Analysis of degradation of proteins labeled with fluorescein isothiocyanate by Sephadex G-25 affinity chromatography.

Fluorescein isothiocyanate (FITC) has strong affinity to Sephadex G-25 gel. Amino acids labeled with FITC were found to have affinity to the gel, their elution from a Sephadex G-25 column being markedly retarded. On the other hand, proteins labeled with FITC had no affinity to the gel and were eluted in the void volume of the column. The affinity of FITC-labeled compounds to a Sephadex G-25 column decreased with increase in their molecular mass. On the basis of these findings, the degradations of various FITC-labeled proteins by disrupted lysosomes in vitro were analyzed using a small Sephadex G-25 column. FITC-labeled degradation products were easily separated from the substrate proteins, and their production was shown to increase with the incubation time and to be suppressed by the proteinase inhibitor leupeptin. This procedure is a simple method for assay of protein degradation and should be useful in analyses of the roles of proteinases in protein degradation.