AvidinOX, the oxidized derivative of
Avidin, is a chemically modified
glycoprotein, being currently under clinical investigation for targeted delivery of radioactive
biotin to inoperable
tumors. AvidinOX is produced by 4-hydroxyazobenzene-2-carboxylic
acid (
HABA)-assisted
sodium periodate oxidation of
Avidin. The peculiar property of the
periodate-generated glycol-split
carbohydrate moieties to form Schiff's bases with amino groups of the tissue
proteins allows to achieve a tissue half-life of 2 weeks compared to 2 h of native
Avidin.
Carbohydrate oxidation, along with possible minor
amino acid modifications, introduces additional microheterogeneity in the
glycoprotein structure, making its characterization even more demanding than for native
glycoproteins. Aiming at the elucidation of the effects of oxidation conditions on the AvidinOX
protein backbone and
sugars, this microheterogeneous
glycoprotein derivative was characterized for the first time using a combination of different analytical methods, including colorimetric methods, mass spectrometry, hollow-fiber flow field-flow fractionation with UV and multi-angle
laser scattering detection (HF5-UV-MALS), and NMR. The proposed integrated approach reveals structural features of AvidinOX relevant for its biological activity, e.g., oxidized sites within both
carbohydrate moieties and
protein backbone and conformational stability, and will be considered as an analytical tool for AvidinOX industrial preparations. It is worth noting that this study enriches also the structural data of native
Avidin published up-to-date (e.g.,
glycan structure and distribution,
peptide fingerprint, etc.). Graphical abstract Scheme of phenylacetic
hydrazide/MALDI-TOF approach for quantification of
aldehydes in AvidinOX based on the determination of the number of
hydrazone adducts between
hydrazide reagent and
aldehyde groups of
protein.