Intracellular binding of fluorescein in lymphocytes

Abstract

The fluorescence characteristics of intracellular fluorescein, formed by 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 < 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 higher affinity and more rigidly (P = 0.43) to albumin than to intracellular macromolecules in general. In order to elucidate the state of intracellular fluorescein further, we have studied the fluorescence characteristics of intracellular fluorescein and compared them to those of fluorescein in phosphate buffered saline (PBS) and in solutions of glycerol and albumin in PBS, glycerol serving as a medium of lower polarity than water and albumin as a model for fluorescein binding proteins. The results are consistent with the assumption that part of the intracellular fluorescein is bound to macromolecules while the rest is dissolved in intracellular water. TheoryTo make a simple model for intracellular fluorescein binding, we assume that the fluorescence from the cells during FDA hydrolysis consists of two components: ( a ) fluorescence from unbound fluores-272

Spectroscopic properties of fluorescein in living lymphocytes

“…The other group is fluorescein molecules bound to intracellular structures, which have slightly red-shifted spectra and a relatively large fluorescence anisotropy. This result is consistent with the model that the degree of polarization is determined by the mixture of free and bound dye molecules (16,251.…”

“…Therefore, in the case of our experiment at 20"C, the leakage of dyes from the cells is considered to be negligible, and it is likely that almost all the fluorescein molecules are present inside the lymphocytes during the measurement. This point is very important in this type of experiment, because the leakage of fluorescein molecules from the cells causes undesirable background fluorescence, which becomes a significant source of error even if we employ compensation techniques, such as filtration (3), centrifugation (16,22), or fluorescence quenching (1).…”

Fluorescence Lifetime Microscopy of the Na+ Indicator Sodium Green in HeLa Cells

“…Indeed, we found that both K d (steady-state measurements) and K d app (lifetime measurements) are higher in the presence of BSA. A red shift in the emission spectra upon adding albumin to the buffer solution has been also observed for fluorescein (29) and the fluorescein-based pH indicator BCECF (30).…”

Analysis of enzyme kinetics in individual living cells utilizing fluorescence intensity and polarization measurements

“…The depolarization pattern, observed in both serum and in live cells, indicates various degrees of association and affinities of the fluorescein to other molecules (27)(28)(29).…”