand adopts a boat-chair conform ation. A weak interaction between the hydroxyl group and the palladium (II) atom seems to improve the stability of the reagent. The yield of cleavage after a 24-h incubation at 60 degrees C increases from 39% to 85% as the pH decreases from 6.2 to 3.2. The protein fragments are se parated by SDS-PAGE electrophoresis and HPLC separation methods, and i dentified by ESIMS and MALDI-TOF mass spectrometric methods and by det ermination of terminal amino-acid sequences. Most of the 13 cleavage s ites are clustered around the methionine, arginine, and some of the hi stidine residues, whose side chains can bind to palladium(II). Cleavag e tends to occur at the peptide bonds one to three positions removed f rom the binding residues; the scissile bonds usually lie on the amino- terminal side, seldom on the carboxy-terminal side, of the binding res idues. Removal of the heme and unfolding of the protein do not drastic ally alter the pattern of cleavage; The ability of palladium(II) aqua complexes to cleave proteins at relatively few sites, with explicable selectivity, with good to very good yield, and in weakly acidic and ne arly neutral solutions, bodes well for their future use in biochemical and bioanalytical practice.