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To overcome the uncertainty of the colorimetric or fluorimetric method so far employed for the evaluation of monomethoxy(polyethylene glycol) (MPEG) covalently bound to protein, a direct method based on amino acid analysis is proposed. The method exploits the use of MPEG, which was bounded with the unnatural amino acid norleucine (MPEG-Nle). MPEG-Nle was activated at its carboxylic group to succinimidyl ester for the binding to the amino groups of protein. After acid hydrolysis, the amino acid content is evaluated by conventional amino acid analyzer or by reverse-phase HPLC as phenylthiocarbamyl derivative. The number of bound MPEG chains is calculated from the amino acid composition, since one norleucine residue is released from each bound polymer chain. The method was verified with several proteins in comparison with colorimetric ones, also in the case of proteins that contain chromophores in the visible range, such cytocrome C. It was observed that in most of the cases, the colorimetric methods give an overestimation of the degree of protein modification.
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