Abstract

Fluorescein is a good fluorescent label for high sensitivity analysis. The molecule has high molar absorptivity, 5 × 10⁴ L mol^-1 cm^-1 at 488 nm and near unit fluorescence quantum yield in the pH range of 8 to 10. Unfortunately, the molecule is not photostable undergoing irreversible photobleaching after absorbance of about 8,000 photons. Fluorescein may be used to label amino groups in amino acids, peptides, and proteins through the isothiocyanate derivative. Under basic conditions, the thiocarbamoyl derivative is formed, with relatively good stability. The reaction between amino acids and fluorescein isothiocyanate is first order in bod the concentration of amino acid and derivative, with an activation energy of about 16 kcal/mol. Under acidic conditions, the cyclic thiohydantoin derivative is formed, cleaving the terminal amino acid from proteins and peptides. This thiohydantoin derivative possesses greater photostability than the thiocarbamoyl derivative, decomposing after absorbance of about 12,000 photons. The thiocarbamoyl-thiohydantoin derivative series is the basis of an Edmon degradation scheme for protein sequencing. In addition to amino acid labeling, fluorescein may be used to label thiols through the bromobimane derivatives; a high sensitivity DNA analysis is based on this compound. Last, succinylfluorescein labeled chain terminating dideoxynucleotides are used in DNA sequencing, these molecules have similar spectral properties as fluorescein.

© 1990 Optical Society of America