Intracellular binding of fluorescein in lymphocytes

The fluorescence characteristics of intracellular fluorescein, formed by the hydrolysis of fluorescein diacetate in peripheral human lymphocytes, were studied by fluorometry on cell suspensions and compared to those of albumin bound and free fluorescein in solution. The absorption and fluorescence spectra of both intracellular fluorescein and fluorescein in aqueous solutions of albumin and glycerol were red shifted by 2-10 nm as compared to the spectra of fluorescein in phosphate buffered saline. The fluorescence polarization (P) of both intracellular fluorescein and a mixture of albumin-bound and free fluorescein showed a decrease towards low emission wavelengths and an increase toward high excitation wavelengths. The results were found to be consistent with a simple model assuming that part of the intracellular fluorescein is dissolved in the aqueous phase of the cytoplasm, giving P less than 0.1, while the rest is bound to macromolecules, giving P = 0.33. The fraction of bound intracellular fluorescein was estimated to be about 70%. Fluorescein was found to bind with high affinity and more rigidly (P = 0.43) to albumin than to intracellular macromolecules in general.