Reaction of hen egg white lysozyme with Fischer-type metallocarbene complexes - Characterization of the conjugates and determination of the metal complex binding sites

The introduction of heavy atoms into protein crystals is sometimes rendered difficult and tedious because of the poor specificity of the available rea gents for particular target residues. On the other hand, transition organom etallic chemistry offers an almost untouched field for this purpose. In par ticular, Fischer-type metallocarbene complexes of the general formula (CO)( 5)W=C(OR1)R-2 may be attractive reagents because they contain the heavy ele ment tungsten and specifically target amino groups to form stable, covalent aminocarbene adducts. With a small protein such as hen egg white lysozyme (HEWL) with a limited number of potential binding sites, it was possible to form protein-aminocarbene conjugates that have an average of one aminocarb ene moiety per protein molecule. RP-BPLC combined with matrix-assisted lase r desorption ionization time-of-flight (MALDI-TOF) MS analysis of the conju gates revealed that they were mixtures of the native protein, monoaminocarb enes and diaminocarbenes. Tryptic proteolysis experiments performed on the protein conjugates combined with MALDI-TOF-MS analysis of the aminocarbenic peptides allowed us to determine that lysines 13, 33, 97 and 116 were invo lved in the reaction of HEWL with (CO)(5)W=C(OMe)Me.