Estimation of the degradation of endocytosed material by flow cytofluorometry using two neoglycoproteins containing different numbers of fluorescein molecules

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Abstract
The fluorescence properties of fluorescein bound to protein are used to quantitate by flow cytofluorometry the degradation of fluorescein-labelled .alpha.-glucosylated serum albumin (fluorescein-labeled neoglycoprotein) after endocytosis by the membrane lectin of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein decreases when the number of fluorescein residues per protein molecule increases; however, after proteolytic digestion the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The extent of the degradation of the endocytosed neoglycoprotein was determined with a flow cytofluorometer by using two neoglycoproteins containing either a small or a large number of fluorescein residues per neoglycoprotein molecule. At 4.degree. C, 3LL cells bind 750,000 molecules of fluorescein-labelled .alpha.-glucosylated serum albumin with an apparent binding constant of 2 .times. 106 1 .times. mole-1. At 37.degree. C, after 4 hr incubation 2.2 .times. 106 molecules of fluorescent .alpha.-glucosylated serum albumin were cell-associated, and of these at least one third were degraded.

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