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.

[1]  A. Habeeb,et al.  Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. , 1966, Analytical biochemistry.

[2]  J. V. Alstine,et al.  Synthesis and characterization of poly(ethylene glycol) derivatives , 1984 .

[3]  Sidney Udenfriend,et al.  Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines in the Picomole Range , 1972, Science.

[4]  A. Sehon,et al.  Suppression of Reaginic Antibodies , 1978, Annales d'immunologie.

[5]  F. Davis,et al.  Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. , 1977, The Journal of biological chemistry.

[6]  B. Mattiasson,et al.  Immunological quantitation of bacterial cells using a partition affinity ligand assay: a model study on the quantitation of streptococci B. , 1982, Analytical biochemistry.

[7]  A. Sehon,et al.  Abrogation of reaginic antibodies with modified allergens , 1977, Nature.

[8]  F. Veronese,et al.  Anti‐inflammatory and pharmacokinetic properties of superoxide dismutase derivatized with polyethylene glycol via active esters , 1983, The Journal of pharmacy and pharmacology.

[9]  C. Stowell,et al.  A fluorescamine assay for submicrogram quantities of protein in the presence of Triton X-100. , 1978, Analytical biochemistry.

[10]  S. Pizzo,et al.  A new procedure for the synthesis of polyethylene glycol-protein adducts; effects on function, receptor recognition, and clearance of superoxide dismutase, lactoferrin, and alpha 2-macroglobulin. , 1983, Analytical biochemistry.

[11]  T. King,et al.  Preparation of protein conjugates with alkoxypolyethylene glycols. , 2009, International journal of peptide and protein research.