Reaction of Fluorescein-Isothiocyanate with Proteins and Amino Acids

I. Covalent and Non-Covalent Binding of Fluorescein-Isothiocyanate and Fluorescein to Proteins

Abstract

1. The reaction of fluorescein-isothiocyanate (FITC) with proteins was studied under various conditions.
2. It was found that the α-amino groups of the N-terminal amino acids were the prime target of the reaction (at pH<9.5) of FITC with neocarzinostatin and insulin although the ε-amino groups became reactive at higher pH.
3. Egg white lysozyme [EC 3. 2. 1. 17] and bovine serum albumin were found to bind with FITC even at low pH, while ovomucoid and neocarzinostatin did not bind below pH 7.
4. Trifluoroacetic acid treatment of fluorescein-thiocarbamylated insulin (FTC-insulin) resulted in the release of terminal FTH-amino acids (Phe and Gly) only. These FTH-amino acids were identified for the first time.
5. Non-covalent binding of this fluorochrome to proteins was studied with fluoro-scein. The results indicated non-covalent dye-binding of the fluorescein with albumin and Iysozyme [EC 3. 2. 1. 17].
6. Above results showed that FITC is a useful reagent but that non-covalent dye-binding should be born in mind when used with some proteins.