Identification of placental transforming growth factor-beta and bikunin metabolites as contaminants of pharmaceutical human chorionic gonadotrophin preparations by proteomic techniques.
A contaminant protein complex found in pharmaceutical urinary human chorionic gonadotrophin preparations is reported to have anti-human immunodeficiency virus-associated Kaposi's sarcoma activity. The aim of this study was to isolate and characterize this protein complex by proteomic approaches. Size exclusion chromatography was used in the isolation of these human chorionic gonadotrophin-associated fragments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of a protein complex that dissociated into two protein bands under reducing conditions. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of this complex showed three polypeptides at approximately 6.2, 11.4, and 15.8 kDa. Peptide mass mapping and N-terminal amino acid sequencing identified two polypeptides as metabolites of placental transforming growth factor-beta (11.4 kDa) and bikunin (15.8 kDa). Subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of the anti-human immunodeficiency virus-associated Kaposi's sarcoma active preparations CG-10 (Sigma), Pregnyl (Organon), and Profasi (Serono) revealed the presence of metabolites of placental transforming growth factor-beta in all three; no other non-human chorionic gonadotrophin-related protein species were observed in these preparations. Our findings present evidence that urinary human chorionic gonadotrophin preparations are contaminated with metabolites of placental transforming growth factor-beta, which may have transforming growth factor-beta agonist actions, and metabolites of bikunin, which is a protease inhibitor. In combination these molecules may be responsible for the anti-human immunodeficiency virus-associated Kaposi's sarcoma activity demonstrated for these urinary human chorionic gonadotrophin preparations.
Malatos
Sotiris
S
Biomedical Sciences, Institute of Social and Health Research, Middlesex University, Enfield EN3 4SA, United Kingdom.
Neubert
Hendrik
H
Kicman
Andrew T
AT
Iles
Ray K
RK
D000595:Amino Acid Sequence; D006063:Chorionic Gonadotropin / Q000032:analysis* / Q000627:therapeutic use; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015658:HIV Infections / Q000150:complications; D006801:Humans; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000378:metabolism; D008969:Molecular Sequence Data; D010920:Placenta / Q000378:metabolism; D040901:Proteomics; D012514:Sarcoma, Kaposi / Q000188:drug therapy / Q000209:etiology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016212:Transforming Growth Factor beta / Q000032:analysis* / Q000378:metabolism; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000032:analysis* / Q000378:metabolism
D006063:Chorionic Gonadotropin; D008562:Membrane Glycoproteins; C109148:SPINT2 protein, human; D016212:Transforming Growth Factor beta; D014360:Trypsin Inhibitor, Kunitz Soybean