Tumor pretargeting with biotinylated antibody/avidin complexes improves the therapeutic index of systemically administered biotin-tumor necrosis factor (TNF) conjugates. Since the number of biotins in this conjugate is known to be critical for activity, we have characterized the structure of different biotin-TNF conjugates, prepared by reaction with d-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester and identified the biotinylation sites by trypsin digestion, reverse-phase chromatography, and electrospray mass spectrometry analyses. The results have shown that N-terminal valine is a preferential biotinylation site at pH 5.8, half of biotins being located on the alpha-amino group of this residue in a conjugate bearing one biotin/trimer (on average). Moreover, evidence has been obtained to suggest that the remaining part of biotins are linked to the epsilon-amino group of lysine 128, 112, and 65, while lysine 11, 90, and 98 were practically unmodified. No evidence of O-biotinylation of serine, threonine and tyrosine was obtained.
Magni, F., Curnis, F., Marazzini, L., Colombo, R., Sacchi, A., Corti, A., et al. (2001). Biotinylation sites of tumor necrosis factor-alpha determined by liquid chromatography-mass spectrometry. ANALYTICAL BIOCHEMISTRY, 298(2), 181-188 [10.1006/abio.2001.5374].
Citazione: | Magni, F., Curnis, F., Marazzini, L., Colombo, R., Sacchi, A., Corti, A., et al. (2001). Biotinylation sites of tumor necrosis factor-alpha determined by liquid chromatography-mass spectrometry. ANALYTICAL BIOCHEMISTRY, 298(2), 181-188 [10.1006/abio.2001.5374]. | |
Tipo: | Articolo in rivista - Articolo scientifico | |
Carattere della pubblicazione: | Scientifica | |
Presenza di un coautore afferente ad Istituzioni straniere: | No | |
Titolo: | Biotinylation sites of tumor necrosis factor-alpha determined by liquid chromatography-mass spectrometry | |
Autori: | Magni, F; Curnis, F; Marazzini, L; Colombo, R; Sacchi, A; Corti, A; Kienle, M | |
Autori: | ||
Data di pubblicazione: | 2001 | |
Lingua: | English | |
Rivista: | ANALYTICAL BIOCHEMISTRY | |
Digital Object Identifier (DOI): | http://dx.doi.org/10.1006/abio.2001.5374 | |
Appare nelle tipologie: | 01 - Articolo su rivista |