STRUCTURAL DETERMINATION OF THE CONJUGATE OF HUMAN SERUM-ALBUMIN WITHA MITOMYCIN-C DERIVATIVE, KW-2149, BY MATRIX-ASSISTED LASER DESORPTION IONIZATION MASS-SPECTROMETRY/

new mitomycin C derivative, KW-2149, is known to form a covalent conju gate with human serum albumin (HSA). This conjugate exhibits 1/20 of t he anticellular activity of unconjugated KW-2149. Structural studies o f this conjugate were carried out using a combination of enzymatic dig estion, high-performance liquid chromatography (HPLC), and matrix-assi sted laser desorption/ionization (MALDI) mass spectrometry. The trypti c peptide T5 (residues 21-41) was the only peptide found to be modifie d by KW-2149 moieties, the [(gamma-L-glutamylamino)ethyl]thio group or the (2-aminoethyl)thio group, through a disulfide bond. Although the latter peptide lost its mitomycin C moiety in the course of tryptic di gestion, these data strongly suggest that KW-2149 was bound to Cys-34, the only free cysteine on KSA.