Analysis of lysosomal degradation of fluorescein isothiocyanate- labelled proteins by Toyopearl HW-40 affinity chromatography.
Fluorescein isothiocyanate (FITC) was found to have a strong affinity to Toyopearl gel, which is used for gel filtration. FITC-labelled amino acids also showed affinity to Toyopearl gel, their elution from a Toyopearl HW-40 column being retarded. On the other hand, FITC-labelled proteins had no affinity to the gel and were recovered in the flow-through fractions. These findings were applied to the analysis of the degradation of various FITC-labelled proteins by lysosomal enzymes in vitro. FITC-labelled degradation products were easily separated from FITC-labelled substrate proteins on a small Toyopearl HW-40 column. Their production increased with the incubation time and was markedly suppressed by the proteinase inhibitor leupeptin. The FITC-labelled degradation product was identified to be mainly lysine with a FITC-labelled epsilon-amino group by its different elution position to those of lysine with a FITC-labelled alpha-amino group and other various FITC-labelled amino acids.
Ohshita
T
T
Institute for Enzyme Research, University of Tokushima, Japan.
Katanuma
N
N
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D008099:Liver / Q000648:ultrastructure*; D008239:Lysine / Q000032:analysis / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D011108:Polymers; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D051381:Rats
D005456:Fluorescent Dyes; D011108:Polymers; D011506:Proteins; C080256:Toyopearl HW 40; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine