Solution- and crystal-phase covalent modification of lysozyme by a purpose-designed organoruthenium complex. A MALDI-TOF MS study of its metal binding sites.
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.
Salmain
Michèle
M
Ecole Nationale Supérieure de Chimie de Paris, Laboratoire de Chimie Organométallique, UMR CNRS 7576, 75231 Paris cedex 05, France. salmain@ext.jussieu.fr
Caro
Bertrand
B
Le Guen-Robin
Françoise
F
Blais
Jean-Claude
JC
Jaouen
Gérard
G
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D004058:Diffusion; D004529:Egg White / Q000032:analysis; D006868:Hydrolysis; D008670:Metals / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry*; D009942:Organometallic Compounds / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011492:Protein Hydrolysates / Q000737:chemistry; D012428:Ruthenium / Q000737:chemistry*; D012996:Solutions; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000737:chemistry
D008670:Metals; D009942:Organometallic Compounds; D010455:Peptides; D011492:Protein Hydrolysates; D012996:Solutions; D012428:Ruthenium; D009113:Muramidase; D014357:Trypsin