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 with the third complex. Its structure was determined by X-ray crystallographic analysis of its precursor, the square-planar complex cis-[Pd(dtco-OH)Cl₂], in which the chelating ligand adopts a boat-chair conformation. 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  °C increases from 39% to 85% as the pH decreases from 6.2 to 3.2. The protein fragments are separated by SDS-PAGE electrophoresis and HPLC separation methods, and identified by ESIMS and MALDI-TOF mass spectrometric methods and by determination of terminal amino-acid sequences. Most of the 13 cleavage sites are clustered around the methionine, arginine, and some of the histidine residues, whose side chains can bind to palladium(II). Cleavage tends to occur at the peptide bonds one to three positions removed from the binding residues; the scissile bonds usually lie on the amino-terminal side, seldom on the carboxy-terminal side, of the binding residues. Removal of the heme and unfolding of the protein do not drastically 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 nearly neutral solutions, bodes well for their future use in biochemical and bioanalytical practice. Received: 23 December 1997 / Accepted: 14 April 1998