A fluorometric assay of the degree of modification of protein primary amines with polyethylene glycol.

An alternative primary amine assay utilizing 4-phenylspiro[furan-2(3H),1'-phthalan]-3,3'-dione (fluorescamine) is compared with the frequently applied trinitrobenzenesulfonic acid assay as a means of estimating protein primary amine modification with cyanuric chloride attachment of polyethylene glycol (PEG). The results of the two assays are compared for three different proteins and several advantages of the fluorescamine assay are emphasized. In particular, the fluorescamine assay was found to be unaffected by free PEG up to 0.08% in the assay mixture. The fluorescamine assay can be used to measure the partition coefficients of native or PEG-modified protein in two-phase aqueous polymer systems.