Solution‐ and Crystal‐Phase Covalent Modification of Lysozyme by a Purpose‐Designed Organoruthenium Complex. A MALDI‐TOF MS Study of its Metal Binding Sites

Abstract

Study of the reaction between the transition organometallic complex 4-ruthenocenyl 2,6-dimethylpyrylium tetrafluoroborate and the enzyme hen egg white lysozyme (HEWL) in solution and by diffusion in crystals was performed by use of a combination of spectroscopic and chromatographic methods. Conjugation involving the lysine residues of lysozyme appeared to occur readily, yielding very stable ruthenocenyl pyridinium adducts with average degrees of incorporation ranging from 0.2 to 1.8 metal complexes per protein molecule, depending on reaction conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that the protein conjugates were in fact mixtures of unmodified, mono-, di- and sometimes tripyridinium adducts. In combination with reversed-phased HPLC, we were able to show that six different monoruthenocenyl pyridinium adducts were formed in solution. This result was confirmed by trypsin digestion of a ruthenocenyl pyridinium conjugate and MALDI-TOF MS analysis of the peptide mixture, which showed that lysines 1, 13, 33, 96, 97 and 116 were involved in the reaction with the pyrylium complex, lysines 13, 33 and 116 being the major binding sites. In the tetragonal crystal state, no binding of the ruthenium complex was shown to occur at lysine 116, owing to steric hindrance at this particular position.

Biophysical characterisation of adducts formed between anticancer metallodrugs and selected proteins: New insights from X-ray diffraction and mass spectrometry studies

“…Just a few additional structures, somehow connected to our topics, had been obtained by Jaouen and coworkers working on specific lysozyme modifications caused by a variety of organometallic agents [36,37]. In contrast, a quite conspicuous number of crystallographic structures were available in the HAD data bank in relation to the heavy atom replacement methodology [38].…”

Structural analysis of organometallic compounds with soft ionization mass spectrometry

“…In addition to iron, other central metals can be present in metallocenes. Polyisotopic titanocene intermediates of the Tebbe olefination reaction (Meurer et al, 2006) and Petasis olefination reaction (Meurer et al, 2003) were characterized with APCI-MS/ MS. MALDI-TOF MS in combination with reversed-phase HPLC was used to screen the reaction between ruthenocene derivate with hen egg white lysozyme (Salmain et al, 2004), and the application of ESI was published for ruthenocenes FIGURE 8. MALDI-TOF mass spectrum of products obtained from the reaction of 1aLi 2 with [FeCl 2 (thf) 1.5 ] (reproduced with permission from Kohler, Schell, & Weber, 2002).…”

Simple Synthesis of Ruthenium π Complexes of Aromatic Amino Acids and Small Peptides

“…In this respect, it is interesting to note that lysine residues located on the surface of lysozyme have been successfully labeled recently by a ruthenocenyl pyrillium salt. [17] Conclusion A simple approach to ruthenium p complexes of aromatic amino acids and small peptides was developed. The synthe-sis was carried out in water at room temperature in the presence of oxygen and concomitant biological substances (nonaromatic amino acids, sugars, and nucleotides).…”